WO2023083298A1 - 抗icosl抗体融合蛋白及用途 - Google Patents

抗icosl抗体融合蛋白及用途 Download PDF

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WO2023083298A1
WO2023083298A1 PCT/CN2022/131362 CN2022131362W WO2023083298A1 WO 2023083298 A1 WO2023083298 A1 WO 2023083298A1 CN 2022131362 W CN2022131362 W CN 2022131362W WO 2023083298 A1 WO2023083298 A1 WO 2023083298A1
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seq
amino acid
acid sequence
icosl
variable region
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PCT/CN2022/131362
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French (fr)
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毛浪勇
应华
李玲玲
黄旋
金薪盛
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to CN202280073112.2A priority Critical patent/CN118176216A/zh
Publication of WO2023083298A1 publication Critical patent/WO2023083298A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

Definitions

  • the present disclosure belongs to the field of biotechnology, and more specifically, the present disclosure relates to an anti-ICOSL antibody and its fusion protein and applications.
  • SLE Systemic lupus erythematosus
  • TLR receptors stimulating pDC cells to secrete a large amount of interferon ⁇ .
  • Interferon further stimulates myeloid cells to secrete various cytokines, respectively promoting the activation of B cells and T cells (Curr Opin Rheumatol. 2017 Mar; 29(2):178-186.).
  • BAFF and APRIL are two activating factors of B cells, which promote the differentiation and maturation of B cells and the production of antibodies by interacting with receptors on the surface of B cells (Nat Rev Rheumatol.2016Nov22; 12(12):716-730.) .
  • the signaling pathways of ICOSL and ICOS play a very important role in the process of T cell activation and T-B cell interaction.
  • the release of pro-inflammatory cytokines after T cell activation leads to the differentiation of pro-inflammatory T cells and disrupts the homeostasis of Treg cells in vivo.
  • T-B cells will further promote the activation of T cells and B cells and the production of pro-inflammatory cytokines to form a continuous immune response cycle (Nat Rev Rheumatol. 2014Jan; 10(1):23-34.).
  • the two immune pathways of T cells and B cells are involved. Simultaneously inhibiting the signaling pathways of T cell and B cell activation and T-B cell interaction will have a better effect on SLE.
  • the ICOS/ICOSL signaling pathway also plays an important role in other immune responses such as inflammatory reactions, allergic reactions, autoimmune diseases, transplant rejection, and B cell or T cell disorders (such as cancer).
  • ICOSL Inducible costimulator-ligand, also known as B7RP1, B7H2, CD275
  • B7RP1, B7H2, CD275 is an immune co-stimulatory molecule belonging to the B7 family.
  • ICOSL is mainly expressed on the surface of antigen-presenting cells, including B cells, macrophages, monocytes and dendritic cells (BioDrugs. 2013 Feb; 27(1): 1-13.).
  • the receptor of ICOSL is ICOS
  • ICOS is mainly expressed on the surface of T cells.
  • the signaling pathways mediated by ICOSL and ICOS mainly play a role in the differentiation of T cells and the activation of B cells dependent on T cells (Nat Rev Cancer.2012Mar 22 ; 12(4):252-64.).
  • ICOSL-ICOS signaling pathway can promote Th2, Th1, Th17 and Tfh cells to secrete and express IL-4, IFN ⁇ , IL-23R and IL-21, respectively, and these cytokines and receptors are involved in maintaining the activity of various T cell subtypes And promote their further amplification (Curr Opin Immunol.2010Jun; 22(3):326-32.).
  • ICOSL also plays a very important function in the process of Tfh cell migration and T-B cell interaction.
  • TACI Transmembrane activator and CAML interactor, also known as TNFRSF13B
  • TNFRSF13B Transmembrane activator and CAML interactor
  • TACI is associated with a subset of B cells and T cells.
  • the TACI receptor binds to BAFF (B-cell activating factor, also known as TNFSF13B) of the tumor necrosis factor ligand family.
  • BAFF is a B-cell activator belonging to the TNF family.
  • BAFF is mainly expressed on the surface of bone marrow cell membrane and exists in the form of trimer.
  • the BAFF on the surface of the cell membrane will be hydrolyzed by protease to form soluble BAFF and enter the blood circulation system.
  • the soluble BAFF has the characteristics of multimerization, and can form up to 60-mers.
  • BAFF can also interact with another protein of the same family, APRIL, to form a heterologous trimer.
  • APRIL another protein of the same family
  • BAFF receptors on the surface of B cells namely BAFF-R, BCMA and TACI.
  • BAFF interacts with these three receptors and participates in the differentiation, maturation, survival and regulation of B cells.
  • APRIL and BAFF have two common receptors, namely BCMA and TACI.
  • APRIL interacts with these two receptors to participate in the survival and regulation of B cells (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 is important for maintaining B cell homeostasis, and overactivation of BAFF signaling leads to the survival of self-reactive B cells and the 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).
  • the present disclosure constructs an anti-ICOSL antibody fusion protein, which comprises an anti-ICOSL antibody and a TACI polypeptide.
  • the anti-ICOSL antibody fusion protein 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 The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein,
  • HCDR1, HCDR2 and HCDR3 of the heavy chain variable region comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 42, 3, 39, 40 or 41, respectively, and the light chain variable region
  • the LCDR1, LCDR2 and LCDR3 comprise the amino acid sequence of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 38, 4, 32, 33, 34, 35, 36 or 37, respectively; or
  • HCDR1, HCDR2 and HCDR3 of the heavy chain variable region comprise the amino acid sequence of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 30, 1, 28 or 29, respectively
  • the LCDR1 of the light chain variable region , LCDR2 and LCDR3 comprise the amino acid sequence of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 22, 2, 20, 21, 23, 24, 25, 26 or 27, respectively.
  • the anti-ICOSL antibody fusion protein according to 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 selected from Kabat , IMGT, Chothia, AbM and Contact numbering rules defined.
  • the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the Kabat numbering convention.
  • 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 IMGT numbering convention. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the Chothia numbering convention. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and 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.
  • the anti-ICOSL antibody fusion protein of any one of the above, 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
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 13
  • the 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
  • LCDR3 comprises the amino acid sequence of SEQ ID NO: 16;
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5
  • HCDR2 comprises the amino acid sequence of SEQ ID NO: 19 or 6
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 7
  • the 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
  • LCDR3 comprises the amino acid sequence of SEQ ID NO:10 or 18.
  • the anti-ICOSL antibody fusion protein of any one of the above, 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
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 13
  • the light chain LCDR1 of the variable region comprises the amino acid sequence of SEQ ID NO: 14
  • LCDR2 comprises the amino acid sequence of SEQ ID NO: 15
  • LCDR3 comprises the amino acid sequence of SEQ ID NO: 16;
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5
  • HCDR2 comprises the amino acid sequence of SEQ ID NO: 19
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 7
  • the light chain LCDR1 of the 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 anti-ICOSL antibody fusion protein of any one of the above, 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: 12
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 13
  • the light chain variable region The LCDR1 comprises the amino acid sequence of SEQ ID NO: 14, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 16.
  • the anti-ICOSL antibody fusion protein of any one of the above, wherein
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5
  • HCDR2 comprises the amino acid sequence of SEQ ID NO: 6
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region
  • the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8
  • the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9
  • the LCDR3 comprises the amino acid sequence of SEQ ID NO: 10; or
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5
  • HCDR2 comprises the amino acid sequence of SEQ ID NO: 6
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region
  • the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8
  • the LCDR2 comprises the amino acid sequence of SEQ ID NO: 17
  • the LCDR3 comprises the amino acid sequence of SEQ ID NO: 10; or
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5
  • HCDR2 comprises the amino acid sequence of SEQ ID NO: 19
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 7
  • the light chain variable region The LCDR1 comprises the amino acid sequence of SEQ ID NO:8, the LCDR2 comprises the amino acid sequence of SEQ ID NO:9, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:10.
  • the anti-ICOSL antibody fusion protein according to any one of the above, is a murine antibody, a chimeric antibody or a humanized antibody.
  • the antibody is a humanized antibody.
  • the anti-ICOSL antibody fusion protein of any one of the above, wherein:
  • the heavy chain variable region comprises at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 42, 39, 40 or 41 and said light chain variable region comprises at least 90% (e.g., at least 90%, 95%, 96%, 97%) of SEQ ID NO: 38, 32, 33, 34, 35, 36 or 37 , 98% or 99%) sequence identity of amino acid sequences; or
  • the heavy chain variable region comprises at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 30, 28 or 29 Amino acid sequence, and said light chain variable region comprises at least 90% (e.g., at least 90%, 95%, 96%, 97%) of SEQ ID NO: 22, 20, 21, 23, 24, 25, 26 or 27 , 98% or 99%) sequence identity amino acid sequence; or (iii) said heavy chain variable region comprises an amino acid sequence having at least 90% (for example at least 90%, 95%, 96%, 97%) with SEQ ID NO: 3 , 98% or 99%) sequence identity amino acid sequence, and said light chain variable region comprises an amino acid sequence with SEQ ID NO: 4 having at least 90% (for example at least 90%, 95%, 96%, 97%, 98%) or 99%) amino acid sequences of sequence identity; or
  • said heavy 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: 1
  • the light chain variable region comprises an amino acid sequence having at least 90% (eg, at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 2.
  • the anti-ICOSL antibody fusion protein comprises a frame region selected from positions 37, 43 and 49 (numbering according to the Kabat numbering system) ) in one or more amino acid mutations, and/or the framework region of the light chain variable region contains a group selected from positions 1, 2, 3, 42, 43, 60 and 85 (numbered according to the Kabat numbering system) One or more amino acid mutations.
  • the anti-ICOSL antibody fusion protein wherein the 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 comprising the amino acid sequence of SEQ ID NO: 13, and LCDR1 of said light chain variable region comprising the amino acid sequence of SEQ ID NO: 14, LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 16 Amino acid sequence; the framework region of the heavy chain variable region of the antibody comprises one or more amino acid mutations selected from: 37I, 43E and 49A (numbering according to the Kabat numbering system), and/or the light chain can be The framework region of the variable region contains one or more amino acid mutations selected from: 1N, 2T, 3V, 42Q, 43S, 60D, 85V (numbered according to the Kabat numbering system).
  • the anti-ICOSL antibody fusion protein according to any one of the above, the framework region of the heavy chain variable region of the anti-ICOSL antibody comprises (numbering according to the Kabat numbering system) in one or more amino acid mutations, and/or the frame region of the light chain variable region comprises one or More amino acid mutations.
  • the anti-ICOSL antibody fusion protein wherein HCDR1 of the heavy chain variable region of the anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 5, and HCDR2 comprises the amino acid sequence of SEQ ID NO: 19 or 6, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 7, and LCDR1 of the light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, LCDR2 comprising the amino acid sequence of SEQ ID NO: 17 or 9, and LCDR3 comprising the amino acid sequence of SEQ ID NO: the amino acid sequence of 10 or 18;
  • the framework region of the heavy chain variable region of the antibody comprises one or more of the following: 1E, 24T, 69L, 71V, 73K and 78A (numbered according to the Kabat numbering system) amino acid mutations, and/or the framework region of the light chain variable region comprises one or more amino acid mutations selected from: 1A, 2V and 4L (numbering according to the Kabat numbering system).
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein,
  • said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 42, 39, 40 or 41, and said light chain variable region comprises SEQ ID NO: 38, 32, 33, 34, 35, 36 or the amino acid sequence of 37; or
  • said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 30, 28 or 29, and said light chain variable region comprises SEQ ID NO: 22, 20, 21, 23, 24, 25, 26 or the amino acid sequence of 27; or
  • said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1, and said light chain variable region comprises the amino acid sequence of SEQ ID NO: 2;
  • said heavy chain variable region comprises the amino acid sequence of SEQ ID NO:3
  • said light chain variable region comprises the amino acid sequence of SEQ ID NO:4.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein,
  • said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 42, and said light chain variable region comprises the amino acid sequence of SEQ ID NO: 38;
  • said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 30, and said 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 the above, wherein the anti-ICOSL antibody comprises an antibody heavy chain constant region and a light chain constant region.
  • the heavy chain constant region is a human IgG heavy chain constant region.
  • the heavy chain constant region is selected from human IgGl, IgG2, IgG3 and IgG4 constant regions.
  • the light chain constant region is selected from a human antibody kappa or lambda chain constant region.
  • 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.
  • the Fc region of the heavy chain constant region has one or more amino acid substitutions that reduce binding of the Fc region to an Fc receptor.
  • the Fc region has L234A, L235A mutations, and/or S228P mutations, and/or YTE mutations (M252Y, S254T, and T256E), and the numbering of the mutations is based on the EU index.
  • the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:43
  • the light chain constant region comprises the amino acid sequence of SEQ ID NO:44.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein,
  • the heavy chain of said anti-ICOSL antibody 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: 47, and a light chain comprising 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; or
  • the heavy chain of the anti-ICOSL antibody 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: 45, and light chains comprising an amino acid sequence having at least 90% (eg, at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 46.
  • the anti-ICOSL antibody fusion protein according to any one of the above,
  • the heavy chain of the anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 47
  • the light chain of the anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 48
  • the heavy chain of the anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 45
  • 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 the above, wherein the TACI polypeptide has a better function of preventing fragmentation.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein the TACI polypeptide is a polypeptide comprising the 48th to 85th amino acid residues of SEQ ID NO: 58 or a variant thereof; wherein , the variant has an amino acid substitution at one or more positions selected from positions 49, 52, 53, 57, 65, 82 and 83, and the position of the amino acid substitution is relative to the sequence SEQ ID NO: 58 amino acid residue positions numbered in natural order.
  • the variant of the TACI polypeptide is one or more amino acids selected from the group consisting of 49T or 49R, 52S, 53E or 53Q, 57E, 65T or 65A, 82A or 82R, and 83Y Replacement, the position of the amino acid replacement is the amino acid residue position numbered relative to the natural sequence of the sequence SEQ ID NO:58.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein the TACI polypeptide is shown as SEQ ID NO: 58 or a truncated fragment of SEQ ID NO: 58 or a variant thereof.
  • the truncated fragment comprises the 48th to 85th amino acid residues of SEQ ID NO:58.
  • the variant has one or more amino acid substitutions selected from positions 49, 52, 53, 57, 65, 82 and 83 on SEQ ID NO: 58 or a truncated fragment thereof, wherein the amino acid substitution The positions are the amino acid residue positions numbered relative to the natural sequence of the sequence SEQ ID NO:58.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein the truncated fragment of the TACI polypeptide comprises: the 48th to the 86th amino acid residues of SEQ ID NO: 58; SEQ ID NO : the 48th to the 87th amino acid residue of 58; or the 48th to the 88th amino acid residue of SEQ ID NO: 58.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein the sequence of the TACI polypeptide is shown in any one of SEQ ID NO: 60-63.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein, the TACI polypeptide, its sequence is a variant of SEQ ID NO: 58 or a truncated fragment of SEQ ID NO: 58 (such as SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or the variant of the truncated fragment of the sequence of SEQ ID NO: 63), said variant has on SEQ ID NO: 58 or its truncated fragment sequence Replacement of any 1, 2, 3, 4, 5, 6 or 7 amino acids selected from positions 49, 52, 53, 57, 65, 82 and 83, the position of the amino acid replacement Dots are amino acid residue positions numbered relative to the natural order of the sequence SEQ ID NO:58.
  • the anti-ICOSL antibody fusion protein according to 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 one or more amino acid substitutions selected from the group consisting of 49T or 49R, 52S, 53E or 53Q, 57E, 65T or 65A, 82A or 82R, and 83Y (for example, 1, 2 , 3, 4, 5, 6 or 7 amino acid substitutions), wherein the position of the amino acid substitution is the amino acid residue position numbered relative to the natural sequence of the sequence SEQ ID NO:58.
  • amino acid substitutions selected from the group consisting of 49T or 49R, 52S, 53E or 53Q, 57E, 65T or 65A, 82A or 82R, and 83Y (for example, 1, 2 , 3, 4, 5, 6 or 7 amino acid substitutions), wherein the position of the amino acid substitution is the amino acid residue position
  • the anti-ICOSL antibody fusion protein according to 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 there is any amino acid substitution selected from 49T, 52S, 53E, 53Q, 57E and 82A on the sequence of SEQ ID NO: 63. There are 49R and 65T amino acid substitutions on the sequence of SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein, the amino acid sequence of the TACI polypeptide is as shown in any one of SEQ ID NO: 51 to 83.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein the amino acid sequence of the TACI polypeptide is shown in any one of SEQ ID NO: 60-63, SEQ ID NO: 66-83.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein the amino acid sequence of the TACI polypeptide is shown in SEQ ID NO: 83.
  • the anti-ICOSL antibody fusion protein as described in any one of the above it comprises:
  • Second chain the light chain of an anti-ICOSL antibody ( Figure 2); or
  • the second chain [light chain of anti-ICOSL antibody] - [linker 2] - [TACI polypeptide 2] ( Figure 3);
  • the TACI polypeptide 1 and the TACI polypeptide 2 are the same or different, and the linker 1 and the linker 2 are the same or different.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein the TACI polypeptide 1 or TACI polypeptide 2 has an amino acid sequence as shown in any one of SEQ ID NO: 51 to 83.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein the amino acid sequence of the TACI polypeptide 1 or TACI polypeptide 2 is as in SEQ ID NO: 60-63, SEQ ID NO: 66-83 either shown.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein the amino acid sequence of the TACI polypeptide 1 or TACI polypeptide 2 is shown in SEQ ID NO: 83.
  • the linker 1 and linker 2 each independently have a structure of (G x S) y , wherein x is selected from an integer of 1-5, and y is selected from an integer of 0-6. In some embodiments, the linker 1 and linker 2 each independently have a structure of (G x S) y , wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6. In some embodiments, the linker 1 and the linker 2 are each independently GGGS (shown in SEQ ID NO: 89) or GGGGSGGGGSGGGGS (shown in SEQ ID NO: 90).
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein, the anti-ICOSL antibody fusion protein has: a first chain comprising the amino acid sequence of SEQ ID NO: 86, and comprising SEQ ID NO : the second chain of the amino acid sequence of 88, 87 or 48; or the anti-ICOSL antibody fusion protein has: the first chain comprising the amino acid sequence of SEQ ID NO: 84, and the amino acid comprising SEQ ID NO: 85 or 46 the second strand of the sequence. In some embodiments, the anti-ICOSL antibody fusion protein has two first chains comprising the amino acid sequence of SEQ ID NO:86, and two second chains comprising the amino acid sequence of SEQ ID NO:88.
  • the anti-ICOSL antibody fusion protein has two first chains comprising the amino acid sequence of SEQ ID NO:86, and two second chains comprising the amino acid sequence of SEQ ID NO:87. In some embodiments, the anti-ICOSL antibody fusion protein has two first chains comprising the amino acid sequence of SEQ ID NO:86, and two second chains comprising the amino acid sequence of SEQ ID NO:48. In some embodiments, the anti-ICOSL antibody fusion protein has two first chains comprising the amino acid sequence of SEQ ID NO:84, and two second chains comprising the amino acid sequence of SEQ ID NO:85. In some embodiments, the anti-ICOSL antibody fusion protein has two first chains comprising the amino acid sequence of SEQ ID NO:84, and two second chains comprising the amino acid sequence of SEQ ID NO:46.
  • the present disclosure provides an anti-ICOSL antibody, 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 The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein,
  • HCDR1, HCDR2 and HCDR3 of the heavy chain variable region comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 42, 3, 39, 40 or 41, respectively, and the light chain variable region
  • the LCDR1, LCDR2 and LCDR3 comprise the amino acid sequence of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 38, 4, 32, 33, 34, 35, 36 or 37, respectively; or
  • HCDR1, HCDR2 and HCDR3 of the heavy chain variable region comprise the amino acid sequence of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 30, 1, 28 or 29, respectively
  • the LCDR1 of the light chain variable region , LCDR2 and LCDR3 comprise the amino acid sequence of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 22, 2, 20, 21, 23, 24, 25, 26 or 27, respectively.
  • the anti-ICOSL antibody according to 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 selected from Kabat, IMGT , Chothia, AbM and Contact numbering rules defined.
  • the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the Kabat numbering convention.
  • 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 IMGT numbering convention.
  • the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the Chothia numbering convention. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and 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.
  • 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
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 13
  • the 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
  • LCDR3 comprises the amino acid sequence of SEQ ID NO: 16;
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5
  • HCDR2 comprises the amino acid sequence of SEQ ID NO: 19 or 6
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 7
  • the 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
  • LCDR3 comprises the amino acid sequence of SEQ ID NO:10 or 18.
  • 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
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 13
  • the light chain LCDR1 of the variable region comprises the amino acid sequence of SEQ ID NO: 14
  • LCDR2 comprises the amino acid sequence of SEQ ID NO: 15
  • LCDR3 comprises the amino acid sequence of SEQ ID NO: 16;
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5
  • HCDR2 comprises the amino acid sequence of SEQ ID NO: 19
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 7
  • the light chain LCDR1 of the 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 anti-ICOSL antibody according to any one of the above, said anti-ICOSL antibody is a murine antibody, a chimeric antibody or a humanized antibody. In some embodiments, the antibody is a humanized antibody.
  • the heavy chain variable region comprises at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 42, 39, 40 or 41 and said light chain variable region comprises at least 90% (e.g., at least 90%, 95%, 96%, 97%) of SEQ ID NO: 38, 32, 33, 34, 35, 36 or 37 , 98% or 99%) sequence identity of amino acid sequences; or
  • the heavy chain variable region comprises at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 30, 28 or 29 Amino acid sequence, and said light chain variable region comprises at least 90% (e.g., at least 90%, 95%, 96%, 97%) of SEQ ID NO: 22, 20, 21, 23, 24, 25, 26 or 27 , 98% or 99%) sequence identity amino acid sequence; or (iii) said heavy chain variable region comprises an amino acid sequence having at least 90% (for example at least 90%, 95%, 96%, 97%) with SEQ ID NO: 3 , 98% or 99%) sequence identity amino acid sequence, and said light chain variable region comprises an amino acid sequence with SEQ ID NO: 4 having at least 90% (for example at least 90%, 95%, 96%, 97%, 98%) or 99%) amino acid sequences of sequence identity; or
  • said heavy 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: 1
  • the light chain variable region comprises an amino acid sequence having at least 90% (eg, at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 2.
  • the anti-ICOSL antibody according to any one of the above, the framework region of the heavy chain variable region of the anti-ICOSL antibody comprises an One or more amino acid mutations of , and/or the frame region of the light chain variable region comprises one or More amino acid mutations.
  • the anti-ICOSL antibody fusion protein wherein the 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 comprising the amino acid sequence of SEQ ID NO: 13, and LCDR1 of said light chain variable region comprising the amino acid sequence of SEQ ID NO: 14, LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 16 Amino acid sequence; the framework region of the heavy chain variable region of the antibody comprises one or more amino acid mutations selected from: 37I, 43E and 49A (numbering according to the Kabat numbering system), and/or the light chain can be The framework region of the variable region contains one or more amino acid mutations selected from: 1N, 2T, 3V, 42Q, 43S, 60D, 85V (numbered according to the Kabat numbering system).
  • the anti-ICOSL antibody according to any one of the above, the framework region of the heavy chain variable region of the anti-ICOSL antibody comprises positions selected from positions 1, 24, 69, 71, 73, and 78 (according to One or more amino acid mutations in the Kabat numbering system numbering), and/or one or more amino acid mutations selected from positions 1, 2 and 4 (numbering according to the Kabat numbering system) on the framework region of the light chain variable region amino acid mutation.
  • the anti-ICOSL antibody fusion protein wherein HCDR1 of the heavy chain variable region of the anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 5, and HCDR2 comprises the amino acid sequence of SEQ ID NO: 19 or 6, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 7, and LCDR1 of the light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, LCDR2 comprising the amino acid sequence of SEQ ID NO: 17 or 9, and LCDR3 comprising the amino acid sequence of SEQ ID NO: the amino acid sequence of 10 or 18;
  • the framework region of the heavy chain variable region of the antibody comprises one or more of the following: 1E, 24T, 69L, 71V, 73K and 78A (numbered according to the Kabat numbering system) amino acid mutations, and/or the framework region of the light chain variable region comprises one or more amino acid mutations selected from: 1A, 2V and 4L (numbering according to the Kabat numbering system).
  • said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 42, 39, 40 or 41, and said light chain variable region comprises SEQ ID NO: 38, 32, 33, 34, 35, 36 or the amino acid sequence of 37; or
  • said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 30, 28 or 29, and said light chain variable region comprises SEQ ID NO: 22, 20, 21, 23, 24, 25, 26 or the amino acid sequence of 27; or
  • said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1, and said light chain variable region comprises the amino acid sequence of SEQ ID NO: 2;
  • said heavy chain variable region comprises the amino acid sequence of SEQ ID NO:3
  • said light chain variable region comprises the amino acid sequence of SEQ ID NO:4.
  • said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 42, and said light chain variable region comprises the amino acid sequence of SEQ ID NO: 38;
  • said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 30, and said light chain variable region comprises the amino acid sequence of SEQ ID NO: 22.
  • the anti-ICOSL antibody according to any one of the above, wherein said anti-ICOSL antibody comprises an antibody heavy chain constant region and a light chain constant region.
  • the heavy chain constant region is a human IgG heavy chain constant region.
  • the heavy chain constant region is selected from human IgG1, IgG2, IgG3 and IgG4 constant regions; in some embodiments, the light chain constant region is selected from human antibody kappa or lambda chain constant regions.
  • 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.
  • the Fc region of the heavy chain constant region has one or more amino acid substitutions that reduce binding of the Fc region to an Fc receptor.
  • the Fc region has L234A, L235A mutations, and/or S228P mutations, and/or YTE mutations (M252Y, S254T, and T256E), and the numbering of the mutations is based on the EU index.
  • the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:43
  • the light chain constant region comprises the amino acid sequence of SEQ ID NO:44.
  • the heavy chain of said anti-ICOSL antibody 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: 47, and a light chain comprising 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; or
  • the heavy chain of the anti-ICOSL antibody 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: 45, and light chains comprising an amino acid sequence having at least 90% (eg, at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 46.
  • the anti-ICOSL antibody of any one of the above is anti-ICOSL antibody of any one of the above.
  • the heavy chain of the anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 47
  • the light chain of the anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 48
  • the heavy chain of the anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 45
  • the light chain of the anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 46.
  • the present disclosure also provides an isolated anti-ICOSL antibody or anti-ICOSL antibody fusion protein, which competes for binding to human ICOSL or its expression with the anti-ICOSL antibody or anti-ICOSL antibody fusion protein described in any one of the preceding bit.
  • the anti-ICOSL antibody or anti-ICOSL antibody fusion protein of any one of the foregoing has one or more of the following characteristics:
  • A. specifically binds to human ICOSL (or its epitope) and cynomolgus monkey ICOSL (or its epitope), and does not specifically bind to mouse ICOSL; 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) KD value combined with human ICOSL, and/or less than 8.00E-09M ( For example, a KD value of 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) combined with cynomolgus ICOSL,
  • the KD value is measured by surface plasmon resonance assay (e.g. surface plasmon resonance assay); in some embodiments, the KD value is detected by the method of Test Example 8 of the present disclosure;
  • B. specifically binds to human APRIL (or its epitope), cynomolgus monkey APRIL (or its epitope) and/or mouse APRIL (or its epitope); preferably, with less than 2.00E-11M (such as less than 2.00 E-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) combined with human APRIL to less than 2.00E-11M KD of 10M (e.g.
  • the KD value is combined with mouse APRIL, and the KD value is measured by surface plasmon resonance assay (for example surface plasmon resonance assay); in some embodiments, the KD value is detected by the method of Test Example 8 of the present disclosure;
  • C. specifically binds to human BAFF (or its epitope), cynomolgus monkey BAFF (or its epitope) and/or mouse BAFF (or its epitope); E-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) combined with human BAFF to less than 4.00E-11M 10M (such as less than 4.00E-10M, less than 3.00E-10M, less than 2.00E-10M, less than 1.30E-10M or less) KD value combined with cynomolgus monkey BAFF, and/or less than 6.00E-11M ( For example, a KD value of less than 6.00E-11M, less than 5.00E-11M, less than 4.00E-11M, less than 3.20E-11M or less) binds to mouse BAFF as measured by surface plasmon resonance assay ( For example surface plasmon resonance assay); In some embodiments, the KD value
  • the IC value of blocking the combination of human ICOSL and ICOS is less than 0.16nM (such as 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), the IC50 value is detected by FACS method; in some embodiments, the IC50 value is tested by the present disclosure Example 3 method detection;
  • the IC value of blocking the combination of APRIL and BCMA is less than 40.00nM (such as 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), the IC50 value is detected by ELISA method; 4 method detection;
  • the IC value of blocking the combination of APRIL and TACI is less than 40.00nM (such as 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 IC50 value is detected by ELISA method; 4 method detection;
  • the IC value of blocking the combination of BAFF and BCMA is less than 2.00nM (such as 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), the IC50 value is detected by ELISA method; in some embodiments, the IC50 value is detected by the method of Test Example 4 of this disclosure;
  • the IC value of blocking the combination of BAFF and BAFF-R is less than 2.00nM (such as 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 is detected by ELISA method; in some embodiments, the IC 50 value is detected by the method of Test Example 4 of this disclosure;
  • the IC value of blocking BAFF and TACI binding is less than 1.00nM (such as less than 1.00nM, less than 0.50nM, less than 0.30nM, less than 0.20nM, less than 0.15nM or Smaller), the IC 50 value is detected by ELISA method; in some embodiments, the IC 50 value is detected by the method of Test Example 4 of the present disclosure;
  • J. has the activity of inhibiting the secretion of cytokines (such as IFN ⁇ , IL-10 and/or IgA); in some embodiments, the IC50 value is detected by the method of Test Example 9 of the present disclosure;
  • cytokines such as IFN ⁇ , IL-10 and/or IgA
  • the K. has the activity of inhibiting B cell proliferation and/or T cell proliferation;
  • the anti-ICOSL antibody or anti-ICOSL antibody fusion protein inhibits the activity of ICOSL-induced T cell proliferation;
  • the The activity of inhibiting T cell proliferation is detected by the method of Test Example 6 of the present disclosure; in some embodiments, the anti-ICOSL antibody fusion protein inhibits the activity of BAFF-induced or APRIL-induced B cell proliferation; in some embodiments, the inhibition The proliferative activity of B cells was detected by the method of Test Example 7 of the present disclosure.
  • the present disclosure also provides a pharmaceutical composition, which comprises any one of the anti-ICOSL antibody fusion protein or anti-ICOSL antibody described above, and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-ICOSL antibody described in any one of the foregoing, and the TACI polypeptide as shown in any one of SEQ ID NO: 51 to 83, and one or more pharmaceutically acceptable acceptable carrier, diluent or excipient;
  • the TACI polypeptide sequence is shown in any one of SEQ ID NO:60-63, SEQ ID NO:66-83;
  • the TACI polypeptide sequence is as shown in SEQ ID NO: 83.
  • the present disclosure provides a nucleic acid molecule encoding the anti-ICOSL antibody fusion protein or anti-ICOSL antibody of any one of the foregoing.
  • the present disclosure provides an expression vector comprising the aforementioned nucleic acid molecule (DNA or RNA).
  • the present disclosure provides a host cell comprising the aforementioned nucleic acid molecule.
  • the present disclosure provides a host cell comprising the aforementioned expression vector.
  • the host cells provided in this disclosure cannot develop into individual animals or plants.
  • 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 administering to a subject in need thereof a therapeutically effective amount of any of the foregoing The step of anti-ICOSL antibody fusion protein or anti-ICOSL antibody or pharmaceutical composition described in item.
  • the methods of the present disclosure are used to treat autoimmune or inflammatory diseases.
  • the disclosed methods are used to treat a B-cell disorder or a T-cell disorder.
  • the present disclosure provides the use of the anti-ICOSL antibody fusion protein or anti-ICOSL antibody, nucleic acid molecule or pharmaceutical composition described in any one of the foregoing in the preparation of a medicament for treating a disease.
  • the present disclosure also provides the anti-ICOSL antibody fusion protein or anti-ICOSL antibody, nucleic acid molecule or composition described in any one of the foregoing for use as a medicine.
  • the medicament is used to treat an autoimmune disease, an inflammatory disease, a B cell disorder, or a T cell disorder.
  • the medicament is used to treat an autoimmune or inflammatory disease.
  • the medicament is used to treat a B cell disorder or a T cell disorder.
  • the disease of any one of the preceding is a disease or disorder associated with high expression of ICOSL.
  • the disease or condition of any of the preceding is an autoimmune disease, an inflammatory disease, a B cell disorder, or a T cell disorder.
  • the autoimmune or inflammatory disease is selected from the group consisting of: systemic lupus erythematosus, rheumatoid arthritis, graft versus host disease, asthma, immune thrombocytopenic purpura, multiple sclerosis, diabetes Inflammatory diseases caused by psoriasis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Graves' disease, and Hashimoto's thyroiditis.
  • the B-cell disorder or T-cell disorder is a tumor.
  • the tumor is selected from the group consisting of 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.
  • the treatment of any of the preceding further comprises administering to the subject an additional therapeutic agent.
  • Figure 1 Schematic structure of anti-ICOSL antibody fusion proteins such as 259H4L7-T11 and 201H3L3-T11.
  • Figure 2 Schematic diagram of the structure of anti-ICOSL antibody fusion proteins such as 201H3L3-T7 and 259H4L7-T7.
  • Figure 3 Schematic diagram of the structure of an anti-ICOSL antibody fusion protein such as 259H4L7-T9.
  • Figure 4 The results of the anti-ICOSL antibody fusion protein inhibiting the secretion of IL-10.
  • Figure 5 The results of the anti-ICOSL antibody fusion protein inhibiting the secretion of IFN ⁇ .
  • Figure 6 The results of the anti-ICOSL antibody fusion protein inhibiting the secretion of IgA.
  • Figure 7 Experimental results of anti-ICOSL antibody fusion protein inhibiting the production of KLH-specific IgG.
  • Figure 8 Experimental results of anti-ICOSL antibody fusion protein inhibiting the production of KLH-specific IgM.
  • cytokine is a generic term for proteins released by a population of cells that act as intercellular mediators on other cells.
  • cytokines include lymphokines, monokines, chemokines and traditional polypeptide hormones.
  • exemplary cytokines include: IL-10, IFN- ⁇ , IL-6, TNF ⁇ , IL-17 and IL-5.
  • TACI described in this disclosure is a membrane-bound receptor.
  • Wild-type human TACI extracellular region (positions 1-165) refers to SEQ ID NO: 51 of the present disclosure.
  • TACI extracellular domain and “TACI extracellular region” can be replaced with each other.
  • ICOSL Inducible costimulator-ligand, also known as B7RP1, B7H2, CD275
  • B7RP1, B7H2, CD275 is an immune co-stimulatory molecule belonging to the B7 family. It is currently known that the receptor of ICOSL is ICOS, and ICOS is mainly expressed on the surface of T cells.
  • the signaling pathways mediated by ICOSL and ICOS mainly play a role in the differentiation of T cells and the activation of B cells dependent on T cells (Nat Rev Cancer.2012Mar 22 ; 12(4):252-64.).
  • ICOSL When “ICOSL” is used as the target of an antibody or its fusion protein, it should be broadly understood and is intended to cover various forms of molecules of ICOSL in various stages of the mammalian body, such as but not limited to ICOSL gene amplification, replication, Molecules produced during 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 covers artificially produced or ICOSL expressed in vitro.
  • ICOSL gene amplification, replication, Molecules produced during 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 covers artificially produced or ICOSL expressed in vitro.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics 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, eg, hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine.
  • Amino acid analogs are compounds that have the same basic chemical structure (i.e., the alpha carbon bonded to a hydrogen, carboxyl, amino group, and R group) as a naturally occurring amino acid, such as homoserine, norleucine, methionine sulfoxide , Methylsulfonium methionine.
  • Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • An amino acid mimetic refers to a chemical compound that has a structure that differs from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid.
  • amino acid mutation includes amino acid substitutions (also called amino acid substitutions), deletions, insertions and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to achieve the final construct so long as the final construct possesses the desired properties, such as reduced binding to Fc receptors. Amino acid sequence deletions and insertions may be at the amino- and/or carboxy-terminal end of the polypeptide chain. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, ie, replacing one amino acid with another amino acid having different structural and/or chemical properties.
  • Amino acid substitutions include substitutions with non-naturally occurring amino acids or with derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine) .
  • Amino acid mutations can be performed using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is anticipated that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modification, may also be available. Various expressions may be used herein to indicate amino acid mutations.
  • amino acid residue at a specific position can be expressed in the form of position + amino acid residue, for example, 366W means that the amino acid residue at position 366 is W.
  • T366W means that the amino acid residue at the 366th position is mutated from the original T to W.
  • the sequence is described as 366W or T366W in the claims, it cannot be understood that the original amino acid residue T at the 366 position constitutes any limitation on the scope of protection.
  • antibody is used in the broadest sense and encompasses various 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) as long as they exhibit the desired antigen-binding activity.
  • Native antibody refers to a naturally occurring immunoglobulin molecule. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of 2 identical light chains and 2 identical heavy chains disulfide-bonded.
  • each heavy chain has a variable region (VH), also known as variable heavy domain, heavy chain variable region, followed by a heavy chain constant region, the natural IgG heavy chain constant region usually contains three Constant domains (CH1, CH2 and CH3).
  • VH variable heavy domain
  • VL variable light domain
  • CL constant light domain
  • Natural complete antibody light chain includes light chain variable region VL and constant region CL, VL is at the amino terminal of light chain, light chain constant region includes ⁇ chain and ⁇ chain; heavy chain includes variable region VH and constant region (CH1, CH2 and CH3), the VH is at the amino-terminus of the heavy chain, the constant region is at the carboxy-terminus, wherein CH3 is closest to the carboxy-terminus of the polypeptide, and the heavy chain can belong to any isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes) , IgA (including IgA1 and IgA2 subtypes), IgM and IgE.
  • IgG including IgG1, IgG2, IgG3 and IgG4 subtypes
  • IgA including IgA1 and IgA2 subtypes
  • IgM and IgE IgE.
  • variable region or “variable domain” of an antibody refers to the domains of the heavy or light chain of an antibody that are involved in the binding of the antibody to antigen.
  • 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 (CDR).
  • FR conserved framework regions
  • CDR complementarity determining region
  • CDR refers to the region in the variable domain that mainly contributes to binding to the antigen
  • framework or "FR” refers to the variable domain residues other than the CDR residues.
  • VH contains 3 CDR regions: HCDR1, HCDR2 and HCDR3; VL contains 3 CDR regions: LCDR1, LCDR2 and LCDR3.
  • Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also known as the N terminus) to the carboxyl terminus (also known as the C terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3 , FR4.
  • amino acid sequence boundaries of CDRs can be determined by various known schemes, for example: “Kabat” numbering convention (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health , Bethesda, MD), “Chothia” numbering sequence, “ABM” numbering sequence, "contact” numbering sequence (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J].
  • antibody fragment refers to a molecule other than an intact antibody that comprises the portion of an intact antibody that binds to the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , single domain antibody, single chain Fab (scFab), diabody, linear antibody, single chain antibody molecule (e.g. scFv), and multispecific antibodies formed from antibody fragments.
  • Fc region or “fragment crystallizable region” is used to define the C-terminal region of an antibody heavy chain, including native and engineered Fc regions.
  • the Fc region comprises two subunits that are the same or different.
  • 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 carboxyl terminus.
  • Suitable Fc regions for use in the antibodies described herein include the Fc regions of human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.
  • the boundaries of the Fc region can also be varied, 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 (residue 447 according to the EU numbering system). system residues 446 and 447).
  • the numbering convention for the Fc region is the EU numbering system, also known as the EU index.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chains is derived from a particular source or species, while the remaining portion of the heavy and/or light chains is derived from another, different source or species.
  • humanized antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts (ie, the constant regions and the framework portion of the variable regions).
  • affinity refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, unless otherwise indicated, binding “affinity” refers to internal binding affinity, which reflects the interaction between members of a binding pair (eg, antibody and 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 routine methods known in the art, including those described herein.
  • the term “kassoc” or “ka” refers to the on-rate of a particular antibody-antigen interaction and the term “kdis” or “kd” refers to the dissociation rate of a particular antibody-antigen interaction.
  • KD refers to the dissociation constant, which is obtained from the ratio of kd to ka (ie, kd/ka) and is expressed as molarity (M).
  • M molarity
  • the KD value of an antibody can be determined using methods well known in the art. For example, using biosensing systems such as systems measuring surface plasmon resonance (eg Biacore), or measuring affinity in solution by solution equilibrium titration (SET).
  • effector function refers to those biological activities attributable to an antibody Fc region (either native sequence Fc region or amino acid sequence mutated Fc region) and which vary with antibody isotype.
  • 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, cell surface receptors (e.g., B cell receptors, body) downregulation; and B cell activation.
  • the term “monoclonal antibody” refers to a population of substantially homogeneous antibodies, ie, the antibody molecules comprised in the population are identical in amino acid sequence, except for natural mutations that may be present in minor amounts.
  • polyclonal antibody preparations typically contain multiple different antibodies with different amino acid sequences in their variable domains, often specific for different epitopes.
  • “Monoclonal” denotes the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method.
  • the antibodies provided by the present disclosure are monoclonal antibodies.
  • antigen refers to a molecule or portion of a molecule 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 capable of interacting with different antigen binding proteins (eg antibodies).
  • epitope refers to an area or region on an antigen to which an antibody (including antigen-binding fragments thereof) can specifically bind.
  • An epitope may be formed from a continuous string of amino acids (linear epitope) or comprise non-contiguous amino acids (conformational epitope), such that non-contiguous amino acids are brought into spatial proximity to one another as a result of folding of the antigen (ie by tertiary folding).
  • the difference between a conformational epitope and a linear epitope is that antibody binding to a conformational epitope is lost in the presence of denaturing solvents.
  • 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 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 antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.
  • the term “capable of specifically binding”, “specifically binds” or “binds” means that an antibody is capable of binding to a certain antigen or epitope thereof with a higher affinity than to other antigens or epitopes.
  • the antibody is prepared in an amount of about 1 ⁇ 10 -7 M or less (e.g., about 1 ⁇ 10 -8 M, 1 ⁇ 10 -9 M, 1 ⁇ 10 -10 M, 1 ⁇ 10 -11 M or less).
  • the antibody binds the antigen with a KD that is 10% or less (eg, 1%) of the antibody's KD for binding to a non-specific antigen (eg, BSA, casein).
  • KD can be measured using known methods, for example by measured by surface plasmon resonance.
  • antibodies that specifically bind to an antigen or an epitope thereof may have cross-reactivity to other related antigens, e.g. , cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp) or marmoset (Callithrix jacchus) (commonmarmoset, marmoset)) are cross-reactive.
  • anti-ICOSL antibody and "ICOSL-binding antibody” refer to antibodies capable of binding ICOSL or an epitope thereof with sufficient affinity.
  • the antibody binds to an unrelated, non-ICOSL protein to an extent less than about 10% of the antibody's binding to ICOSL as measured by a surface plasmon resonance assay (e.g., ).
  • an antibody that binds to an ICOSL protein has a dissociation constant (KD) of ⁇ about 1 ⁇ M, ⁇ about 100 nM, ⁇ about 10 nM, ⁇ about 1 nM, ⁇ about 0.1 nM, ⁇ about 0.01 nM, or ⁇ about 0.001 nM.
  • the anti-ICOSL antibody binds a human or cynomolgus ICOSL epitope.
  • linker refers to a connecting unit connecting two polypeptide fragments, usually with a certain degree of flexibility, and the use of the linker will not lose the original function of the protein domain.
  • linkers appearing 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.
  • antibody-dependent cellular cytotoxicity is mechanisms for inducing cell death that rely on antibody coating of target cells with lytically active effector cells ( Cells such as natural killer (NK), monocytes, macrophages and neutrophils) interact via Fc ⁇ receptors (Fc ⁇ Rs) expressed on effector cells.
  • NK cells express FcyRIIIa
  • monocytes express FcyRI, FcyRII, and FcyRIIIa.
  • the ADCC activity of the antibodies provided herein can be assessed using an in vitro assay using antigen-expressing cells as target cells and NK cells as effector cells. Cell lysis is detected based on labels released from lysed cells, such as radioactive substrates, fluorescent dyes, or native intracellular proteins.
  • ADCP antibody-dependent cellular phagocytosis
  • complement-dependent cytotoxicity refers to a cell death-inducing mechanism in which the Fc effector domain of a target-binding antibody binds and activates the complement component C1q, which in turn activates the complement cascade, resulting in target cell death.
  • Activation of complement can also result in the deposition of complement components on the surface of target cells that promote CDC by binding to complement receptors (eg, CR3) on leukocytes.
  • complement receptors eg, CR3
  • nucleic acid is used herein interchangeably with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, synthetic, naturally occurring and non-naturally occurring, having similar binding properties to the reference nucleic acid, and defined in Metabolized in a manner similar to the reference nucleotide.
  • 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 other than 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 in separate vectors, and present in a host cell Such one or more nucleic acid molecules at one or more positions.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences as well as the explicitly indicated sequence.
  • degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is mixed with bases and/or deoxygenated Inosine residue substitution.
  • 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 stated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • sequence identity refers to the degree (percentage) to which the amino acids/nucleic acids of the two sequences are identical at equivalent positions when the two sequences are optimally aligned; where necessary, gaps are introduced in the optimal alignment to obtain the maximum percent sequence identity and not to consider any conservative substitutions as part of the sequence identity.
  • alignment can be achieved by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • the amino acid residue position numbered with respect to the natural order of the sequence SEQ ID NO: XX means that the subject sequence is optimally aligned with the sequence of SEQ ID NO: XX to obtain the highest percentage identity, at this time
  • the site corresponding to the target sequence and the SEQ ID NO: XX sequence is the relative site of the two sequences.
  • the amino acid residue positions on the extracellular region sequences SEQ ID NO: 51 and SEQ ID NO: 60 of TACI relative to the natural sequence numbering of SEQ ID NO: 58 are shown in Table 2:
  • the 2nd (natural sequence) residue site of SEQ ID NO: 60 and the 49th (natural sequence) residue site of the sequence SEQ ID NO: 58 are corresponding sites.
  • fused or “linked” refer to the joining of components (eg, TACI polypeptide and antibody heavy/light chains) by covalent bonds, either directly or via one or more linkers.
  • linker is a peptide linker
  • the covalent bond is a peptide bond.
  • anti-ICOSL antibody fusion protein refers to a protein in which an anti-ICOSL antibody is fused to an active protein.
  • a TACI polypeptide is fused to the N- or C-terminus of the heavy or light chain of an anti-ICOSL antibody to form a protein.
  • vector means a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked.
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be ligated into the viral genome.
  • AAV adeno-associated viral vector
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors).
  • vectors can integrate into the genome of the host cell after introduction into the host cell, thereby replicating along with the host genome.
  • expression vector or "expression construct” refers to a vector that can transform a host cell and contains a vector that directs and/or controls (along with the host cell) the expression of one or more heterologous coding regions operably linked thereto.
  • Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, when an intron is present, RNA splicing of the coding region to which it is operably linked.
  • host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical to the parental cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as the cells screened or selected in the originally transformed cells are included herein.
  • 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, dog, monkey, pig, goat, cow, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster cells Kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.
  • Fungal cells include yeast and filamentous fungal cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia puntiae, Pichia thermotolerans, Pichia willow salictaria), Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia, Saccharomycescerevisiae, Saccharomyces cerevisiae , Hansenula polymorpha, Kluyveromyces, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fus
  • composition means a mixture comprising one or more anti-ICOSL antibody fusion proteins described herein and other chemical components, such as physiological/pharmaceutically acceptable carriers and excipients.
  • pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical formulation that is distinct from the active ingredient and that is nontoxic to the subject; for example, but not limited to, buffers, excipients, stabilizing preservatives or preservatives.
  • subject or “individual” includes humans and non-human animals.
  • Non-human animals include all vertebrates (eg, mammals and non-mammals) such as non-human primates (eg, cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles.
  • patient or “subject” are used interchangeably herein unless otherwise indicated.
  • cyno or “cynomolgus” refers to Macaca fascicularis.
  • the individual or subject is a human.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to the interaction of an exogenous drug, therapeutic agent, diagnostic agent or composition with an animal, human , subjects, cells, tissues, organs or biological fluids.
  • sample refers to a collection of fluid, cells, or tissue isolated from a subject, as well as fluid, cells, or tissue present in a subject.
  • samples are biological fluids such as blood, serum, serosal fluid, plasma, lymph, urine, saliva, cyst fluid, tears, faeces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites , pleura, pericardium, peritoneum, peritoneal and other body cavity fluids, fluid collected from bronchial lavage, synovial fluid, liquid solutions in contact with subjects or biological sources, such as cell and organ culture media (including cell or organ condition media), lavage fluid, etc., tissue biopsy samples, fine needle aspirations, surgically resected tissues, organ cultures, or cell cultures.
  • biological fluids such as blood, serum, serosal fluid, plasma, lymph, urine, saliva, cyst fluid, tears, faeces, sputum, mucosal secretions of secretory tissues and organs
  • Treatment and “management” refer to clinical intervention that attempts to alter the course of the individual being treated, and may be performed for prophylaxis or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, prevention of occurrence or recurrence of disease, alleviation of symptoms, alleviation/reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of rate of disease progression, amelioration or palliation of disease state, and regression or amelioration of prognosis.
  • the antibodies or fusion proteins of the present disclosure are used to delay the development of a disease or slow the progression of a disease.
  • an “effective amount” is generally sufficient to reduce the severity and/or frequency of symptoms, eliminate these symptoms and/or underlying causes, prevent the occurrence of symptoms and/or their underlying causes, and/or ameliorate or ameliorate the impairment caused by or associated with the disease state amount.
  • the effective amount is a therapeutically or prophylactically effective amount.
  • a “therapeutically effective amount” is sufficient to treat a disease state or symptom, especially a state or symptom associated with the disease state, or otherwise prevent, hinder, delay or reverse the disease state or any other adverse effect in any way related to the disease state. The amount of progression of the desired symptoms.
  • 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 associated symptoms .
  • Complete therapeutic or prophylactic effect does not necessarily occur after administration of one dose, but may occur after administration of a series of doses.
  • a therapeutically or prophylactically effective amount may be administered in one or more administrations.
  • “Therapeutically effective amount” and “prophylactically effective amount” can vary depending on 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 an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of a patient.
  • Anti-ICOSL antibodies of the present disclosure are provided.
  • the present disclosure contemplates a novel anti-ICOSL antibody. It has one or more of the following properties:
  • A. specifically binds to human ICOSL and cynomolgus monkey ICOSL or its epitope, and does not specifically bind to mouse ICOSL; E-09M, less than 2.00E-09M, less than 1.50E-09M, 9.00E-10M or less) KD value combined with human ICOSL, and/or less than 8.00E-09M (such as less than 8.00E-09M, less than A KD value of 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) binds to cynomolgus ICOSL by surface plasmon measured by resonance assays (e.g. ); In some embodiments, the KD value is detected by the method of Test Example 8 of the present disclosure;
  • the IC value of blocking human ICOSL and ICOS binding is less than 0.16nM (such as 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), the IC50 value is detected by FACS method; 3 method detection;
  • C. has the activity of inhibiting the secretion of IFN ⁇ , IL-10 and/or IgA cytokines; in some embodiments, the IC50 value is detected by the method of Test Example 9 of the present disclosure;
  • D. has the activity of inhibiting T cell proliferation; in some embodiments, the anti-ICOSL antibody or anti-ICOSL antibody fusion protein inhibits the activity of ICOSL-induced T cell proliferation; in some embodiments, the activity of inhibiting T cell proliferation is passed through this Disclosure test example 6 method detection.
  • the present disclosure provides an anti-ICOSL antibody, 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, and the light chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises The chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein,
  • 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 selected from the Kabat numbering convention Defined.
  • HCDR1 of the heavy chain variable region is shown in SEQ ID NO: 11
  • HCDR2 is shown in SEQ ID NO: 31 or 12
  • HCDR3 is shown in SEQ ID NO: 13
  • the light chain can be LCDR1 of the variable region is set forth in SEQ ID NO: 14
  • LCDR2 is set forth in SEQ ID NO: 15
  • LCDR3 is set forth in SEQ ID NO: 16; or
  • HCDR1 of the heavy chain variable region is shown in SEQ ID NO: 5
  • HCDR2 is shown in SEQ ID NO: 19 or 6
  • HCDR3 is shown in SEQ ID NO: 7
  • the light chain can be LCDR1 of the variable region is set forth in SEQ ID NO:8, LCDR2 is set forth in SEQ ID NO:17 or 9, and LCDR3 is set forth in SEQ ID NO:10 or 18.
  • HCDR1 of the heavy chain variable region is shown in SEQ ID NO: 11
  • HCDR2 is shown in SEQ ID NO: 31
  • HCDR3 is shown in SEQ ID NO: 13
  • the light chain variable region LCDR1 is set forth in SEQ ID NO: 14
  • LCDR2 is set forth in SEQ ID NO: 15
  • LCDR3 is set forth in SEQ ID NO: 16;
  • HCDR1 of the heavy chain variable region is shown in SEQ ID NO: 5
  • HCDR2 is shown in SEQ ID NO: 19
  • HCDR3 is shown in SEQ ID NO: 7
  • the light chain variable region LCDR1 is set forth in SEQ ID NO:8, LCDR2 is set forth in SEQ ID NO:17, and LCDR3 is set forth in SEQ ID NO:10.
  • the anti-ICOSL antibody of any one of the preceding items said anti-ICOSL antibody is a murine, chimeric or humanized antibody. In some embodiments, the anti-ICOSL antibody is humanized.
  • the anti-ICOSL antibody of any one of the preceding is a humanized antibody.
  • the heavy chain variable region of the anti-ICOSL antibody has FR1, FR2, FR3 derived from IGHV3-74*03 and FR4 derived from IGHJ1*01, and is unsubstituted or has selected One or more amino acid substitutions 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 and FR4 derived from IGKJ2*01 and which are unsubstituted or have a number selected from the group consisting of 1N, 2T, 3V, 42Q, 43S, 60D, 85V (numbering according to the Kabat numbering system) One or more amino acid substitutions.
  • the anti-ICOSL antibody wherein the HCDR1 of the heavy chain variable region is shown in SEQ ID NO: 11, HCDR2 is shown in SEQ ID NO: 31 or 12, and HCDR3 is shown in SEQ ID NO : 13, and the LCDR1 of the light chain variable region is as shown in SEQ ID NO: 14, the LCDR2 is as shown in SEQ ID NO: 15, and the LCDR3 is as shown in SEQ ID NO: 16.
  • the above variable regions and CDRs are defined according to the Kabat numbering convention.
  • the anti-ICOSL antibody as described in any one of the preceding is a humanized antibody
  • the heavy chain variable region of the anti-ICOSL antibody has FR1 derived from IGHV1-46*01, FR2, FR3 and FR4 derived from IGHJ6*01 and which are unsubstituted or have one or more selected from the group consisting of 1E, 24T, 69L, 71V, 73K and 78A (numbering according to the Kabat numbering system) amino acid substitutions; and/or the light chain variable region has FR1, FR2, FR3 derived from IGKV2-28*01 and FR4 derived from IGKJ4*01, and it is unsubstituted or has a group selected from 1A, One or more amino acid substitutions in the group consisting of 2V and 4L (numbering according to the Kabat numbering system).
  • the anti-ICOSL antibody wherein the HCDR1 of the heavy chain variable region is shown in SEQ ID NO: 5, HCDR2 is shown in SEQ ID NO: 19 or 6, and HCDR3 is shown in SEQ ID NO : 7, and the LCDR1 of the light chain variable region is as shown in SEQ ID NO: 8, LCDR2 is as shown in SEQ ID NO: 17 or 9, and LCDR3 is as shown in SEQ ID NO: 10 or 18.
  • the above variable regions and CDRs are defined according to the Kabat numbering convention.
  • said heavy chain variable region is as shown in SEQ ID NO: 42, 39, 40 or 41, and said light chain variable region is as SEQ ID NO: 38, 32, 33, 34, 35, 36 or 37; or
  • said heavy chain variable region is as shown in SEQ ID NO: 30, 28 or 29, and said light chain variable region is as SEQ ID NO: 22, 20, 21, 23, 24, 25, 26 or 27; or
  • said heavy chain variable region is as shown in SEQ ID NO: 1, and said light chain variable region is as shown in SEQ ID NO: 2; or
  • the heavy chain variable region is as shown in SEQ ID NO:3, and the light chain variable region is as shown in SEQ ID NO:4.
  • said heavy chain variable region is shown in SEQ ID NO: 42, and said light chain variable region is shown in SEQ ID NO: 38;
  • said heavy chain variable region is shown in SEQ ID NO: 30, and said light chain variable region is shown in SEQ ID NO: 22.
  • the anti-ICOSL antibody according to any one of the above, wherein said anti-ICOSL antibody comprises an antibody heavy chain constant region and a light chain constant region.
  • the heavy chain constant region is a human IgG heavy chain constant region.
  • the heavy chain constant region is selected from human IgG1, IgG2, IgG3 and IgG4 constant regions; in some embodiments, the light chain constant region is selected from human antibody kappa or lambda chain constant regions.
  • 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.
  • the Fc region of the heavy chain constant region has one or more amino acid substitutions that reduce binding of the Fc region to an Fc receptor.
  • the Fc region has L234A, L235A mutations, and/or S228P mutations, and/or YTE mutations (M252Y, S254T, and T256E), and the numbering of the mutations is based on the EU index.
  • the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:43
  • the light chain constant region comprises the amino acid sequence of SEQ ID NO:44.
  • the heavy chain of the anti-ICOSL antibody has at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 47, and the light chain to SEQ ID NO: 48 has at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity; or
  • the heavy chain of the anti-ICOSL antibody has at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 45, and the light chain to SEQ ID NO: 46 has at least 90% (eg, at least 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the anti-ICOSL antibody of any one of the above is anti-ICOSL antibody of any one of the above.
  • Anti-ICOSL antibody fusion protein of the present disclosure is provided.
  • the disclosure constructs an anti-ICOSL antibody fusion protein, which comprises an anti-ICOSL antibody and a TACI polypeptide, wherein the anti-ICOSL antibody specifically binds to human ICOSL or its epitope.
  • the anti-ICOSL antibody fusion protein according to any one of the foregoing has one or more of the following properties:
  • A. specifically binds to human ICOSL and cynomolgus monkey ICOSL, and does not specifically bind to mouse ICOSL; Less than 2.00E-09M, less than 1.50E-09M, 9.00E-10M or less) KD value combined with human ICOSL, and/or less than 8.00E-09M (for example, 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) KD value combined with cynomolgus monkey ICOSL, said KD value is determined by surface plasmon resonance measurement (e.g. ); In some embodiments, the KD value is detected by the method of Test Example 8 of the present disclosure;
  • KD value is measured by surface plasmon resonance assay (e.g. ); In
  • C. specifically binds to human BAFF, cynomolgus monkey BAFF and/or mouse BAFF; E-11M, less than 2.00E-11M, less than 1.00E-11M or less) KD value combined with human BAFF to less than 4.00E-10M (eg, less than 4.00E-10M, less than 3.00E-10M, less than 2.00E -10M, less than 1.30E-10M or less), binds to cynomolgus BAFF 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) binding to mouse BAFF with a KD value as measured by a surface plasmon resonance assay (e.g. );
  • the KD value is detected by the method of Test Example 8 of the present disclosure;
  • the IC value of blocking human ICOSL and ICOS binding is less than 0.16nM (such as 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), the IC50 value is detected by FACS method; 3 method detection;
  • the IC value of blocking APRIL and BCMA binding is less than 40.00nM (such as 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), the IC50 value is detected by ELISA method; method detection;
  • the F. have blocking APRIL and TACI binding activity;
  • the IC value of blocking APRIL and TACI binding is less than 40.00nM (such as 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 IC50 value is detected by ELISA method; method detection;
  • the IC value of blocking the binding of BAFF to BCMA is less than 2.00nM (such as 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), the IC50 value is detected by ELISA method; in some embodiments, the IC50 value is detected by the method of Test Example 4 of this disclosure;
  • the H. have blocking BAFF and BAFF-R binding activity;
  • the IC value of blocking BAFF and BAFF-R binding is less than 2.00nM (such as 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 is detected by ELISA method; in some embodiments, the IC 50 value is detected by the method of Test Example 4 of this disclosure;
  • the IC value of blocking BAFF binding to TACI is less than 1.00nM (such as less than 1.00nM, less than 0.50nM, less than 0.30nM, less than 0.20nM, less than 0.15nM or more Small), the IC50 value is detected by ELISA method; in some embodiments, the IC50 value is detected by the method of Test Example 4 of the present disclosure;
  • J. has the activity of inhibiting the secretion of IFN ⁇ , IL-10 and/or IgA cytokines; in some embodiments, the IC50 value is detected by the method of Test Example 9 of the present disclosure;
  • the K. has the activity of inhibiting B cell proliferation and/or T cell proliferation;
  • the anti-ICOSL antibody or anti-ICOSL antibody fusion protein inhibits the activity of ICOSL-induced T cell proliferation;
  • the The activity of inhibiting T cell proliferation is detected by the method of Test Example 6 of the present disclosure; in some embodiments, the anti-ICOSL antibody fusion protein inhibits the activity of B cell proliferation induced by BAFF or APRIL; in some embodiments, the inhibition of B cell The proliferative activity was detected by the method of Test Example 7 of the present disclosure.
  • anti-ICOSL antibody fusion proteins Exemplary anti-ICOSL antibody fusion proteins
  • the anti-ICOSL antibody fusion protein wherein the TACI polypeptide is less likely to be broken than the wild-type TACI polypeptide (sequence shown in SEQ ID NO: 51).
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein the TACI polypeptide is shown as SEQ ID NO: 58 or a truncated fragment of SEQ ID NO: 58 or SEQ ID NO: A variant of 58 or a variant of a truncated fragment of SEQ ID NO: 58; wherein the truncated fragment comprises the 48th to 85th amino acid residues of SEQ ID NO: 58, and the variant is in SEQ ID NO: 58 ID NO: 58 or its truncated fragment has 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 substitution site is Amino acid residue positions numbered relative to the natural sequence of the sequence SEQ ID NO:58.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein the TACI polypeptide, wherein the truncated fragment of the TACI polypeptide comprises: the 48th to the 86th of SEQ ID NO:58 Amino acid residues; the 48th to 87th amino acid residues of SEQ ID NO:58; or the 48th to 88th amino acid residues of SEQ ID NO:58.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein the sequence of the TACI polypeptide is shown in any one of SEQ ID NO: 60-63.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein, the TACI polypeptide, its sequence is a variant of SEQ ID NO: 58 or a truncated fragment of SEQ ID NO: 58 (such as SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63), said variant is at SEQ ID NO: 58 or its truncated fragment sequence (such as SEQ ID NO: 60 , SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63) has any one, two, three, 4, 5, 6 or 7 amino acid substitutions, the position of the amino acid substitution is the amino acid residue position numbered relative to the natural sequence of the sequence SEQ ID NO:58.
  • the TACI polypeptide its sequence is a variant of SEQ ID NO: 58 or a truncated fragment of SEQ ID NO: 58 (such as SEQ ID NO: 60, SEQ ID NO: 61,
  • the anti-ICOSL antibody fusion protein according to 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 one or more amino acid substitutions selected from the group consisting of 49T or 49R, 52S, 53E or 53Q, 57E, 65T or 65A, 82A or 82R, and 83Y (for example, 1, 2 , 3, 4, 5, 6 or 7 amino acid substitutions), wherein the amino acid substitution position is the amino acid residue position numbered relative to the natural sequence of the sequence SEQ ID NO:58.
  • amino acid substitution position is the amino acid residue position numbered relative to the natural sequence of the sequence SEQ ID NO:58.
  • the anti-ICOSL antibody fusion protein according to 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 there is any amino acid substitution selected from 49T, 52S, 53E, 53Q, 57E and 82A on the sequence of SEQ ID NO: 63. There are 49R and 65T amino acid substitutions on the sequence of SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein the sequence of the TACI polypeptide is shown in any one of SEQ ID NO: 60-63, SEQ ID NO: 66-83. In some embodiments, the anti-ICOSL antibody fusion protein according to any one of the above, wherein the TACI polypeptide sequence is shown in SEQ ID NO:83.
  • the anti-ICOSL antibody fusion protein according to any one of the above, which consists of 2 identical first chains and 2 identical second chains, wherein,
  • the first chain from N-terminus to C-terminus: [heavy chain of anti-ICOSL antibody] - [linker 1] - [TACI polypeptide 1], and
  • the second chain, from the N-terminal to the C-terminal is: [TACI polypeptide 2]-[Linker 2]-[light chain of anti-ICOSL antibody]; or the second chain, from the N-terminal to the C-terminal is [ The light chain of anti-ICOSL antibody]-[Linker 2]-[TACI polypeptide 2]; or the second chain, which is the light chain of anti-ICOSL antibody;
  • the TACI polypeptide 1 and the TACI polypeptide 2 may be the same or different, and the linker 1 and the 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 the linker 2 are independently (G x S) y linkers, wherein x is selected from an integer of 1-5, and y is selected from an integer of 0-6. More preferably, the linker 1 and linker 2 are independently the linker shown in SEQ ID NO: 89 or 90.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein the anti-ICOSL antibody is the anti-ICOSL antibody according to any one of the above.
  • the anti-ICOSL antibody fusion protein according to any one of the above, wherein, the first chain of the anti-ICOSL antibody fusion protein is as shown in SEQ ID NO: 86, and the second chain is as shown in SEQ ID NO : 88, 87 or 48. In some embodiments, the anti-ICOSL antibody fusion protein according to any one of the above, wherein, the first chain of the anti-ICOSL antibody fusion protein is as shown in SEQ ID NO: 84, and the second chain is as shown in SEQ ID NO : 85 or 46.
  • amino acid sequence variants of the anti-ICOSL antibodies provided herein, or fusion proteins thereof are contemplated.
  • 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, and/or insertions, and/or substitutions of residues within the amino acid sequence of the anti-ICOSL antibody or its fusion protein. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics, such as antigen-binding properties.
  • antibody variants having one or more amino acid substitutions are provided.
  • Sites of interest for substitution mutagenesis include CDRs and FRs.
  • Conservative substitutions are shown in Table 3 under the heading "Preferred Substitutions”. More substantial changes are provided in Table 3 under the heading "Exemplary Substitutions" and are described further below with reference to amino acid side chain classes.
  • Amino acid substitutions can be introduced into an antibody of interest, and the products screened for desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
  • amino acids can be grouped as follows:
  • Non-conservative substitutions would entail replacing a member of one of these classes for a member of another class.
  • substitutional variant involves substituting one or more CDR residues of a parent antibody (eg, a humanized or human antibody).
  • a parent antibody eg. a humanized or human antibody
  • the resulting variant selected for further study will have an altered (e.g. improved) certain biological property (e.g. increased affinity, reduced immunogenicity) relative to the parent antibody, and/or will be substantially Some of the biological properties of the parental antibody are retained.
  • An 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 antibodies are displayed on phage and screened for specific biological activity (eg, binding affinity).
  • Alterations can be made to the CDRs, eg, to improve antibody affinity. Such changes can be made to CDR "hot spots", i.e. residues encoded by codons that undergo mutations at high frequency during the somatic maturation process, and/or to residues that contact antigen, while making changes to the resulting variant VH or VL test for binding affinity.
  • affinity maturation diversity is introduced into the variable genes selected for maturation by any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis middle. Then, create secondary libraries. The library is then screened to identify any antibody variants with the desired affinity.
  • CDR-directed approach involves a CDR-directed approach, in which several CDR residues (eg, 4-6 residues) are randomized.
  • CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling.
  • HCDR3 and LCDR3 are frequently targeted.
  • substitutions, insertions or deletions may be made within one or more CDRs, so long as such changes do not substantially reduce the ability of the antibody to bind antigen.
  • conservative changes eg, conservative substitutions, as provided in Table 3
  • Such changes may eg be outside antigen contacting residues in the CDRs.
  • each CDR is unchanged, or contains no more than 1, 2 or 3 amino acid substitutions.
  • alanine scanning mutagenesis One method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis".
  • residues e.g. charged residues such as Arg, Asp, His, Lys and Glu
  • neutral or negatively charged amino acids e.g. Ala or Polypropylene
  • Amino acid amino acids
  • substitutions may be introduced at amino acid positions showing functional sensitivity to the initial substitution.
  • contact points between antibody and antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain desired properties.
  • Amino acid sequence insertions include amino- and/or carboxy-terminal fusions of polypeptides ranging in length from 1 residue to 100 or more residues, and intrasequence insertions of single or multiple amino acid residues.
  • 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 the antibody to an enzyme (or a polypeptide that extends the half-life of the antibody).
  • 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, such as its binding to an Fc ⁇ receptor binding and reduce or eliminate effector function.
  • a native IgG Fc region specifically an IgG 1 Fc region or an IgG 4 Fc region, may cause the fusion proteins of the present disclosure to target cells expressing Fc receptors, rather than cells expressing antigens.
  • an engineered Fc region of the present disclosure exhibits reduced binding affinity for an Fc receptor and/or reduced effector function.
  • the engineered Fc region has a binding affinity for Fc receptors that is reduced by more than 50%, 80%, 90%, or 95% compared to a native Fc region.
  • the Fc receptor is an Fc gamma receptor.
  • the Fc receptor is a human Fc ⁇ receptor, eg, Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIB, Fc ⁇ RIIIa.
  • the engineered Fc region also has reduced binding affinity for complement, such as C1q, compared to a native Fc region.
  • the engineered Fc region has no reduced binding affinity for neonatal Fc receptor (FcRn) compared to a native Fc region.
  • the engineered Fc region has reduced effector function, which may include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced Antibody-dependent cell-mediated cytotoxicity (ADCC), decreased antibody-dependent cellular phagocytosis (ADCP), decreased cytokine secretion, decreased immune complex-mediated antigen uptake by antigen-presenting cells, decreased interaction with NK cells decreased binding to macrophages, decreased binding to monocytes, decreased binding to polymorphonuclear cells, decreased direct signaling-induced apoptosis, decreased dendritic cell maturation, or decreased T cells primed.
  • CDC complement-dependent cytotoxicity
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • cytokine secretion decreased immune complex-mediated antigen uptake by antigen-presenting cells
  • decreased interaction with NK cells decreased binding to macrophages
  • monocytes decreased binding to monocytes
  • polymorphonuclear cells
  • amino acid residue substitutions at positions 238, 265, 269, 270, 297, 327, and 329 may reduce effector function.
  • the Fc region is a human IgG 1 Fc region, and the amino acid residues at positions 234 and 235 are A, and the numbering is based on the EU index.
  • amino acid residue substitutions at positions such as 228 may reduce effector function.
  • Anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins may comprise different binding domains fused to the two subunits of the Fc region, thus potentially leading to undesired homodimerization.
  • the Fc region of the present disclosure comprises modifications according to the knob-into-hole (KIH) technique, which involves the introduction of a knob at the interface of the first subunit and the introduction of a knob at the interface of the second subunit.
  • KH knob-into-hole
  • the bulge structure is constructed by replacing small amino acid side chains from the interface of the first subunit with larger side chains such as tyrosine or tryptophan. Instead, the pore structure is created in the interface of the second subunit by replacing large amino acid side chains with smaller ones, such as alanine or threonine.
  • the protrusion structure and hole structure are prepared by changing the nucleic acid encoding the polypeptide, and the optional amino acid substitutions are shown in Table 4 below:
  • knob-and-hole technique other techniques for modifying the CH3 domain of the heavy chain of an antibody to achieve heterodimerization are also known in the art, for example 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 a disulfide bond engineered, for example, the first subunit of the Fc region comprises a 354C mutation and the second subunit comprises a 349C mutation, resulting in an engineered disulfide bond between the first subunit and the second subunit of the Fc region. Sulfur bond, promotes heterodimerization of the first and second subunits of the Fc region.
  • the Fc region of the antigen-binding molecule can further introduce other amino acid modifications, such as allotype amino acid residue mutations to reduce immunogenicity.
  • the Fc of IgG1 introduces the 356E and 358M mutations.
  • the C-terminus of the Fc region may be a complete C-terminus ending with the amino acid residue PGK; it may also be a shortened C-terminus in which, for example, one or two C-terminal amino acid residues have been removed.
  • the C-terminus of the heavy chain is a shortened C-terminus ending in PG.
  • whole antibodies may comprise antibody mixtures from which all K447 residues and/or G446+K447 residues have been removed.
  • whole antibodies can include antibody mixtures from which the K447 residue and/or the G446+K447 residues have not been removed.
  • intact antibodies have a mixture of antibodies with and without the K447 residue and/or G446+K447 residues.
  • Anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins can be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding the antibody or fusion protein are provided.
  • the present disclosure provides an isolated nucleic acid encoding an anti-ICOSL antibody or an anti-ICOSL antibody fusion protein as previously described. Such nucleic acid may be derived from an independent polypeptide chain encoding any of the foregoing.
  • the present disclosure provides one or more vectors (eg, expression vectors) comprising such nucleic acids.
  • the disclosure provides host cells comprising such nucleic acids.
  • an antibody or fusion protein comprising, under conditions suitable for expression, culturing a host cell comprising a nucleic acid encoding said antibody or fusion protein, as provided above, and The anti-ICOSL antibody or anti-ICOSL antibody fusion protein is optionally recovered from the host cell (or host cell culture medium).
  • nucleic acid encoding the protein is isolated and inserted into one or more vectors for further cloning and/or expression in host cells.
  • 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 described herein. For example, it can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. After expression, it can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • 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 for expression of antibodies or fusion proteins may also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells.
  • a number of baculovirus strains have been identified for use in combination with insect cells, particularly for the transfection of Spodoptera frugiperda cells; plant cell cultures can also be used as hosts, e.g.
  • vertebrate cells can also be used as hosts, eg mammalian cell lines adapted for growth in suspension.
  • suitable mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7); the human embryonic kidney line (293 or 293T cells); baby hamster kidney cells (BHK); Sertoli) cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells ( BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells.
  • Suitable mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines, such as YO, NSO and Sp2/0.
  • CHO Chinese Hamster Ovary
  • myeloma cell lines such as YO, NSO and Sp2/0.
  • Anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins provided herein can be identified, screened or characterized for their physical/chemical characteristics and/or biological activity by various assays known in the art.
  • the anti-ICOSL antibody or anti-ICOSL antibody fusion protein of the present disclosure is tested for activity, eg, by known methods such as ELISA, Western blot, and the like.
  • anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins provided herein can be used in methods of treatment.
  • the present disclosure provides the use of an anti-ICOSL antibody or an anti-ICOSL antibody fusion protein in the manufacture or preparation of a medicament.
  • the medicament is for the treatment of an autoimmune disease, an inflammatory disease, a B-cell disorder, or a T-cell disorder, the disease being a disease associated with ICOSL overexpression.
  • the autoimmune or inflammatory disease is selected from the group consisting of: systemic lupus erythematosus, rheumatoid arthritis, graft versus host disease, asthma, immune thrombocytopenic purpura, multiple sclerosis, diabetes Inflammatory diseases caused by psoriasis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Graves' disease and Hashimoto's thyroiditis, etc.
  • the B-cell disorder or T-cell disorder is a neoplastic disease.
  • the tumor is selected from the group consisting of 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.
  • the autoimmune disease is systemic lupus erythematosus.
  • 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 agent).
  • a "subject" according to any of the above embodiments may be a human.
  • a pharmaceutical composition comprising the anti-ICOSL antibody or anti-ICOSL antibody fusion protein, for example, for any of the above pharmaceutical uses or methods of treatment.
  • a pharmaceutical composition comprises any antibody or fusion protein provided herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises at least one additional therapeutic agent.
  • the anti-ICOSL antibody or anti-ICOSL antibody fusion protein of the present disclosure can be used alone or in combination with other agents for therapy.
  • an anti-ICOSL antibody or anti-ICOSL antibody fusion protein of the present disclosure can be administered in combination with at least one additional therapeutic agent.
  • Combined administration refers to a method of providing two or more active compounds to a subject simultaneously or sequentially for therapeutic purposes.
  • time interval between each administration is sufficient to achieve a synergistic effect between the active compounds administered.
  • Two or more active compounds are in the same separate container.
  • the anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins of the present disclosure can be administered by any suitable means, including parenteral, intrapulmonary, intranasal, and if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, eg, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic.
  • a variety of 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, dosed, and administered in a manner consistent with Good Manufacturing Practice. Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and others known to the medical practitioner. factor.
  • a polypeptide or fusion protein may or may not be formulated with 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 generally 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 any route empirically/clinically determined to be appropriate.
  • Effective amounts in specific embodiments can be derived from dose-response curves derived from animal model test systems and are allowed to be determined according to the judgment of the physician and each patient's circumstances.
  • the amount of drug required for one administration of the subject can be conveniently obtained by calculating the body weight of the subject and the dose per body weight required for one administration.
  • the dose can be determined by the equivalent dose conversion relationship between the dose per unit body weight of experimental animals and humans (Freireich et al. 1966, Cancer Chemother Rep 50: 219). For example, it can be based on the guidance issued by FDA, SFDA and other drug regulatory agencies.
  • the human and mouse doses can be converted by using conversion coefficients based on the body surface areas of humans and mice (eg refer to the doses in Test Examples 9-11).
  • the appropriate dose of the 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 disease to be treated type of drug, type of therapeutic molecule, severity and course of the disease, whether administered for prophylactic or therapeutic purposes, previous therapy, patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician.
  • the therapeutic molecule is suitably administered to the patient at one time or over a series of treatments.
  • an article of manufacture comprising materials useful for the treatment, prevention and/or diagnosis of the above-mentioned disorders.
  • the article comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like.
  • Containers can be formed from various materials such as glass or plastic.
  • the container holds the antibody or its fusion protein alone, or in combination with another component, and can have a sterile access port (eg, the container can be an intravenous solution bag or vial with a stopper).
  • At least one active agent in the container is an anti-ICOSL antibody or anti-ICOSL antibody fusion protein of the present disclosure.
  • the label or package insert indicates that the antibody or fusion protein thereof is used to treat the condition of choice.
  • the article of manufacture may comprise: (a) a first container having a composition 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 the composition therein A container, wherein the composition comprises an additional cytotoxic or other therapeutic agent.
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer. It may further comprise other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
  • Embodiment 1 Preparation of mouse anti-human ICOSL monoclonal antibody
  • mice were immunized with human ICOSL protein (Sino Biological, 11559-H08H). After 3 times of immunization, blood was taken to measure the titer of the antibody in the serum, and the mice with high antibody titer in the serum and the titer tended to plateau were selected for splenocyte fusion, and the fused hybridoma cells were plated in a 96-well cell culture plate , placed in a 37°C, 5% CO 2 incubator for cultivation. The cell culture supernatant was taken for detection by enzyme-linked immunosorbent assay (ELISA). The screened positive clones were expanded, cryopreserved and subcloned two to three times until single-cell clones were obtained.
  • ELISA enzyme-linked immunosorbent assay
  • Selected hybridoma clones were further prepared and purified using serum-free cell culture methods.
  • the resulting hybridoma antibody was detected by FACS for the binding of the antibody to the human ICOSL protein on the cell surface and the blocking of the receptor (see test example 1 and test example 3 of the present disclosure for the method), and the hybridization with good binding activity and blocking activity was selected. tumor cell lines.
  • VH/VL sequences of the above mAb201 and mAb259 candidate molecules were amplified by PCR, and then homologously recombined with the expression vector pHr (with signal peptide and hIgG4/hkappa constant region gene (CH1-Fc/CL) fragment).
  • the human heavy chain IgG4 constant region sequence is shown in SEQ ID NO: 43
  • the human light chain ⁇ constant region sequence is shown in SEQ ID NO: 44
  • the recombinant chimeric antibody full-length expression plasmid VH-CH1-Fc is constructed -pHr/VL-CL-pHr, and then obtain its chimeric antibodies Ch201 and Ch259.
  • variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 is formed, and then the variable region sequence is fused with a human constant region sequence to obtain a humanized antibody.
  • the humanization of mAb201 and mAb259 murine antibodies is exemplarily described below, in which the CDR amino acid residues of the antibodies are determined and annotated by the Kabat numbering system.
  • the heavy and light chain variable region germline genes with high homology were selected as templates.
  • the humanized light chain template of the murine antibody mAb201 is selected from IGKV2-28*01 and IGKJ4*01, that is, the FR1, FR2, FR3 of the human germline light chain IGKV2-28*01, and the JK4 region of IGKJ4*01 ( As FR4) as the humanized antibody light chain framework region;
  • the humanized heavy chain template is selected from IGHV1-46*01 and IGHJ6*01, that is, FR1, FR2, and FR3 of the human germline heavy chain IGHV1-46*01 are selected, and the JH6 region of IGHJ6*01 (as FR4) as the humanized antibody heavy chain framework region.
  • the CDRs of the murine antibody mAb201 were grafted into the corresponding human templates to replace the CDR regions of the human templates; then, the 1st, 2nd and/or 4th positions of the light chain variable regions of the humanized antibodies (numbering according to the Kabat numbering system) amino acid residues 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; in addition, The first amino acid residue in the LCDR2 of the light chain variable region: LVSNRFS (SEQ ID NO: 9) was mutated from L to K to obtain a new LCDR2: KVSNRFS (SEQ ID NO: 17); for light chain variable The 8th amino acid residue in the LCDR3 of the region: FQSNYLPLT (SEQ ID NO: 10) was mutated from L to P to obtain a new LCDR3: FQSNYLPPT (SEQ ID NO: 18);
  • Amino acid sequence (SEQ ID NO: 25) of >hAb201VL6 (Graft+D1A, I2V, M4L+L96P)
  • Amino acid sequence (SEQ ID NO: 27) of >hAb201VL8 (Graft+D1A, I2V, M4L+L50K, L96P)
  • the above sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in sequence, the underlined part in the sequence is the CDR sequence (confirmed according to the Kabat numbering system), and the rest is the FR sequence, and the italic bold part indicates the mutation site.
  • the humanized light chain template of the mouse antibody mAb259 is selected from IGKV4-1*01/IGKV1-39*01 and IGKJ2*01, namely Select human germline light chain IGKV4-1*01 or FR1, FR2, FR3 of IGKV1-39*01, and JK4 region of IGKJ2*01 (as FR4) as humanized antibody light chain framework region; humanized heavy chain
  • the template is selected from IGHV3-74*03 and IGHJ1*01, that is, select the FR1, FR2, FR3 of the human germline heavy chain IGHV3-74*03, and the JH1 region of IGHJ1*01 (as FR4) as the humanized antibody heavy chain frame area.
  • the CDRs of the murine antibody mAb259 were grafted into the corresponding human templates to replace the CDR regions of the human templates; then, the 1st, 2nd, 3rd, 42nd, 42nd, Amino acid residues 43, 60 and/or 85 (numbered according to the Kabat numbering system) are 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.
  • HCDR2 of the heavy chain variable region YISSSSGKVYADAVKG (SEQ ID NO: 12) was mutated from S to D to obtain a new HCDR2: YIDSSSGKVYADAVKG (SEQ ID NO: 31).
  • the humanized sequence of the mouse antibody mAb259 is as follows:
  • Amino acid sequence (SEQ ID NO: 33) of >hAb259VL2 (Graft(IGKV4-1*01-IGKJ2*01)+D1N, I2T, P43S)
  • Amino acid sequence (SEQ ID NO: 35) of >hAb259VL4 (Graft(IGKV1-39*01-IGKJ2*01+D1N, I2T, A43S, S60D, T85V)
  • Amino acid sequence (SEQ ID NO: 42) of >hAb259VH4 (Graft+V37I, K43E, S49A+S52D)
  • sequence sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where the underlined part is the CDR sequence (confirmed according to the Kabat numbering system), and the rest is the FR sequence, and the italic bold part indicates the mutation site.
  • the expression vectors of antibody light chain and heavy chain were respectively constructed, and the humanized antibody light and heavy chains were cross-paired and combined, and the culture supernatant was collected and purified after transfection into 293E cells to obtain the humanized full-length antibody.
  • the heavy chain constant region of the humanized antibody can be selected from constant regions of IgG1, IgG2, IgG3, IgG4 and variants thereof.
  • the human heavy chain IgG4 constant region (as shown in SEQ ID NO: 43) and the aforementioned human Fusion of the variable region of the heavy chain of the humanized heavy chain to form the full-length heavy chain of the antibody;
  • the constant region of the light chain of the humanized antibody can be selected from the constant region of the human ⁇ , ⁇ chain or its variants, for example, the constant region of the human light chain is used
  • the ⁇ chain (as shown in SEQ ID NO: 44) is fused to the aforementioned humanized light chain variable region to form the full-length light chain of the antibody.
  • Exemplary antibody constant region sequences are as follows:
  • Amino acid sequence of the light chain constant region of human kappa chain (SEQ ID NO: 44):
  • the carboxy-terminal of the heavy chain variable region of the humanized antibody of mAb201 is connected to the amino-terminal of the human heavy chain IgG4 constant region (such as SEQ ID NO: 43) to form the full-length heavy chain of the antibody, and the humanized antibody is simultaneously
  • the carboxy-terminus of the variable region of the light chain was connected to the amino-terminus of the constant region of the human light chain kappa (such as SEQ ID NO: 44) to form the full-length light chain of the antibody, and the humanized antibody of mAb201 as shown in the following table 7-1 was obtained:
  • 201-H1L1 means that the heavy chain variable region is hAb201VH1 (as shown in SEQ ID NO: 28), the light chain variable region is hAb201VL1 (as shown in SEQ ID NO: 20), and the heavy chain constant region It is IgG4 constant region (as shown in SEQ ID NO: 43), and the light chain constant region is a humanized antibody of kappa constant region (as shown in SEQ ID NO: 44).
  • the carboxy-terminal of the heavy chain variable region of the humanized antibody derived from mAb259 was linked to the amino-terminal of the human heavy chain IgG4 constant region to form a full-length heavy chain of the antibody, and the carboxy-terminal of the light chain variable region of the humanized antibody The amino terminus of the light chain ⁇ constant region was connected to form the full-length light chain of the antibody, and the mAb259 humanized antibody as shown in the following table 7-2 was obtained:
  • 259-H1L1 indicates that the heavy chain variable region is hAb259VH1 (as shown in SEQ ID NO: 39), the light chain variable region is hAb259VL1 (as shown in SEQ ID NO: 32), and the heavy chain constant region It is IgG4 constant region (as shown in SEQ ID NO: 43), and the light chain constant region is a humanized antibody of kappa constant region (as shown in SEQ ID NO: 44).
  • Embodiment 3 Construction of anti-ICOSL antibody fusion protein
  • the aforementioned anti-ICOSL antibody is fused with the TACI polypeptide to construct an anti-ICOSL antibody fusion protein.
  • the anti-ICOSL antibody is humanized antibody 259-H4L7 or 201-H3L3.
  • the TACI polypeptide can be any suitable TACI polypeptide.
  • the TACI polypeptide is derived from the patent application PCT/CN2022/084256 and its priority 202110348497.6 (invention name: a new type of TACI polypeptide, its fusion protein and its use; filing date: 2021/3/31) (by citing the TACI polypeptides described in this paper in its entirety), including, for example, the TACI polypeptides shown in Table 11 below:
  • the TACI polypeptide (such as TACI-9-15c) is fused to the C-terminal of the heavy chain, the N-terminal or the C-terminal of the light chain of an anti-ICOSL antibody (such as 201-H3L3 or 259-H4L7), and the fusion antibody heavy chain
  • an anti-ICOSL antibody such as 201-H3L3 or 259-H4L7
  • the C-terminus of the TACI polypeptide at the C-terminus of the chain or light chain adds QK as a protective amino acid to prevent the amino acid at the TACI-terminus from being cut by carboxypeptidase.
  • anti-ICOSL antibody fusion proteins were constructed, among which, the structural schematic diagrams of 259H4L7-T11 and 201H3L3-T11 are shown in Figure 1, the structural schematic diagrams of 201H3L3-T7 and 259H4L7-T7 are shown in Figure 2, and the structural schematic diagrams of 259H4L7-T9 are shown in Figure 3.
  • the anti-ICOSL antibody fusion protein includes 2 identical first chains and 2 identical second chains, specifically as follows:
  • the amino acid sequence of the second chain of 201H3L3-T7 is the same as that of the light chain of 201-H3L3 (SEQ ID NO: 46).
  • amino acid sequence of the first chain of 201H3L3-T11 is the same as the amino acid sequence of the first chain of 201H3L3-T7 (SEQ ID NO: 84);
  • amino acid sequence of the second chain of 259H4L7-T7 is the same as that of the light chain of 259-H4L7 (SEQ ID NO: 48).
  • the amino acid sequence of the first chain of 259H4L7-T9 is the same as the amino acid sequence of the first chain of 259H4L7-T7 (SEQ ID NO: 86);
  • amino acid sequence of the first chain of 259H4L7-T11 is the same as the amino acid sequence of the first chain of 259H4L7-T7 (SEQ ID NO: 86);
  • the single underline is the TACI sequence
  • the double underline is the antibody variable region sequence
  • the italic is the antibody constant region sequence
  • the bold is the linker sequence.
  • control molecule used in the test example of this disclosure is AMG-570 (an antibody fusion protein fused with an anti-ICOSL antibody and a BAFF polypeptide) is as follows:
  • the single underline in the sequence is the variable region
  • the double underline is the BAFF-binding polypeptide
  • the bold is the connecting amino acid
  • the rest are the constant region.
  • hICOSL-CHOK1 In order to detect the binding of the test molecule to ICOSL, its binding activity to hICOSL-CHOK1 in CHOK1 cells (ATCC, CCL-61) overexpressing human ICOSL was detected by FACS (flow cytometry). Digest hICOSL-CHOK1 cells with 0.25% trypsin, centrifuge at 300g for 5 minutes, and resuspend the cells with PBS (Biosun, B320) + 2% FBS (Biosun, BS-0002-500). 50 ⁇ L of the cell suspension was added to a 96-well U-plate (Corning, 3795), so that the number of cells per well was 10 5 .
  • the binding activity of the test molecule to BAFF and APRIL protein is detected by ELISA method (coating test molecule).
  • the specific method is as follows:
  • the molecule to be tested was diluted to 2 ⁇ g/mL with PBS buffer at pH 7.4, added to a 96-well microtiter plate (Corning, 3590) at a volume of 100 ⁇ L/well, and incubated overnight at 4°C. After the liquid was discarded, 300 ⁇ L of 1% Casein (37528, Thermo) diluted with PBS was added to each well for blocking, and incubated at 37° C. for 2 hours.
  • PBST buffer pH 7.4PBS containing 0.1% tween-20
  • human BAFF ACROBiosystems, BAF-H52D4
  • human APRIL R&D Systems, 5860-AP-010/CF
  • TMB chromogenic substrate KPL, 5120-00757
  • 50 ⁇ l 1M H 2 SO 4 50 ⁇ l 1M H 2 SO 4
  • the absorbance at 450nm was used to fit the binding curve between the molecule to be tested and the antigen by software, and the EC 50 value was calculated.
  • Test Example 3 Blocking the binding experiment of ICOSL and ICOS
  • the blocking activity of the molecules to be tested on the combination of ICOSL and ICOS was detected by FACS. Digest hICOSL-CHOK1 cells (same as Test Example 1) with 0.25% trypsin, centrifuge at 300g for 5 minutes, and resuspend the cells with PBS (Biosun, B320) + 2% FBS (Biosun, BS-0002-500). Add 50 ⁇ L of cell suspension to 96-well plate
  • the number of cells per well is 10 5 .
  • the molecules to be tested were added into a 96-well plate in a gradient dilution, mixed gently, and incubated at 4°C in the dark for 40 minutes.
  • the plate was washed twice with PBS+2% FBS, 100 ⁇ L of 20 nM hICOS-rabbit Fc protein (Sino Biological, 10344-H31H) was added to each well, and incubated at 4° C. in the dark for 1 hour.
  • the plate was washed twice, and 100 ⁇ L of goat anti-rabbit IgG Fc FITC fluorescent secondary antibody (Invitrogen, A16125, diluted 1:500) was added to each well, and incubated at 4°C in the dark for 40 minutes. After washing the plate twice, add 100 ⁇ L PBS to each well to resuspend the cells, and read the fluorescence value with a flow cytometer. Use software to fit the curve of inhibitory ligand and receptor binding, and calculate the IC 50 value. The experimental results are shown in Table 15 below.
  • Test Example 4 Binding experiment of blocking BAFF, APRIL and receptors
  • the blocking activity of the anti-ICOSL antibody fusion protein on the binding of BAFF or APRIL to its receptors was detected by ELISA method.
  • the specific method is as follows:
  • the receptor protein was diluted to 2 ⁇ g/mL with PBS buffer of pH 7.4, added to a 96-well microtiter plate at a volume of 100 ⁇ L/well, and incubated overnight at 4°C. After the liquid was discarded, 200 ⁇ L of 1% Casein blocking solution (Thermo, 37528) was added to each well for blocking, and incubated at 37° C. for 2 hours. After the blocking, the blocking solution was discarded, and the plate was washed 3 times with PBST buffer (pH 7.4, PBS containing 0.1% tween-20) before use.
  • PBST buffer pH 7.4, PBS containing 0.1% tween-20
  • a fixed concentration of biotin-labeled ligand protein was mixed with a gradiently diluted fusion protein, pre-incubated at 37°C for 30 minutes, then added to the blocked microtiter plate, and incubated at 37°C for 1.5 hours. After the incubation, the plate was washed 3 times with PBST, 100 ⁇ L streptavidin-HRP (Invitrogen, 434323, diluted 1:4000) was added to each well, and incubated at 37° C. for 1 hour.
  • the sources of the receptor proteins used are as follows: BAFF-R (Sino biological, 16079-H02H), BCMA (Sino biological, 10620-H02H), TACI (ACROBiosystems, TAI-H5256).
  • BAFF-R Sesino biological, 16079-H02H
  • BCMA Sesino biological, 10620-H02H
  • TACI ACROBiosystems, TAI-H5256.
  • Test Example 5 Jurkat-ICOS-NFAT reporter gene experiment
  • the inhibitory activity of the tested molecules on the ICOSL-ICOS signaling pathway was detected by the Jurkat-ICOS-NFAT reporter gene assay.
  • the experimental method is as follows:
  • the CHOK1 cells stably expressing human ICOSL hICOSL-CHOK1 were resuspended, and 12,500 cells were plated in 96-well cell plates at 40 ⁇ L per well.
  • the full-length sequence of ICOS was fused with the intracellular region of CD3zeta, and stably expressed on the surface of Jurkat cells (ATCC, TIB-152) to obtain Jurkat-NFAT-ICOS-CD3zeta cells.
  • 40 ⁇ L per well of 2 ⁇ 10 5 cells were plated in a 96-well cell plate containing hICOSL-CHOK1.
  • the sample to be tested was serially diluted, 20 ⁇ L per well was added to the cell plate, and incubated in a 37°C incubator for 48 hours.
  • the cell culture plate was taken out, 50 ⁇ L of One-glo luciferase detection solution (Promega, E6120) was added to each well, and the bioluminescent signal was detected with a microplate reader (PerkinElmer, EnVision 2105).
  • the fluorescence value was fitted to the inhibition curve by software, and the IC 50 value was calculated.
  • Table 19 The experimental results are shown in Table 19 below.
  • the activity of anti-ICOSL antibody fusion protein in inhibiting ICOSL-induced T cell proliferation was detected by T cell proliferation assay.
  • the experimental method is as follows:
  • sample name IC 50 (nM) for inhibition of T cell proliferation 201H3L3-T7 0.6146 201H3L3-T11 0.6429 259H4L7-T7 0.6007 259H4L7-T9 0.3753 259H4L7-T11 0.5890
  • the activity of anti-ICOSL antibody fusion protein in inhibiting the proliferation of B cells induced by BAFF and APRIL was detected by B cell proliferation assay.
  • the experimental method is as follows:
  • the mouse spleen was taken for grinding, centrifuged at 4°C for 5 minutes to collect the cells in the lower layer, washed once with washing solution (PBS+2% FBS+2mM EDTA) and centrifuged, after removing the supernatant, red blood cell lysate (RBC Lysis Buffer, Invitrogen, 00 -4333-57), let it stand at room temperature for 5 minutes until the red blood cells were completely lysed. Centrifuge again and resuspend cells for counting.
  • the cell suspension was sorted with a B Cell Isolation Kit (B Cell Isolation Kit, Miltenyi Biotec, 130-090-862), and the isolated B cells were sorted with RPMI 1640 medium (Gibco, 11875119)+10% FBS (Gibco, 10099-141)+50 ⁇ M 2-mercaptoethanol (Sigma-Aldrich, M6250), resuspended and counted, and the cells were plated in 96-well cell plates for use.
  • B Cell Isolation Kit B Cell Isolation Kit, Miltenyi Biotec, 130-090-862
  • RPMI 1640 medium Gibco, 11875119
  • FBS Gibco, 10099-141
  • 2-mercaptoethanol Sigma-Aldrich, M6250
  • Biosensing chip Protein A (GE, 29127556) to affinity capture a certain amount of the sample to be tested, and then flow through a series of concentration gradient antigens on the surface of the chip, and use Biacore (GE, 8K) to detect the reaction signal in real time to obtain the binding and dissolving away from the curve.
  • Biacore GE, 8K
  • the biochip was cleaned and regenerated with 10 mM glycine-hydrochloric acid solution pH 1.5 (GE, BR-1003-54).
  • the experimental data was fitted with a 1:1 model using BIAevaluation version 4.1 software to obtain the affinity value.
  • the relevant antigen 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), crab-eating Monkey ICOSL (Sino biological, 90800-C08H), cynomolgus monkey BAFF (Kactus, BAF-CM412), cynomolgus monkey APRIL (Kactus, APR-CM410B), mouse ICOSL (Sino biological, 50190-M08H), mouse BAFF (Acro Biosystems, BAF-M521y), mouse APRIL (R&D Systems, 7907-AP/CF).
  • the test results of affinity are shown in Table 23 to Table 30 below:
  • the anti-ICOSL antibody fusion protein constructed in this disclosure has strong affinity to APRIL of human, monkey and mouse, while the positive control molecule AMG-570 does not bind to APRIL of human, monkey and mouse.
  • the anti-ICOSL antibody fusion protein constructed in the present disclosure has a higher affinity with human, monkey and mouse BAFF than the positive control molecule AMG-570.
  • the anti-ICOSL antibody and its fusion protein constructed in this disclosure have good binding activity to human ICOSL and monkey ICOSL, but have no cross-binding activity to mouse ICOSL.
  • mice were stimulated with human hICOSL-CHOK1 cells and human BAFF protein to induce the production of cytokines such as IFN ⁇ , IL-10 and IgA in the mice, and the in vivo activity of the anti-ICOSL antibody fusion protein was evaluated by detecting the levels of these cytokines.
  • the experimental process is as follows:
  • mice SPF grade female C57BL/6 mice (Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.), 8 weeks old, were randomly divided into 4 groups, 8 mice in each group, and injected with hICOSL-CHOK1 cells (10 6 cells/mouse) and 1 mg/kg human BAFF protein (Sino biological, 10056-HNCH), injected once a day for four days.
  • Each group positive control (AMG-570 12mpk) group, 259H4L7-T7 11.7mpk group, 259H4L7-T11 12.4mpk group, negative control (PBS) group
  • PBS negative control
  • mice in each group were collected, and the levels of IFN ⁇ , IL-10 and IgA were detected respectively.
  • AMG-570 is used as a positive control
  • PBS is used as a negative control.
  • the drug molar concentrations of AMG-570 12mpk (mpk, that is, the dosage is mg/kg), 259H4L7-T7 11.7mpk and 259H4L7-T11 12.4mpk are the same.
  • the percentages in the table are the percentages of reductions in plasma concentrations of IFN ⁇ , IL-10, and IgA in each test group compared with the negative control.
  • mice SPF grade female human ICOSL/human ICOS C57BL/6 transgenic mice (Biocytogen Jiangsu Gene Biotechnology Co., Ltd.), mice weighing 15-18g, aged 6 to 8 weeks. On day 1, mice were emulsified and mixed with 1 mg/kg KLH (Keyhole Limpet Hemocyanin, hemocyanin) (Sigma, H7017) and adjuvant (Thermo, 77161), and mice were immunized with 5 mg/kg KLH alone on day 21 .
  • KLH is a protein with strong immunogenicity, which can cause a strong immune response in mice and produce KLH-specific antibodies.
  • Dosing started the day before KLH immunization and was administered every 5 days for a total of 35 days.
  • the dosage is AMG-570 15mpk, 259H4L7-T7 14.6mpk, 259H4L7-T11 15.6mpk, the drug molar concentration of each group is the same, AMG-570 is used as positive control, and PBS is used as negative control.
  • Serum samples of the mice in each group were taken every week, and the intensity of the fusion protein to inhibit the immune response was evaluated by detecting the KLH-specific IgG and IgM contents in the mouse serum.
  • the detection kits used in this test example are as follows: Mouse anti-KLH IgG ELISA Kit (Life diagnostics, KLHG-1), Mouse anti-KLH IgM ELISA Kit (Life Diagnostics, KLHM-1). The experimental results are shown in Figure 7 and Figure 8.
  • the detection results of KLH-specific IgG showed that all the administration groups could significantly inhibit the production of KLH-specific IgG at the two time points of day 28 and day 35, and the 259H4L7-T7 and 259H4L7-T11 groups were completely absent An immune response appeared, the level of KLH-specific IgG was the same as that of normal mice, and the immunosuppressive activity was stronger than that of AMG-570.
  • the detection results of KLH-specific IgM showed that AMG-570 only had significant inhibitory activity on days 7-14, while 259H4L7-T7 and 259H4L7-T11 showed significant inhibitory activity at all time points, and the inhibitory activity was greater than Significantly stronger than AMG-570.

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Abstract

涉及抗ICOSL抗体融合蛋白及用途。具体而言，涉及抗ICOSL抗体、其融合蛋白及用途。

Description

抗ICOSL抗体融合蛋白及用途
本申请要求2021年11月11日提交的专利申请(申请号CN202111330908.5)的优先权。
技术领域
本披露属于生物技术领域，更具体地，本披露涉及一种抗ICOSL抗体及其融合蛋白和应用。
背景技术
这里的陈述仅是提供与本披露有关的背景信息，而不必然地构成现有技术。
系统性红斑狼疮(systemic lupus erythematosus，SLE)是一种影响多种器官的自身免疫性疾病。在遗传和环境等因素的作用下，导致人体失去免疫耐受，产生自身抗体和核苷酸免疫复合物进入血液循环系统，免疫复合物激活TLR受体，刺激pDC细胞分泌大量的干扰素α。干扰素进一步刺激髓系细胞分泌多种细胞因子，分别促进B细胞和T细胞的激活(Curr Opin Rheumatol.2017Mar；29(2):178-186.)。已知BAFF和APRIL是两种B细胞的激活因子，它们通过与B细胞表面的受体作用促进B细胞的分化成熟和产生抗体(Nat Rev Rheumatol.2016Nov22；12(12):716-730.)。而ICOSL和ICOS的信号通路在T细胞激活和T-B细胞相互作用的过程中发挥了非常重要的作用。T细胞激活后释放促炎细胞因子导致促炎T细胞的分化并破坏Treg细胞在体内的平衡。此外，T-B细胞之间的相互作用会进一步促进T细胞和B细胞的活化并产生促炎细胞因子进而形成持续的免疫反应循环(Nat Rev Rheumatol.2014Jan；10(1):23-34.)。在SLE的发病过程中，T细胞和B细胞这2条免疫途径都参与其中，同时抑制T细胞和B细胞激活以及T-B细胞相互作用的信号通路，对SLE会有比较好的作用效果。其它研发还发现，ICOS/ICOSL信号途径在其它机体炎症反应、过敏反应、自身免疫性疾病、移植排斥等免疫应答以及B细胞或T细胞障碍(例如癌症)发生过程中也发挥了重要的作用。
ICOSL(Inducible costimulator-ligand，也称为B7RP1，B7H2，CD275)，是属于B7家族的一个免疫共刺激分子。ICOSL主要在抗原递呈细胞表面表达，包括B细胞，巨噬细胞，单核细胞和树突状细胞(BioDrugs.2013Feb；27(1):1-13.)。目前已知ICOSL的受体是ICOS，ICOS主要在T细胞表面表达，ICOSL和ICOS介导的信号通路主要在T细胞的分化和T细胞依赖的B细胞活化中发挥作用(Nat Rev Cancer.2012Mar 22；12(4):252-64.)。ICOSL-ICOS信号通路的激活可以分别促进Th2、Th1、Th17和Tfh细胞分泌表达IL-4，IFNγ，IL-23R和IL-21，这些细胞因子和受体参与维持多种T细胞亚型的活性并且促进他们进一步扩增(Curr Opin Immunol.2010Jun；22(3):326-32.)。此外，ICOSL在Tfh细胞迁移和T-B细胞相互 作用的过程中也发挥了非常重要的功能。
TACI(Transmembrane activator and CAML interactor，也称为TNFRSF13B)是一种膜结合受体，具有包含二个富含半胱氨酸的假性重复片断(cysteine-rich pseudo-repeats)的胞外区、一个跨膜区和与CAML(钙调节剂和亲环蛋白配体)相互作用的胞质区。TACI与B细胞和T细胞的一种亚型相关。TACI受体与肿瘤坏死因子配体家族的BAFF(B-cell activating factor，也称为TNFSF13B)相结合。BAFF是属于TNF家族的一种B细胞激活因子。BAFF主要在骨髓细胞膜表面表达，以三聚体的形式存在。在细胞膜表面的BAFF会被蛋白酶水解形成可溶性的BAFF进入血液循环系统，可溶性的BAFF具有多聚化的特征，最多可以形成60聚体。另外BAFF也可以跟同家族的另外一个蛋白APRIL作用形成异源的三聚体。目前已知在B细胞表面有三个BAFF的受体，分别为BAFF-R、BCMA和TACI。BAFF与这三个受体作用，参与B细胞的分化成熟、存活和调节。APRIL与BAFF有两个共同的受体，分别为BCMA和TACI。APRIL与这两个受体作用参与B细胞的存活和调节(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对维持B细胞的体内平衡非常重要，BAFF信号通路的过度激活会导致自反应B细胞的存活并产生自身抗体促进自身免疫反应(Cancro,M.P.,D.P.D'Cruz,and M.A.Khamashta,J Clin Invest,2009.119:p.1066-73)。
发明内容
本披露构建了一种抗ICOSL抗体融合蛋白，其包含抗ICOSL抗体和TACI多肽。
在一些实施方案中，所述抗ICOSL抗体融合蛋白，其中所述抗ICOSL抗体包含重链可变区和轻链可变区，其中，所述重链可变区包含HCDR1、HCDR2和HCDR3，所述轻链可变区包含LCDR1、LCDR2和LCDR3，其中，
(i)所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：42、3、39、40或41中的HCDR1、HCDR2和HCDR3的氨基酸序列，和所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：38、4、32、33、34、35、36或37中的LCDR1、LCDR2和LCDR3的氨基酸序列；或
(ii)所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：30、1、28或29中的HCDR1、HCDR2和HCDR3的氨基酸序列，和所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：22、2、20、21、23、24、25、26或27中的LCDR1、LCDR2和LCDR3的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据选自Kabat、IMGT、Chothia、AbM和Contact的编号规则定义的。 在一些实施方案中，所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据Kabat编号规则定义的。在一些实施方案中，所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据IMGT编号规则定义的。在一些实施方案中，所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据Chothia编号规则定义的。在一些实施方案中，所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据AbM编号规则定义的。在一些实施方案中，所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据Contact编号规则定义的。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中：
(i)所述重链可变区的HCDR1包含SEQ ID NO：11的氨基酸序列，HCDR2包含SEQ ID NO：31或12的氨基酸序列，和HCDR3包含SEQ ID NO：13的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：14的氨基酸序列，LCDR2包含SEQ ID NO：15的氨基酸序列，和LCDR3包含SEQ ID NO：16的氨基酸序列；或
(ii)所述重链可变区的HCDR1包含SEQ ID NO：5的氨基酸序列，HCDR2包含SEQ ID NO：19或6的氨基酸序列，和HCDR3包含SEQ ID NO：7的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：8的氨基酸序列，LCDR2包含SEQ ID NO：17或9的氨基酸序列，和LCDR3包含SEQ ID NO：10或18的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中
(i)所述重链可变区的HCDR1包含SEQ ID NO：11的氨基酸序列，HCDR2包含SEQ ID NO：31的氨基酸序列，和HCDR3包含SEQ ID NO：13的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：14的氨基酸序列，LCDR2包含SEQ ID NO：15的氨基酸序列，和LCDR3包含SEQ ID NO：16的氨基酸序列；或
(ii)所述重链可变区的HCDR1包含SEQ ID NO：5的氨基酸序列，HCDR2包含SEQ ID NO：19的氨基酸序列，和HCDR3包含SEQ ID NO：7的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：8的氨基酸序列，LCDR2包含SEQ ID NO：17的氨基酸序列，和LCDR3包含SEQ ID NO：10的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中
所述重链可变区的HCDR1包含SEQ ID NO：11的氨基酸序列，HCDR2包含SEQ ID NO：12的氨基酸序列，和HCDR3包含SEQ ID NO：13的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：14的氨基酸序列，LCDR2包含SEQ ID NO：15的氨基酸序列，和LCDR3包含SEQ ID NO：16的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中
所述重链可变区的HCDR1包含SEQ ID NO：5的氨基酸序列，HCDR2包含SEQ ID NO：6的氨基酸序列，和HCDR3包含SEQ ID NO：7的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：8的氨基酸序列，LCDR2包含SEQ ID NO：9的氨基酸序列，和LCDR3包含SEQ ID NO：10的氨基酸序列；或
所述重链可变区的HCDR1包含SEQ ID NO：5的氨基酸序列，HCDR2包含SEQ ID NO：6的氨基酸序列，和HCDR3包含SEQ ID NO：7的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：8的氨基酸序列，LCDR2包含SEQ ID NO：17的氨基酸序列，和LCDR3包含SEQ ID NO：10的氨基酸序列；或
所述重链可变区的HCDR1包含SEQ ID NO：5的氨基酸序列，HCDR2包含SEQ ID NO：19的氨基酸序列，和HCDR3包含SEQ ID NO：7的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：8的氨基酸序列，LCDR2包含SEQ ID NO：9的氨基酸序列，和LCDR3包含SEQ ID NO：10的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，所述的抗ICOSL抗体为鼠源抗体、嵌合抗体或人源化抗体。
在一些实施方案中，所述抗体是人源化抗体。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中：
(i)所述重链可变区包含与SEQ ID NO：42、39、40或41具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含与SEQ ID NO：38、32、33、34、35、36或37具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或
(ii)所述重链可变区包含与SEQ ID NO：30、28或29具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含与SEQ ID NO：22、20、21、23、24、25、26或27具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或(iii)所述重链可变区包含与SEQ ID NO：3具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含与SEQ ID NO：4具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或
(iv)所述重链可变区包含与SEQ ID NO：1具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含与SEQ ID NO：2具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，所述抗ICOSL抗体的重链可变区的框架区上包含选自第37、43和49位(根据Kabat编号系统编号)中的一个或更多个氨基酸突变，和/或轻链可变区的框架区上包含选自第1、2、3、42、43、60和85位(根据Kabat编号系统编号)中的一个或更多个氨基酸突 变。
在一些实施方案中，所述的抗ICOSL抗体融合蛋白，其中所述重链可变区的HCDR1包含SEQ ID NO：11的氨基酸序列，HCDR2包含SEQ ID NO：31或12的氨基酸序列，和HCDR3包含SEQ ID NO：13的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：14的氨基酸序列，LCDR2包含SEQ ID NO：15的氨基酸序列，和LCDR3包含SEQ ID NO：16的氨基酸序列；所述抗体的重链可变区的框架区上包含选自：37I、43E和49A(根据Kabat编号系统编号)中的一个或更多个氨基酸突变，和/或所述轻链可变区的框架区上包含选自：1N、2T、3V、42Q、43S、60D、85V(根据Kabat编号系统编号)中的一个或更多个氨基酸突变。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，所述抗ICOSL抗体的重链可变区的框架区上包含选自第1、24、69、71、73、78位(根据Kabat编号系统编号)中的一个或更多个氨基酸突变，和/或轻链可变区的框架区上包含选自第1、2和4位(根据Kabat编号系统编号)中的一个或更多个氨基酸突变。
在一些实施方案中，所述的抗ICOSL抗体融合蛋白，其中抗ICOSL抗体的重链可变区的HCDR1包含SEQ ID NO：5的氨基酸序列，HCDR2包含SEQ ID NO：19或6的氨基酸序列，和HCDR3包含SEQ ID NO：7的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：8的氨基酸序列，LCDR2包含SEQ ID NO：17或9的氨基酸序列，和LCDR3包含SEQ ID NO：10或18的氨基酸序列；所述抗体的重链可变区的框架区上包含选自：1E、24T、69L、71V、73K和78A(根据Kabat编号系统编号)中的一个或更多个氨基酸突变，和/或所述轻链可变区的框架区上包含选自：1A、2V和4L(根据Kabat编号系统编号)中的一个或更多个氨基酸突变。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中，
(i)所述重链可变区包含SEQ ID NO：42、39、40或41的氨基酸序列，和所述轻链可变区包含SEQ ID NO：38、32、33、34、35、36或37的氨基酸序列；或
(ii)所述重链可变区包含SEQ ID NO：30、28或29的氨基酸序列，和所述轻链可变区包含SEQ ID NO：22、20、21、23、24、25、26或27的氨基酸序列；或
(iii)所述重链可变区包含SEQ ID NO：1的氨基酸序列，和所述轻链可变区包含SEQ ID NO：2的氨基酸序列；或
(iv)所述重链可变区包含SEQ ID NO：3的氨基酸序列，和所述轻链可变区包含SEQ ID NO：4的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中，
(i)所述重链可变区包含SEQ ID NO：42的氨基酸序列，和所述轻链可变区 包含SEQ ID NO：38的氨基酸序列；或
(ii)所述重链可变区包含SEQ ID NO：30的氨基酸序列，和所述轻链可变区包含SEQ ID NO：22的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中所述的抗ICOSL抗体包含抗体重链恒定区和轻链恒定区。在一些实施方案中，所述重链恒定区为人IgG重链恒定区。在一些实施方案中，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区。在一些实施方案中，所述轻链恒定区选自人抗体κ或λ链恒定区。在一些实施方案中，所述重链恒定区为人IgG4重链恒定区，所述轻链恒定区为人κ轻链恒定区。在一些实施方案中，所述重链恒定区的Fc区具有一个或更多个能够减少Fc区与Fc受体结合的氨基酸取代。在一些实施方案中，所述Fc区具有L234A、L235A突变，和/或S228P突变，和/或YTE突变(M252Y、S254T和T256E)，所述突变编号依据为EU索引。在一些实施方案中，所述重链恒定区包含SEQ ID NO：43的氨基酸序列，所述轻链恒定区包含SEQ ID NO：44的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中，
(i)所述抗ICOSL抗体的重链包含与SEQ ID NO：47具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和轻链包含与SEQ ID NO：48具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或
(ii)所述抗ICOSL抗体的重链包含与SEQ ID NO：45具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和轻链包含与SEQ ID NO：46具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，
(i)所述抗ICOSL抗体的重链包含SEQ ID NO：47的氨基酸序列，和所述抗ICOSL抗体的轻链包含SEQ ID NO：48的氨基酸序列；或
(ii)所述抗ICOSL抗体的重链包含SEQ ID NO：45的氨基酸序列，和所述抗ICOSL抗体的轻链包含SEQ ID NO：46的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中，所述TACI多肽具有更好的防止断裂功能。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中所述TACI多肽为包含SEQ ID NO：58的第48位至第85位氨基酸残基的多肽或其变体；其中，所述变体为选自第49、52、53、57、65、82和83位中的一个或更多个位点上具有氨基酸替换，所述氨基酸替换的位点为相对于序列SEQ ID NO：58的自然顺序编号的氨基酸残基位点。在一些实施方案中，所述TACI多肽的变体为在选自由49T或49R、52S、53E或53Q、57E、65T或65A、82A或82R、和83Y组 成的组中的一个或更多个氨基酸替换，所述氨基酸替换的位点为相对于序列SEQ ID NO：58的自然顺序编号的氨基酸残基位点。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中，所述TACI多肽如SEQ ID NO：58所示或是SEQ ID NO：58的截短片段或其变体。所述截短片段包含SEQ ID NO：58的第48位至第85位氨基酸残基。所述变体为在SEQ ID NO：58或其截短片段上具有选自第49、52、53、57、65、82和83位中的一个或更多个氨基酸替换，其中所述氨基酸替换的位点为相对于序列SEQ ID NO：58的自然顺序编号的氨基酸残基位点。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中所述TACI多肽的截短片段包含：SEQ ID NO：58的第48位至第86位氨基酸残基；SEQ ID NO：58的第48位至第87位氨基酸残基；或SEQ ID NO：58的第48位至第88位氨基酸残基。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中，所述TACI多肽，序列如SEQ ID NO：60-63任一项所示。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中，所述TACI多肽，其序列为SEQ ID NO：58变体或者SEQ ID NO：58的截短片段(例如SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列的截短片段)的变体，所述变体为在SEQ ID NO：58或其截短片段序列上具有选自第49、52、53、57、65、82和83位中的任意1个、2个、3个、4个、5个、6个或7个氨基酸的替换，所述氨基酸替换的位点为相对于序列SEQ ID NO：58的自然顺序编号的氨基酸残基位点。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中所述TACI多肽为在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有选自由49T或49R、52S、53E或53Q、57E、65T或65A、82A或82R、和83Y组成的组中的一个或更多个氨基酸替换(例如1个、2个、3个、4个、5个、6个或7个氨基酸替换)，其中所述氨基酸替换的位点为相对于序列SEQ ID NO：58的自然顺序编号的氨基酸残基位点。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中所述TACI多肽为：在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有选自49T、52S、53E、53Q、57E和82A中的任一个氨基酸替换。在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有49R和65T氨基酸替换。在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有49R和65A氨基酸替换。在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有49R、65T和82R氨基酸替换。在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID  NO：63序列上具有53E和57E氨基酸替换。在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有52S、53E和57E氨基酸替换。在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有49T和82A氨基酸替换。在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有49T和83Y氨基酸替换。在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有49T、82A和83Y氨基酸替换。或者在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有49T、53E、57E和82A氨基酸替换。其中前述氨基酸替换的位点为相对于序列SEQ ID NO：58的自然顺序编号的氨基酸残基位点。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中，所述TACI多肽，其氨基酸序列如SEQ ID NO：51至83中任一所示。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中所述TACI多肽的氨基酸序列如SEQ ID NO：60-63、SEQ ID NO：66-83中的任一所示。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中所述TACI多肽的氨基酸序列如SEQ ID NO：83所示。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其包括：
(A)第一链：[抗ICOSL抗体的重链]-[连接子1]-[TACI多肽1]，和
第二链：[TACI多肽2]-[连接子2]-[抗ICOSL抗体的轻链](图1)；
(B)第一链：[抗ICOSL抗体的重链]-[连接子1]-[TACI多肽1]，和
第二链：抗ICOSL抗体的轻链(图2)；或
(C)第一链：[抗ICOSL抗体的重链]-[连接子1]-[TACI多肽1]，和
第二链：[抗ICOSL抗体的轻链]-[连接子2]-[TACI多肽2](图3)；
其中，所述(A)、(B)或(C)中，所述TACI多肽1和TACI多肽2是相同或不相同的，所述连接子1和连接子2是相同或不相同的。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中，所述TACI多肽1或TACI多肽2，其氨基酸序列如SEQ ID NO：51至83中任一所示。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中所述TACI多肽1或TACI多肽2的氨基酸序列如SEQ ID NO：60-63、SEQ ID NO：66-83中的任一所示。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中所述TACI多肽1或TACI多肽2的氨基酸序列如SEQ ID NO：83所示。
本披露中的序数词“第一”、“第二”、“1”、“2”(如“连接子2”中的“2”)仅用于区分不同的特征、要素、组件或步骤，不意图限制数量、顺序、水平。
在一些实施方案中，所述连接子1和连接子2各自独立地具有(G xS) y的结构，其中，x选自1-5的整数，y选自0-6的整数。在一些实施方案中，所述连接 子1和连接子2各自独立地具有(G xS) y的结构，其中，x选自1-5的整数，y选自1-6的整数。在一些实施方案中，所述连接子1和连接子2各自独立地为GGGS(如SEQ ID NO：89所示)或GGGGSGGGGSGGGGS(如SEQ ID NO：90所示)。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中，所述的抗ICOSL抗体融合蛋白具有：包含SEQ ID NO：86的氨基酸序列的第一链，和包含SEQ ID NO：88、87或48的氨基酸序列的第二链；或所述的抗ICOSL抗体融合蛋白具有：包含SEQ ID NO：84的氨基酸序列的第一链，和包含SEQ ID NO：85或46的氨基酸序列的第二链。在一些实施方案中，所述的抗ICOSL抗体融合蛋白具有2条包含SEQ ID NO：86的氨基酸序列的第一链，和2条包含SEQ ID NO：88的氨基酸序列的第二链。在一些实施方案中，所述的抗ICOSL抗体融合蛋白具有2条包含SEQ ID NO：86的氨基酸序列的第一链，和2条包含SEQ ID NO：87的氨基酸序列的第二链。在一些实施方案中，所述的抗ICOSL抗体融合蛋白具有2条包含SEQ ID NO：86的氨基酸序列的第一链，和2条包含SEQ ID NO：48的氨基酸序列的第二链。在一些实施方案中，所述的抗ICOSL抗体融合蛋白具有2条包含SEQ ID NO：84的氨基酸序列的第一链，和2条包含SEQ ID NO：85的氨基酸序列的第二链。在一些实施方案中，所述的抗ICOSL抗体融合蛋白具有2条包含SEQ ID NO：84的氨基酸序列的第一链，和2条包含SEQ ID NO：46的氨基酸序列的第二链。
另一方面，本披露提供一种抗ICOSL抗体，其中所述抗ICOSL抗体包含重链可变区和轻链可变区，其中，所述重链可变区包含HCDR1、HCDR2和HCDR3，所述轻链可变区包含LCDR1、LCDR2和LCDR3，其中，
(i)所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：42、3、39、40或41中的HCDR1、HCDR2和HCDR3的氨基酸序列，和所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：38、4、32、33、34、35、36或37中的LCDR1、LCDR2和LCDR3的氨基酸序列；或
(ii)所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：30、1、28或29中的HCDR1、HCDR2和HCDR3的氨基酸序列，和所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：22、2、20、21、23、24、25、26或27中的LCDR1、LCDR2和LCDR3的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，其中所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据选自Kabat、IMGT、Chothia、AbM和Contact的编号规则定义的。在一些实施方案中，所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据Kabat编号规则定义的。在一些实施方案中，所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据IMGT编号规则定义的。在一些实施方案中，所述重链可变区的 HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据Chothia编号规则定义的。在一些实施方案中，所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据AbM编号规则定义的。在一些实施方案中，所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据Contact编号规则定义的。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，其中：
(i)所述重链可变区的HCDR1包含SEQ ID NO：11的氨基酸序列，HCDR2包含SEQ ID NO：31或12的氨基酸序列，和HCDR3包含SEQ ID NO：13的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：14的氨基酸序列，LCDR2包含SEQ ID NO：15的氨基酸序列，和LCDR3包含SEQ ID NO：16的氨基酸序列；或
(ii)所述重链可变区的HCDR1包含SEQ ID NO：5的氨基酸序列，HCDR2包含SEQ ID NO：19或6的氨基酸序列，和HCDR3包含SEQ ID NO：7的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：8的氨基酸序列，LCDR2包含SEQ ID NO：17或9的氨基酸序列，和LCDR3包含SEQ ID NO：10或18的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，其中
(i)所述重链可变区的HCDR1包含SEQ ID NO：11的氨基酸序列，HCDR2包含SEQ ID NO：31的氨基酸序列，和HCDR3包含SEQ ID NO：13的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：14的氨基酸序列，LCDR2包含SEQ ID NO：15的氨基酸序列，和LCDR3包含SEQ ID NO：16的氨基酸序列；或
(ii)所述重链可变区的HCDR1包含SEQ ID NO：5的氨基酸序列，HCDR2包含SEQ ID NO：19的氨基酸序列，和HCDR3包含SEQ ID NO：7的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：8的氨基酸序列，LCDR2包含SEQ ID NO：17的氨基酸序列，和LCDR3包含SEQ ID NO：10的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，所述的抗ICOSL抗体为鼠源抗体、嵌合抗体或人源化抗体。在一些实施方案中，所述抗体是人源化抗体。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，其中：
(i)所述重链可变区包含与SEQ ID NO：42、39、40或41具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含与SEQ ID NO：38、32、33、34、35、36或37具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或
(ii)所述重链可变区包含与SEQ ID NO：30、28或29具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻 链可变区包含与SEQ ID NO：22、20、21、23、24、25、26或27具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或(iii)所述重链可变区包含与SEQ ID NO：3具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含与SEQ ID NO：4具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或
(iv)所述重链可变区包含与SEQ ID NO：1具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含与SEQ ID NO：2具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，所述抗ICOSL抗体的重链可变区的框架区上包含选自第37、43和49位(根据Kabat编号系统编号)中的一个或更多个氨基酸突变，和/或轻链可变区的框架区上包含选自第1、2、3、42、43、60和85位(根据Kabat编号系统编号)中的一个或更多个氨基酸突变。在一些实施方案中，所述的抗ICOSL抗体融合蛋白，其中所述重链可变区的HCDR1包含SEQ ID NO：11的氨基酸序列，HCDR2包含SEQ ID NO：31或12的氨基酸序列，和HCDR3包含SEQ ID NO：13的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：14的氨基酸序列，LCDR2包含SEQ ID NO：15的氨基酸序列，和LCDR3包含SEQ ID NO：16的氨基酸序列；所述抗体的重链可变区的框架区上包含选自：37I、43E和49A(根据Kabat编号系统编号)中的一个或更多个氨基酸突变，和/或所述轻链可变区的框架区上包含选自：1N、2T、3V、42Q、43S、60D、85V(根据Kabat编号系统编号)中的一个或更多个氨基酸突变。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，所述抗ICOSL抗体的重链可变区的框架区上包含选自第1、24、69、71、73、78位(根据Kabat编号系统编号)中的一个或更多个氨基酸突变，和/或轻链可变区的框架区上包含选自第1、2和4位(根据Kabat编号系统编号)中的一个或更多个氨基酸突变。在一些实施方案中，所述的抗ICOSL抗体融合蛋白，其中抗ICOSL抗体的重链可变区的HCDR1包含SEQ ID NO：5的氨基酸序列，HCDR2包含SEQ ID NO：19或6的氨基酸序列，和HCDR3包含SEQ ID NO：7的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：8的氨基酸序列，LCDR2包含SEQ ID NO：17或9的氨基酸序列，和LCDR3包含SEQ ID NO：10或18的氨基酸序列；所述抗体的重链可变区的框架区上包含选自：1E、24T、69L、71V、73K和78A(根据Kabat编号系统编号)中的一个或更多个氨基酸突变，和/或所述轻链可变区的框架区上包含选自：1A、2V和4L(根据Kabat编号系统编号)中的一个或更多个氨基酸突变。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，其中，
(i)所述重链可变区包含SEQ ID NO：42、39、40或41的氨基酸序列，和所述轻链可变区包含SEQ ID NO：38、32、33、34、35、36或37的氨基酸序列；或
(ii)所述重链可变区包含SEQ ID NO：30、28或29的氨基酸序列，和所述轻链可变区包含SEQ ID NO：22、20、21、23、24、25、26或27的氨基酸序列；或
(iii)所述重链可变区包含SEQ ID NO：1的氨基酸序列，和所述轻链可变区包含SEQ ID NO：2的氨基酸序列；或
(iv)所述重链可变区包含SEQ ID NO：3的氨基酸序列，和所述轻链可变区包含SEQ ID NO：4的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，其中，
(i)所述重链可变区包含SEQ ID NO：42的氨基酸序列，和所述轻链可变区包含SEQ ID NO：38的氨基酸序列；或
(ii)所述重链可变区包含SEQ ID NO：30的氨基酸序列，和所述轻链可变区包含SEQ ID NO：22的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，其中所述的抗ICOSL抗体包含抗体重链恒定区和轻链恒定区。在一些实施方案中，所述重链恒定区为人IgG重链恒定区。在一些实施方案中，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区；在一些实施方案中，所述轻链恒定区选自人抗体κ或λ链恒定区。在一些实施方案中，所述重链恒定区为人IgG4重链恒定区，所述轻链恒定区为人κ轻链恒定区。在一些实施方案中，所述重链恒定区的Fc区具有一个或更多个能够减少Fc区与Fc受体结合的氨基酸取代。在一些实施方案中，所述Fc区具有L234A、L235A突变，和/或S228P突变，和/或YTE突变(M252Y、S254T和T256E)，所述突变编号依据为EU索引。在一些实施方案中，所述重链恒定区包含SEQ ID NO：43的氨基酸序列，所述轻链恒定区包含SEQ ID NO：44的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，其中，
(i)所述抗ICOSL抗体的重链包含与SEQ ID NO：47具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和轻链包含与SEQ ID NO：48具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或
(ii)所述抗ICOSL抗体的重链包含与SEQ ID NO：45具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和轻链包含与SEQ ID NO：46具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，
(i)所述抗ICOSL抗体的重链包含SEQ ID NO：47的氨基酸序列，和所述抗ICOSL抗体的轻链包含SEQ ID NO：48的氨基酸序列；或
(ii)所述抗ICOSL抗体的重链包含SEQ ID NO：45的氨基酸序列，和所述抗ICOSL抗体的轻链包含SEQ ID NO：46的氨基酸序列。
在一些实施方案中，本披露还提供一种分离的抗ICOSL抗体或抗ICOSL抗体融合蛋白，其与前面任一项所述的抗ICOSL抗体或抗ICOSL抗体融合蛋白竞争性结合人ICOSL或其表位。
在一些实施方案中，前面任一项所述的抗ICOSL抗体或抗ICOSL抗体融合蛋白，其具有一种或更多种以下特性：
A.与人ICOSL(或其表位)和食蟹猴ICOSL(或其表位)特异性结合，不与鼠ICOSL特异性结合；优选地，以小于5.00E-09M(例如小于5.00E-09M、小于4.00E-09M、小于3.00E-09M、小于2.00E-09M、小于1.50E-09M、9.00E-10M或更小)的KD值与人ICOSL结合，和/或以小于8.00E-09M(例如小于8.00E-09M、小于7.00E-09M、小于6.00E-09M、小于5.00E-09M、小于4.00E-09M、小于3.00E-09M或更小)的KD值与食蟹猴ICOSL结合，所述KD值通过表面等离子体共振测定法测量(例如
Figure PCTCN2022131362-appb-000001
表面等离子体共振测定法测量)；在一些实施方案中，所述KD值通过本披露测试例8方法检测；
B.与人APRIL(或其表位)、食蟹猴APRIL(或其表位)和/或鼠APRIL(或其表位)特异性结合；优选地，以小于2.00E-11M(例如小于2.00E-11M、小于1.80E-11M、小于1.70E-11M、小于1.60E-11M、小于1.20E-11M、小于4.60E-12M或更小)的KD值与人APRIL结合，以小于2.00E-10M(例如小于2.00E-10M、小于1.80E-10M、小于1.70E-10M、小于1.60E-10M、小于1.50E-10M、小于1.40E-10M、小于1.20E-10M或更小)的KD值与食蟹猴APRIL结合，和/或以小于2.00E-11M(例如小于2.00E-11M、小于1.80E-11M、小于1.60E-11M、小于1.40E-11M、小于1.20E-11M、小于1.00E-11M、小于8.00E-12M或更小)的KD值与鼠APRIL结合，所述KD值通过表面等离子体共振测定法所测量(例如
Figure PCTCN2022131362-appb-000002
表面等离子体共振测定法测量)；在一些实施方案中，所述KD值通过本披露测试例8方法检测；
C.与人BAFF(或其表位)、食蟹猴BAFF(或其表位)和/或鼠BAFF(或其表位)特异性结合；优选地，以小于7.00E-11M(例如小于7.00E-11M、小于5.00E-11M、小于4.00E-11M、小于3.00E-11M、小于2.00E-11M、小于1.00E-11M或更小)的KD值与人BAFF结合，以小于4.00E-10M(例如小于4.00E-10M、小于3.00E-10M、小于2.00E-10M、小于1.30E-10M或更小)的KD值与食蟹猴BAFF结合，和/或以小于6.00E-11M(例如小于6.00E-11M、小于5.00E-11M、小于4.00E-11M、小于3.20E-11M或更小)的KD值与鼠BAFF结合，所述KD值通过表面等离子体共振测定法所测量(例如
Figure PCTCN2022131362-appb-000003
表面等离子体共振测定法测量)； 在一些实施方案中，所述KD值通过本披露测试例8方法检测；
D.具有阻断ICOSL与ICOS结合的活性；优选地，阻断人ICOSL与ICOS结合的IC 50值小于0.16nM(例如小于0.16nM、小于0.14nM、小于0.12nM、小于0.10nM、小于0.09nM、小于0.08nM、小于0.07nM、小于0.06nM、小于0.05nM、小于0.04nM或更小)，所述IC 50值通过FACS方法检测；在一些实施方案中，所述IC 50值通过本披露测试例3方法检测；
E.具有阻断APRIL与BCMA结合的活性；优选地，阻断APRIL与BCMA结合的IC 50值小于40.00nM(例如小于40.00nM、小于35.00nM、小于30.00nM、小于20.00nM、小于10.00nM、小于5.00nM、小于2.00nM、小于0.10nM、小于0.06nM、小于0.02nM或更小)，所述IC 50值通过ELISA方法检测；在一些实施方案中，所述IC 50值通过本披露测试例4方法检测；
F.具有阻断APRIL与TACI结合的活性；优选地，阻断APRIL与TACI结合的IC 50值小于40.00nM(例如小于40.00nM、小于37.00nM、小于33.00nM、小于30.00nM、小于25.00nM、小于10.00nM、小于5.00nM、小于2.00nM、小于1.00nM、小于0.50nM或更小)，所述IC 50值通过ELISA方法检测；在一些实施方案中，所述IC 50值通过本披露测试例4方法检测；
G.具有阻断BAFF与BCMA结合的活性；优选地，阻断BAFF与BCMA结合的IC 50值小于2.00nM(例如小于2.00nM、小于1.50nM、小于1.00nM、小于0.90nM、小于0.80nM、小于0.70nM、小于0.60nM、小于0.50nM、小于0.40nM或更小)，所述IC 50值通过ELISA方法检测；在一些实施方案中，所述IC 50值通过本披露测试例4方法检测；
H.具有阻断BAFF与BAFF-R结合的活性；优选地，阻断BAFF与BAFF-R结合的IC 50值小于2.00nM(例如小于2.00nM、小于1.70nM、小于1.60nM、小于1.00nM、小于0.80nM、小于0.70nM或更小)，所述IC 50值通过ELISA方法检测；在一些实施方案中，所述IC 50值通过本披露测试例4方法检测；
I.具有阻断BAFF与TACI结合的活性；优选地，阻断BAFF与TACI结合的IC 50值小于1.00nM(例如小于1.00nM、小于0.50nM、小于0.30nM、小于0.20nM、小于0.15nM或更小)，所述IC 50值通过ELISA方法检测；在一些实施方案中，所述IC 50值通过本披露测试例4方法检测；
J.具有抑制细胞因子(如IFNγ、IL-10和/或IgA)分泌的活性；在一些实施方案中，所述IC 50值通过本披露测试例9方法检测；
K.具有抑制B细胞增殖和/或T细胞增殖的活性；在一些实施方案中，所述抗ICOSL抗体或抗ICOSL抗体融合蛋白抑制ICOSL诱导T细胞增殖的活性；在一些实施方案中，所述抑制T细胞增殖活性通过本披露测试例6方法检测；在一些实施方案中，所述抗ICOSL抗体融合蛋白抑制BAFF诱导的或APRIL诱导的B细胞增殖的活性；在一些实施方案中，所述抑制B细胞增殖活性通过本披露测试 例7方法检测。
本披露还提供一种药物组合物，其包含前面任一项所述的抗ICOSL抗体融合蛋白或抗ICOSL抗体，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
本披露还提供一种药物组合物，其包含前面任一项所述的抗ICOSL抗体，和如SEQ ID NO：51至83中任一所示的TACI多肽，以及一种或多种药学上可接受的载体、稀释剂或赋形剂；
优选地，所述的TACI多肽序列如SEQ ID NO：60-63、SEQ ID NO：66-83中的任一所示；
更优选地，所述的TACI多肽序列如如SEQ ID NO：83所示。
在一些实施方案中，本披露提供一种核酸分子，其编码前面任一项所述的抗ICOSL抗体融合蛋白或抗ICOSL抗体。
在一些实施方案中，本披露提供一种表达载体，其包含前述的核酸分子(DNA或RNA)。
在一些实施方案中，本披露提供一种宿主细胞，其含前述的核酸分子。
在一些实施方案中，本披露提供一种宿主细胞，其含前述的表达载体。
本披露提供的宿主细胞，不能发育成动物或植物个体。
在一些实施方案中，本披露提供一种治疗自身免疫性疾病、炎性疾病、B细胞障碍或T细胞障碍的方法，所述方法包括向有需要的受试者施用治疗有效量的前面任一项所述的抗ICOSL抗体融合蛋白或抗ICOSL抗体或药物组合物的步骤。在一些实施方案中，本披露方法用于治疗自身免疫性疾病或炎性疾病。在一些实施方案中，本披露方法用于治疗B细胞障碍或T细胞障碍。
在一些实施方案中，本披露提供前述任一项所述的抗ICOSL抗体融合蛋白或抗ICOSL抗体、核酸分子或药物组合物在制备用于治疗疾病的药物中的用途。
在另一个方面，本披露还提供用作药物的前述任一项所述的抗ICOSL抗体融合蛋白或抗ICOSL抗体、核酸分子或组合物。在一些实施方案中，药物用于治疗自身免疫性疾病、炎性疾病、B细胞障碍或T细胞障碍。在一些实施方案中，药物用于治疗自身免疫性疾病或炎性疾病。在一些实施方案中，药物用于治疗B细胞障碍或T细胞障碍。
在一些实施方案中，前面任一项所述的疾病是与ICOSL高表达相关的疾病或病症。在一些实施方案中，前面任一项所述的疾病或病症是自身免疫性疾病、炎性疾病、B细胞障碍或T细胞障碍。在一些实施方案中，所述自身免疫性疾病或炎性疾病选自：系统性红斑狼疮、类风湿性关节炎、移植物抗宿主病、哮喘、免疫性血小板减少性紫癜、多发性硬化、糖尿病引起的炎性疾病、银屑病、炎性肠病、克罗恩病、溃疡性结肠炎、格雷夫斯病和桥本氏甲状腺炎。在一些实施方案中，所述B细胞障碍或T细胞障碍为肿瘤。在一些实施方案中，所述肿瘤选自：头颈癌、非小细胞肺癌、尿路上皮癌、白血病、肉瘤、黑色素瘤、腺癌、结直肠 癌、前列腺肿瘤、乳腺癌和小细胞肺癌。在一些实施方案中，所述自身免疫性疾病为系统性红斑狼疮。
在一些实施方案中，前面任一项所述的治疗，进一步包括向受试者施用另外的治疗药物。
附图说明
图1：抗ICOSL抗体融合蛋白如259H4L7-T11和201H3L3-T11的结构示意图。
图2：抗ICOSL抗体融合蛋白如201H3L3-T7和259H4L7-T7的结构示意图。
图3：抗ICOSL抗体融合蛋白如259H4L7-T9的结构示意图。
图4：抗ICOSL抗体融合蛋白抑制IL-10的分泌实验结果。
图5：抗ICOSL抗体融合蛋白抑制IFNγ的分泌实验结果。
图6：抗ICOSL抗体融合蛋白抑制IgA的分泌实验结果。
图7：抗ICOSL抗体融合蛋白抑制KLH特异性IgG的产生实验结果。
图8：抗ICOSL抗体融合蛋白抑制KLH特异性IgM的产生实验结果。
具体实施方式
术语
为了更容易理解本披露，以下对某些技术和科学术语进行了描述。除非在本文中另有明确定义，本文使用的全部技术和科学术语具有与本领域的普通技术人员通常所理解的相同含义。
说明书和权利要求书中所用的单数形式“一个”、“一种”和“所述”包括复数指代，除非上下文清楚表明并非如此。
除非上下文另外清楚要求，否则在专利说明书和权利要求书中，应将词语“包含”、“具有”、“包括”等理解为“包括但不仅限于”的意义，而不是排他性或穷举性意义。
术语“细胞因子”是由一个细胞群体释放的、作为细胞间介质作用于其它细胞的蛋白质的统称。这样的细胞因子的例子包括淋巴因子、单核因子、趋化因子和传统的多肽激素。示例性的细胞因子包括：IL-10、IFN-γ、IL-6、TNFα、IL-17和IL-5。
本披露所述的TACI是一种膜结合受体。野生型人TACI胞外区(第1-165位)参见本披露的SEQ ID NO：51。本披露中的“TACI胞外结构域”与“TACI胞外区”可相互替换。
ICOSL(Inducible costimulator-ligand，也称为B7RP1，B7H2，CD275)，是属于B7家族的一个免疫共刺激分子。目前已知ICOSL的受体是ICOS，ICOS主要在T细胞表面表达，ICOSL和ICOS介导的信号通路主要在T细胞的分化和T细胞依赖的B细胞活化中发挥作用(Nat Rev Cancer.2012Mar 22；12(4):252-64.)。
“ICOSL”作为抗体或其融合蛋白的靶点时，应作广泛的理解，旨在涵盖ICOSL 在哺乳动物体内各阶段中的各种形式的分子，例如但不限于ICOSL基因在扩增、复制、转录、剪接、加工、翻译、修饰过程中所产生的分子(例如前体BCMA、成熟ICOSL、膜表达的ICOSL、ICOSL剪接变体、修饰的ICOSL、或其片段)；该术语也涵盖人工制备的或体外表达的ICOSL。
术语“和/或”，意指包含“和”与“或”两种含义。例如短语“A、B和/或C”旨在涵盖以下方面中的任一个：A、B和C；A、B或C；A或C；A或B；B或C；A和C；A和B；B和C；A(单独)；B(单独)；和C(单独)。
本披露所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
术语“氨基酸”是指天然存在的和合成的氨基酸，以及以与天然存在的氨基酸类似的方式起作用的氨基酸类似物和氨基酸模拟物。天然存在的氨基酸是由遗传密码编码的那些氨基酸，以及后来修饰的那些氨基酸，例如羟脯氨酸、γ-羧基谷氨酸和O-磷酸丝氨酸。氨基酸类似物是指与天然存在的氨基酸具有相同基本化学结构(即与氢、羧基、氨基和R基团结合的α碳)的化合物，例如高丝氨酸、正亮氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍。此类类似物具有修饰的R基团(例如，正亮氨酸)或修饰的肽骨架，但保留与天然存在的氨基酸相同的基本化学结构。氨基酸模拟物是指具有与氨基酸的一般化学结构不同的结构，但是以与天然存在的氨基酸类似的方式起作用的化学化合物。
术语“氨基酸突变”包括氨基酸取代(也称氨基酸替换)、缺失、插入和修饰。可以进行取代、缺失、插入和修饰的任意组合来实现最终构建体，只要最终构建体拥有期望的特性，例如降低对Fc受体的结合。氨基酸序列缺失和插入可以位于多肽链的氨基端和/或羧基端。在一个实施方案中，氨基酸突变是非保守性的氨基酸取代，即将一个氨基酸用具有不同结构和/或化学特性的另一种氨基酸替换。氨基酸取代包括由非天然存在的氨基酸或由20种天然氨基酸的衍生物(例如4-羟脯氨酸、3-甲基组氨酸、鸟氨酸、高丝氨酸、5-羟赖氨酸)替换。可以使用本领域中公知的遗传或化学方法进行氨基酸突变。遗传方法可以包括定点诱变、PCR，基因合成等。预计基因工程以外的改变氨基酸侧链基团的方法，如化学修饰也可能是可用的。本文中可使用各种表述来指示氨基酸突变。本文中，可采用位置+氨基酸残基的方式表示特定位点的氨基酸残基，例如366W，表示在366位点上的氨基酸残基为W。T366W则表示第366位点上的氨基酸残基由原来的T突变为了W。作为一个示例，当在权利要求中以366W或T366W来描述序列时，不能理解为366位点处的原始氨基酸残基T对保护范围构成任何限制。
术语“抗体”以最广义使用，并且涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体；单特异性抗体，多特异性抗体(例如双特异性抗体)；全长抗体和抗体片段(或抗原结合片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。“天然抗体”指天然存在的免疫球蛋白分子。例如，天然IgG抗体是约 150,000道尔顿的异四聚糖蛋白，由二硫键结合的2条相同轻链和2条相同重链构成。从N至C端，每条重链具有一个可变区(VH)，又称作可变重域、重链可变区，接着是重链恒定区，天然IgG重链恒定区通常含三个恒定域(CH1、CH2和CH3)。类似地，从N至C端，每条轻链具有一个可变区(VL)，又称作可变轻域，或轻链可变域，接着是一个恒定轻域(轻链恒定区、CL)。术语“全长抗体”、“完整抗体”和“全抗体”在本文可互换使用，指具有与天然抗体结构基本类似的结构或具有如本文所限定的Fc区的重链的抗体。天然完整抗体轻链包括轻链可变区VL及恒定区CL，VL处于轻链的氨基末端，轻链恒定区包括κ链及λ链；重链包括可变区VH及恒定区(CH1、CH2及CH3)，VH处于重链的氨基末端，恒定区处于羧基末端，其中CH3最接近多肽的羧基末端，重链可属于任何同种型，包括IgG(包括IgG1、IgG2、IgG3及IgG4亚型)、IgA(包括IgA1及IgA2亚型)、IgM及IgE。
术语抗体“可变区”或“可变域”指抗体重链或轻链中参与抗体结合抗原的域。本文中，抗体重链可变区(VH)和轻链可变区(VL)各包含四个保守的框架区(FR)和三个互补决定区(CDR)。其中，术语“互补决定区”或“CDR”指可变结构域内主要促成与抗原结合的区域；“框架”或“FR”是指除CDR残基之外的可变结构域残基。VH包含3个CDR区：HCDR1、HCDR2和HCDR3；VL包含3个CDR区：LCDR1、LCDR2和LCDR3。每个VH和VL由从氨基末端(也称N末端)排到羧基末端(也称C末端)按以下顺序排列的三个CDR和四个FR构成：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。
可以通过各种公知方案来确定CDR的氨基酸序列边界，例如：“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则、“ABM”编号规则、“contact”编号规则(参见Martin,ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001)和ImMunoGenTics(IMGT)编号规则(Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)；Front Immunol.2018 Oct 16；9:2278)等；各种编号系统之间的对应关系是本领域技术人员熟知的，示例性的，如下表1中所示。
表1.CDR编号系统之间的关系
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
除非另有说明，本披露实施例中的可变区和CDR均适用“Kabat”编号规则。
术语“抗体片段”指不同于完整抗体的分子，其包含完整抗体的部分，所述部分与完整抗体所结合的抗原相结合。抗体片段的实例包括但不限于Fv、Fab、Fab′、Fab′-SH、F(ab′) 2、单域抗体、单链Fab(scFab)、双抗体、线性抗体、单链抗体分子(例如scFv)，以及由抗体片段形成的多特异性抗体。
术语“Fc区”或“片段可结晶区”用于定义抗体重链的C末端区域，包括天然Fc区和改造的Fc区。在一些实施方案中，Fc区包含了相同或不同的两个亚基。在一些实施方案中，人IgG重链的Fc区定义为从Cys226位置处的氨基酸残基或从Pro230延伸至其羧基末端。用于本文所述抗体的合适Fc区包括人IgG1、IgG2(IgG2A、IgG2B)、IgG3和IgG4的Fc区。在一些实施方案中，Fc区的边界还可以变化，例如缺失Fc区的C末端赖氨酸(根据EU编号系统的残基447)或缺失Fc区的C末端甘氨酸和赖氨酸(根据EU编号系统的残基446和447)。除非另有说明，Fc区的编号规则为EU编号系统，又称作EU索引。
术语“嵌合抗体”指抗体中的重链和/或轻链的一部分自特定的来源或物种衍生，而重和/或轻链的剩余部分自另外的不同来源或物种衍生的抗体。
术语“人源化抗体”是保留非人抗体的反应性同时在人中具有较低免疫原性的抗体。例如，可以通过保留非人CDR区并用其人对应物(即，恒定区以及可变区的框架区部分)替换抗体的其余部分来实现。
术语“亲和力”是指分子(例如，抗体)的单个结合部位与其结合配体(例如，抗原)之间非共价相互作用的总体的强度。除非另外指明，如本文所用，结合“亲和力”是指内部结合亲和力，其反映出结合对(例如，抗体与抗原)成员之间的相互作用。分子X对其配体Y的亲和力通常可以由解离常数(KD)表示。亲和力可以通过本领域已知的常规方法(包括本文所述的那些方法)测量。
如本文所使用的，术语“kassoc”或“ka”指特定抗体-抗原相互作用的缔合速率，术语“kdis”或“kd”指特定抗体-抗原相互作用的解离速率。术语“KD”指解离常数，其获得自kd与ka的比率(即kd/ka)并且表示为摩尔浓度(M)。可以使用本领域公知的方法测定抗体的KD值。例如，使用生物传感系统例如系统测量表面等离子体共振(例如Biacore)，或通过溶液平衡滴定法(SET)测量溶液中的亲和力。
术语“效应子功能”指那些可归于抗体Fc区(天然序列Fc区或氨基酸序列突变的Fc区)且随抗体同种型而变化的生物学活性。抗体效应子功能的例子包括但不限于：C1q结合和补体依赖性细胞毒性、Fc受体结合、抗体依赖性细胞介导的细胞毒性(ADCC)、吞噬作用、细胞表面受体(例如B细胞受体)下调；和B细胞活化。
术语“单克隆抗体”指基本上均质的抗体的群，即在该群中包含的抗体分子的氨基酸序列是相同的，除了可能少量存在的天然突变以外。相比之下，多克隆抗体制剂通常包含在其可变结构域具有不同氨基酸序列的多种不同抗体，其通常特异 性针对不同表位。“单克隆”表示从基本上均质的抗体群体获得的抗体的特征，并且不应解释为要求通过任何特定方法来生产抗体。在一些实施方案中，本披露提供的抗体是单克隆抗体。
术语“抗原”是指能够由诸如抗原结合蛋白(包括例如抗体)选择性结合的分子或分子部分。抗原可具有一个或多个能够与不同的抗原结合蛋白(例如抗体)相互作用的表位。
术语“表位”指能够与抗体(包括其抗原结合片段)特异性结合的抗原上的区域(area或region)。表位可以由连续氨基酸串(线性表位)形成或包含非连续氨基酸(构象表位)，例如因抗原的折叠(即通过三级折叠)而使得非连续氨基酸在空间上互相接近。构象表位和线性表位的差别在于：在变性溶剂的存在下，抗体对构象表位的结合丧失。表位包含处于独特空间构象的至少3，至少4，至少5，至少6，至少7，或8-10个氨基酸。筛选结合特定表位的抗体(即那些结合相同表位的)可以使用本领域例行方法来进行，例如但不限于丙氨酸扫描，肽印迹，肽切割分析，表位切除，表位提取，抗原的化学修饰(见Prot.Sci.9(2000)487-496)，和交叉阻断。
术语“能够特异性结合”、“特异性结合”或“结合”是指相比其他抗原或表位，抗体能够以更高的亲和力结合至某个抗原或其表位。通常地，抗体以约1×10 -7M或更小(例如约1×10 -8M、1×10 -9M、1×10 -10M、1×10 -11M或更小)的平衡解离常数(KD)结合抗原或其表位。在一些实施方案中，抗体与抗原结合的KD为该抗体结合至非特异性抗原(例如BSA、酪蛋白)的KD的10％或更低(例如1％)。可使用已知的方法来测量KD，例如通过
Figure PCTCN2022131362-appb-000004
表面等离子体共振测定法所测量的。然而，特异性结合至抗原或其表位的抗体可能对其它相关的抗原具有交叉反应性，例如，对来自其它物种(同源)(诸如人或猴，例如食蟹猕猴(Macaca fascicularis)(cynomolgus，cyno)、黑猩猩(Pan troglodytes)(chimpanzee，chimp)或狨猴(Callithrix jacchus)(commonmarmoset，marmoset))的相应抗原具有交叉反应性。
术语“抗ICOSL抗体”和“结合ICOSL的抗体”是指能够以足够的亲和力结合ICOSL或其表位的抗体。在一个实施例中，与无关的、非ICOSL蛋白的抗体的结合程度小于该抗体与ICOSL结合的约10％，所述结合通过表面等离子体共振测定法测量(例如
Figure PCTCN2022131362-appb-000005
)。在某些实施例中，与ICOSL蛋白结合的抗体具有以下解离常数(KD)<约1μM、<约100nM、<约10nM、<约1nM、<约0.1nM、<约0.01nM或<约0.001nM。在某些实施例中，抗ICOSL抗体结合人或食蟹猴ICOSL抗原表位。
术语“连接子”、“Linker”或“接头”指连接两个多肽片段的连接单元，通常具有一定的柔性，接头的使用不会使蛋白质结构域原有的功能丧失。在本文中，同一结构中出现的连接子可以是相同或不同的。连接子可以是肽连接子，其包含 一个或多个氨基酸，典型的约1-30个、2-24个或3-15个氨基酸。应用于本文的连接子可以是相同或不同的。
术语“抗体依赖性细胞的细胞毒性”、“抗体依赖性细胞介导的细胞毒性”或“ADCC”是诱导细胞死亡的机制，该机制依赖于抗体包被靶细胞与具有裂解活性的效应细胞(诸如自然杀伤细胞(NK)、单核细胞、巨噬细胞和中性粒细胞)经由效应细胞上表达的Fcγ受体(FcγR)发生的相互作用。例如，NK细胞表达FcγRIIIa，而单核细胞表达FcγRI、FcγRII和FcγRIIIa。本文提供的抗体的ADCC活性可使用体外测定，使用表达抗原的细胞作为靶细胞和NK细胞作为效应细胞进行评定。根据从裂解的细胞中释放的标记物(例如放射性底物、荧光染料或天然胞内蛋白)来检测细胞裂解。
术语“抗体依赖性细胞吞噬作用”(“ADCP”)是指通过吞噬细胞(诸如巨噬细胞或树突状细胞)的内化作用消除抗体包被的靶细胞的机制。
术语“补体依赖性细胞毒性”或“CDC”是指诱导细胞死亡的机制，其中靶结合抗体的Fc效应域结合并激活补体成分C1q，C1q继而激活补体级联，从而导致靶细胞死亡。补体的激活也可导致补体成分沉积在靶细胞表面上，这些补体成分通过结合白细胞上的补体受体(例如，CR3)来促进CDC。
术语“核酸”在本文中可与术语“多核苷酸”互换使用，并且是指呈单链或双链形式的脱氧核糖核苷酸或核糖核苷酸及其聚合物。所述术语涵盖含有已知核苷酸类似物或修饰的骨架残基或连接的核酸，所述核酸是合成的、天然存在的和非天然存在的，具有与参考核酸相似的结合特性，并且以类似于参考核苷酸的方式代谢。此类类似物的实例包括但不限于硫代磷酸酯、氨基磷酸酯、甲基膦酸酯、手性-甲基膦酸酯、2-O-甲基核糖核苷酸、肽-核酸(PNA)。“分离的”核酸指已经与其天然环境的组分分开的核酸分子。分离的核酸包括在下述细胞中含有的核酸分子，所述细胞通常含有该核酸分子，但该核酸分子存在于染色体外或存在于不同于其天然染色体位置的染色体位置处。编码多肽或融合蛋白的分离的核酸指编码多肽或融合蛋白的一个或更多个核酸分子，包括在单一载体或分开的载体中的这样的一个或更多个核酸分子，和存在于宿主细胞中一个或更多个位置的这样的一个或更多个核酸分子。除非另有说明，否则特定的核酸序列还隐含地涵盖其保守修饰的变体(例如，简并密码子取代)和互补序列以及明确指明的序列。具体地，如下详述，简并密码子取代可以通过产生如下序列而获得，在这些序列中，一个或多个所选的(或全部)密码子的第三位被混合碱基和/或脱氧肌苷残基取代。
术语“多肽”和“蛋白质”在本文中可互换使用，指氨基酸残基的聚合物。该术语适用于氨基酸聚合物，其中一个或多个氨基酸残基是相应天然存在的氨基酸或其人工化学模拟物，以及天然存在的氨基酸聚合物和非天然存在的氨基酸聚合物。除非另外说明，否则特定的多肽序列还隐含地涵盖其保守修饰的变体。
术语序列“同一性”指，当对2条序列进行最佳比对时，2条序列的氨基酸/核酸 在等价位置相同的程度(百分比)；其中，必要时在最佳比对中引入间隙以获取最大序列同一性百分比，且不将任何保守性取代视为序列同一性的一部分。为测定序列同一性百分比，比对可以通过本领域技术已知的技术来实现，例如使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
本披露中，例如“相对于序列SEQ ID NO：XX的自然顺序编号的氨基酸残基位点”意指对象序列与SEQ ID NO：XX序列进行最佳比对，获得最高百分比同一性，此时对象序列与SEQ ID NO：XX序列对应位置的位点为2条序列的相对位点。例如TACI的胞外区序列SEQ ID NO：51和SEQ ID NO：60上相对于SEQ ID NO：58的自然顺序编号的氨基酸残基位点如表2所示：
表2.SEQ ID NO：58位点在其他序列上的相应位点
(以SEQ ID NO：51或/SEQ ID NO：60为例)
Figure PCTCN2022131362-appb-000006
备注：例如SEQ ID NO：60上第2位(自然顺序)残基位点与序列SEQ ID NO：58的第49位(自然顺序)残基位点为相应位点。
术语“融合”或“连接”是指部件(例如TACI多肽和抗体重/轻链)直接地或经由一个或多个连接子通过共价键连接。当连接子是肽连接子时，所述共价键是肽键。
术语“抗ICOSL抗体融合蛋白”是指抗ICOSL抗体与活性蛋白融合的蛋白质。例如，TACI多肽融合到抗ICOSL抗体的重链或轻链的N末端或C末端而形成蛋白。
术语“载体”意指能够转运与其连接的另一多核苷酸的多核苷酸分子。一种类型的载体是“质粒”，其是指环状双链DNA环，其中可以连接附加的DNA区段。另一种类型的载体是病毒载体，例如腺相关病毒载体(AAV或AAV2)，其中另外的DNA区段可以连接到病毒基因组中。某些载体能够在引入它们的宿主细 胞中自主复制(例如，具有细菌复制起点的细菌载体和附加型哺乳动物载体)。其他载体(例如，非附加型哺乳动物载体)可以在引入宿主细胞中后整合到宿主细胞的基因组中，从而与宿主基因组一起复制。术语“表达载体”或“表达构建体”是指可对宿主细胞进行转化，且含有指导和/或控制(连同宿主细胞一起)与其可操作地连接的一个或多个异源编码区的表达的核酸序列的载体。表达构建体可以包括但不限于影响或控制转录、翻译且在存在内含子时影响与其可操作地连接的编码区的RNA剪接的序列。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可互换使用，并且指已经导入外源核酸的细胞，包括此类细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞及自其衍生的后代，而不考虑传代的次数。后代在核酸内容物上可以与亲本细胞不完全相同，而是可以含有突变。本文中包括与在初始转化细胞中筛选或选择的细胞具有相同功能或生物学活性的突变体后代。宿主细胞包括原核和真核宿主细胞，其中真核宿主细胞包括但不限于哺乳动物细胞、昆虫细胞系、植物细胞和真菌细胞。哺乳动物宿主细胞包括人、小鼠、大鼠、犬、猴、猪、山羊、牛、马和仓鼠细胞，包括但不限于中国仓鼠卵巢(CHO)细胞、NSO、SP2细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞(例如，Hep G2)、A549细胞、3T3细胞和HEK-293细胞。真菌细胞包括酵母和丝状真菌细胞，包括例如巴氏毕赤酵母(Pichiapastoris)、芬兰毕赤酵母(Pichia finlandica)、海藻毕赤酵母(Pichia trehalophila)、科克拉马毕赤酵母(Pichia koclamae)、膜状毕赤酵母(Pichia membranaefaciens)、小毕赤酵母(Pichia minuta)(Ogataea minuta、Pichia lindneri)、仙人掌毕赤酵母(Pichiaopuntiae)、耐热毕赤酵母(Pichia thermotolerans)、柳毕赤酵母(Pichia salictaria)、Pichia guercuum、皮杰普毕赤酵母(Pichia pijperi)、具柄毕赤酵母(Pichia stiptis)、甲醇毕赤酵母(Pichia methanolica)、毕赤酵母属、酿酒酵母(Saccharomycescerevisiae)、酿酒酵母属、多形汉逊酵母(Hansenula polymorpha)、克鲁维酵母属、乳酸克鲁维酵母(Kluyveromyces lactis)、白色念珠菌(Candida albicans)、构巢曲霉(Aspergillus nidulans)、黑曲霉(Aspergillus niger)、米曲霉(Aspergillus oryzae)、里氏木霉(Trichoderma reesei)、勒克氏菌(Chrysosporium lucknowense)、镰刀菌属(Fusarium sp.)、禾谷镰刀菌(Fusarium gramineum)、菜镰刀菌(Fusarium venenatum)、小立碗藓(Physcomitrella patens)和粗糙脉孢菌(Neurospora crassa)、曲霉属、、解脂耶氏酵母(Yarrowia lipolytica)。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。
术语“药物组合物”表示含有一种或多种本文所述的抗ICOSL抗体融合蛋白与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。
术语“药学上可接受的载体、稀释剂或赋形剂”指药学配制剂中与活性成分不 同的，且对受试者无毒的成分；例如，但不限于缓冲剂、赋形剂、稳定剂或防腐剂。
术语“受试者”或“个体”包括人类和非人类动物。非人动物包括所有脊椎动物(例如哺乳动物和非哺乳动物)例如非人灵长类(例如，食蟹猴)、绵羊、狗、牛、鸡、两栖动物和爬行动物。除非指出时，否则所述术语“患者”或“受试者”在本文中可互换地使用。如本文所使用的，术语“食蟹猴(cyno)”或“食蟹猴(cynomolgus)”是指食蟹猴(Macaca fascicularis)。在某些实施方案中，个体或受试者是人。
“施用”或“给予”，当其应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。
术语“样品”是指从受试者分离的流体、细胞、或组织的采集物，以及存在于受试者体内的流体、细胞或组织。示例性样品为生物流体，诸如血液、血清、浆膜液、血浆、淋巴液、尿液、唾液、囊液、泪液、排泄物、痰、分泌组织和器官的粘膜分泌物、阴道分泌物、腹水、胸膜、心包、腹膜、腹腔和其它体腔的流体、由支气管灌洗液收集的流体、滑液、与受试者或生物来源接触的液体溶液，例如细胞和器官培养基(包括细胞或器官条件培养基)、灌洗液等，组织活检样品、细针穿刺、手术切除的组织、器官培养物或细胞培养物。
“治疗(treatment或treat)”和“处理”(及其语法变型)指试图改变所治疗个体的过程的临床干预，并且可以为了预防或者在临床病理学的过程期间实施。治疗的期望效果包括但不限于预防疾病的发生或再发生，减轻症状，减轻/减少疾病的任何直接或间接病理后果，预防转移，降低疾病进展速率，改善或减轻疾病状态，和消退或改善的预后。在一些实施方案中，使用本披露的抗体或融合蛋白来延迟疾病的形成或减缓疾病的进展。
“有效量”一般是足以降低症状的严重程度及/或频率、消除这些症状及/或潜在病因、预防症状及/或其潜在病因出现及/或改良或改善由疾病状态引起或与其相关的损伤的量。在一些实施例中，有效量是治疗有效量或预防有效量。“治疗有效量”是足以治疗疾病状态或症状、尤其与该疾病状态相关的状态或症状，或者以其他方式预防、阻碍、延迟或逆转该疾病状态或以任何方式与该疾病相关的任何其他不理想症状的进展的量。“预防有效量”是当给予受试者时将具有预定预防效应，例如预防或延迟该疾病状态的发作(或复发)，或者降低该疾病状态或相关症状的发作(或复发)可能性的量。完全治疗或预防效未必在给予一个剂量之后便发生，可能在给予一系列剂量之后发生。因而，治疗或预防有效量可以一次或多次给予的方式给予。“治疗有效量”和“预防有效量”可取决于多种因素变化：诸如个体的疾病状态、年龄、性别和体重，以及治疗剂或治疗剂组合在个体中引发期望的应答的能力。有效治疗剂或治疗剂组合的示例性指标包括例如患者改善的健康状况。
本披露的抗ICOSL抗体
在一个方面中，本披露设计了一种新的抗ICOSL抗体。其具有以下一种或更多种特性：
A.与人ICOSL和食蟹猴ICOSL或其表位特异性结合，不与鼠ICOSL特异性结合；优选地，以小于5.00E-09M(例如小于5.00E-09M、小于4.00E-09M、小于3.00E-09M、小于2.00E-09M、小于1.50E-09M、9.00E-10M或更小)的KD值与人ICOSL结合，和/或以小于8.00E-09M(例如小于8.00E-09M、小于7.00E-09M、小于6.00E-09M、小于5.00E-09M、小于4.00E-09M、小于3.00E-09M或更小)的KD值与食蟹猴ICOSL结合，所述KD值通过表面等离子体共振测定法所测量(例如
Figure PCTCN2022131362-appb-000007
)；在一些实施方案中，所述KD值通过本披露测试例8方法检测；
B.具有阻断ICOSL与ICOS结合活性；优选地，阻断人ICOSL与ICOS结合的IC 50值小于0.16nM(例如小于0.16nM、小于0.14nM、小于0.12nM、小于0.10nM、小于0.09nM、小于0.08nM、小于0.07nM、小于0.06nM、小于0.05nM、小于0.04nM或更小)，所述IC 50值通过FACS方法检测；在一些实施方案中，所述IC 50值通过本披露测试例3方法检测；
C.具有抑制IFNγ、IL-10和/或IgA细胞因子分泌活性；在一些实施方案中，所述IC50值通过本披露测试例9方法检测；
D.具有抑制T细胞增殖活性；在一些实施方案中，所述抗ICOSL抗体或抗ICOSL抗体融合蛋白抑制ICOSL诱导T细胞增殖的活性；在一些实施方案中，所述抑制T细胞增殖活性通过本披露测试例6方法检测。
示例性的抗ICOSL抗体
一方面，本披露提供一种抗ICOSL抗体，其中所述抗ICOSL抗体包含重链可变区和轻链可变区，其中，所述重链可变区包含HCDR1、HCDR2和HCDR3，所述轻链可变区包含LCDR1、LCDR2和LCDR3，其中，
在一些实施方案中，如上任一项所述的抗ICOSL抗体，其中所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据选自Kabat编号规则定义的。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，其中：
(i)所述重链可变区的HCDR1如SEQ ID NO：11所示，HCDR2如SEQ ID NO：31或12所示，和HCDR3如SEQ ID NO：13所示，和所述轻链可变区的LCDR1如SEQ ID NO：14所示，LCDR2如SEQ ID NO：15所示，和LCDR3如SEQ ID NO：16所示；或
(ii)所述重链可变区的HCDR1如SEQ ID NO：5所示，HCDR2如SEQ ID NO：19或6所示，和HCDR3如SEQ ID NO：7所示，和所述轻链可变区的LCDR1如SEQ ID NO：8所示，LCDR2如SEQ ID NO：17或9所示，和LCDR3如SEQ ID NO：10或18所示。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，其中
(i)所述重链可变区的HCDR1如SEQ ID NO：11所示，HCDR2如SEQ ID NO：31所示，和HCDR3如SEQ ID NO：13所示，和所述轻链可变区的LCDR1如SEQ ID NO：14所示，LCDR2如SEQ ID NO：15所示，和LCDR3如SEQ ID NO：16所示；或
(ii)所述重链可变区的HCDR1如SEQ ID NO：5所示，HCDR2如SEQ ID NO：19所示，和HCDR3如SEQ ID NO：7所示，和所述轻链可变区的LCDR1如SEQ ID NO：8所示，LCDR2如SEQ ID NO：17所示，和LCDR3如SEQ ID NO：10所示。
在一些实施方案中，如前任一项所述的抗ICOSL抗体，所述抗ICOSL抗体是鼠源、嵌合或人源化的抗体。在一些实施方案中，所述抗ICOSL抗体是人源化的。
在一些实施方案中，如前任一项所述的抗ICOSL抗体是人源化抗体。在一些实施方案中，所述抗ICOSL抗体的重链可变区具有来源于IGHV3-74*03的FR1、FR2、FR3和来源于IGHJ1*01的FR4，并且其是未被取代的或具有选自37I、43E和49A(根据Kabat编号系统编号)组成的组中的一个或更多个氨基酸取代；和/或所述轻链可变区具有来源于IGKV4-1*01/IGKV1-39*01的FR1、FR2、FR3和来源于IGKJ2*01的FR4，并且其是未被取代的或具有选自1N、2T、3V、42Q、43S、60D、85V(根据Kabat编号系统编号)组成的组中的一个或更多个氨基酸取代。在一些实施方案中，所述的抗ICOSL抗体，其中所述重链可变区的HCDR1如SEQ ID NO：11所示，HCDR2如SEQ ID NO：31或12所示，和HCDR3如SEQ ID NO：13所示，和所述轻链可变区的LCDR1如SEQ ID NO：14所示，LCDR2如SEQ ID NO：15所示，和LCDR3如SEQ ID NO：16所示。上述可变区和CDR是根据Kabat编号规则定义的。
在一些实施方案中，如前任一项所述的抗ICOSL抗体是人源化抗体，在一些实施方案中，所述抗ICOSL抗体的重链可变区具有来源于IGHV1-46*01的FR1、FR2、FR3和来源于IGHJ6*01的FR4，并且其是未被取代的或具有选自1E、24T、69L、71V、73K和78A(根据Kabat编号系统编号)组成的组中的一个或更多个氨基酸取代；和/或所述轻链可变区具有来源于IGKV2-28*01的FR1、FR2、FR3和来源于IGKJ4*01的FR4，并且其是未被取代的或具有选自1A、2V和4L(根据Kabat编号系统编号)组成的组中的一个或更多个氨基酸取代。在一些实施方案中，所述的抗ICOSL抗体，其中所述重链可变区的HCDR1如SEQ ID NO：5所示，HCDR2如SEQ ID NO：19或6所示，和HCDR3如SEQ ID NO：7所示，和所述轻链可变区的LCDR1如SEQ ID NO：8所示，LCDR2如SEQ ID NO：17或9所示，和LCDR3如SEQ ID NO：10或18所示。上述可变区和CDR是根据Kabat编号规则定义的。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，其中，
(i)所述重链可变区如SEQ ID NO：42、39、40或41所示，和所述轻链可变区如SEQ ID NO：38、32、33、34、35、36或37所示；或
(ii)所述重链可变区如SEQ ID NO：30、28或29所示，和所述轻链可变区如SEQ ID NO：22、20、21、23、24、25、26或27所示；或
(iii)所述重链可变区如SEQ ID NO：1所示，和所述轻链可变区如SEQ ID NO：2所示；或
(iv)所述重链可变区如SEQ ID NO：3所示，和所述轻链可变区如SEQ ID NO：4所示。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，其中，
(i)所述重链可变区如SEQ ID NO：42所示，和所述轻链可变区如SEQ ID NO：38所示；或
(ii)所述重链可变区如SEQ ID NO：30所示，和所述轻链可变区如SEQ ID NO：22所示。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，其中所述的抗ICOSL抗体包含抗体重链恒定区和轻链恒定区。在一些实施方案中，所述重链恒定区为人IgG重链恒定区。在一些实施方案中，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区；在一些实施方案中，所述轻链恒定区选自人抗体κ或λ链恒定区。在一些实施方案中，所述重链恒定区为人IgG4重链恒定区，所述轻链恒定区为人κ轻链恒定区。在一些实施方案中，所述重链恒定区的Fc区具有一个或更多个能够减少Fc区与Fc受体结合的氨基酸取代。在一些实施方案中，所述Fc区具有L234A、L235A突变，和/或S228P突变，和/或YTE突变(M252Y、S254T和T256E)，所述突变编号依据为EU索引。在一些实施方案中，所述重链恒定区包含SEQ ID NO：43的氨基酸序列，所述轻链恒定区包含SEQ ID NO：44的氨基酸序列。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，其中，
(i)所述抗ICOSL抗体的重链与SEQ ID NO：47具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性，和轻链与SEQ ID NO：48具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性；或
(ii)所述抗ICOSL抗体的重链与SEQ ID NO：45具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性，和轻链与SEQ ID NO：46具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性。
在一些实施方案中，如上任一项所述的抗ICOSL抗体，
(i)所述抗ICOSL抗体的重链如SEQ ID NO：47所示，和所述抗ICOSL抗体的轻链如SEQ ID NO：48所示；或
(ii)所述抗ICOSL抗体的重链如SEQ ID NO：45所示，和所述抗ICOSL抗体的轻链如SEQ ID NO：46所示。
本披露的抗ICOSL抗体融合蛋白
本披露构建了一种抗ICOSL抗体融合蛋白，其包含抗ICOSL抗体和TACI多肽，其中所述抗ICOSL抗体特异性结合人ICOSL或其表位。
在一些实施方案中，前面任一项所述抗ICOSL抗体融合蛋白，其具有一种或更多种以下特性：
A.与人ICOSL和食蟹猴ICOSL特异性结合，不与鼠ICOSL特异性结合；优选地，以小于5.00E-09M(例如小于5.00E-09M、小于4.00E-09M、小于3.00E-09M、小于2.00E-09M、小于1.50E-09M、9.00E-10M或更小)的KD值与人ICOSL结合，和/或以小于8.00E-09M(例如小于8.00E-09M、小于7.00E-09M、小于6.00E-09M、小于5.00E-09M、小于4.00E-09M、小于3.00E-09M或更小)的KD值与食蟹猴ICOSL结合，所述KD值通过表面等离子体共振测定法所测量(例如
Figure PCTCN2022131362-appb-000008
)；在一些实施方案中，所述KD值通过本披露测试例8方法检测；
B.与人APRIL、食蟹猴APRIL和/或鼠APRIL特异性结合；优选地，以小于2.00E-11M(例如小于2.00E-11M、小于1.80E-11M、小于1.70E-11M、小于1.60E-11M、小于1.20E-11M、小于4.60E-12M或更小)的KD值与人APRIL结合，以小于2.00E-10M(例如小于2.00E-10M、小于1.80E-10M、小于1.70E-10M、小于1.60E-10M、小于1.50E-10M、小于1.40E-10M、小于1.20E-10M或更小)的KD值与食蟹猴APRIL结合，和/或以小于2.00E-11M(例如小于2.00E-11M、小于1.80E-11M、小于1.60E-11M、小于1.40E-11M、小于1.20E-11M、小于1.00E-11M、小于8.00E-12M或更小)的KD值与鼠APRIL结合，所述KD值通过表面等离子体共振测定法所测量(例如
Figure PCTCN2022131362-appb-000009
)；在一些实施方案中，所述KD值通过本披露测试例8方法检测；
C.与人BAFF、食蟹猴BAFF和/或鼠BAFF特异性结合；优选地，以小于7.00E-11M(例如小于7.00E-11M、小于5.00E-11M、小于4.00E-11M、小于3.00E-11M、小于2.00E-11M、小于1.00E-11M或更小)的KD值与人BAFF结合，以小于4.00E-10M(例如小于4.00E-10M、小于3.00E-10M、小于2.00E-10M、小于1.30E-10M或更小)的KD值与食蟹猴BAFF结合，和/或以小于6.00E-11M(例如小于6.00E-11M、小于5.00E-11M、小于4.00E-11M、小于3.20E-11M或更小)的KD值与鼠BAFF结合，所述KD值通过表面等离子体共振测定法所测量(例如
Figure PCTCN2022131362-appb-000010
)；在一些实施方案中，所述KD值通过本披露测试例8方法检测；
D.具有阻断ICOSL与ICOS结合活性；优选地，阻断人ICOSL与ICOS结合的IC 50值小于0.16nM(例如小于0.16nM、小于0.14nM、小于0.12nM、小于0.10nM、小于0.09nM、小于0.08nM、小于0.07nM、小于0.06nM、小于0.05nM、小于0.04nM或更小)，所述IC 50值通过FACS方法检测；在一些实施方案中，所述IC 50值通过本披露测试例3方法检测；
E.具有阻断APRIL与BCMA结合活性；优选地，阻断APRIL与BCMA结合的IC 50值小于40.00nM(例如小于40.00nM、小于35.00nM、小于30.00nM、 小于20.00nM、小于10.00nM、小于5.00nM、小于2.00nM、小于0.10nM、小于0.06nM、小于0.02nM或更小)，所述IC 50值通过ELISA方法检测；在一些实施方案中，所述IC 50值通过本披露测试例4方法检测；
F.具有阻断APRIL与TACI结合活性；优选地，阻断APRIL与TACI结合的IC 50值小于40.00nM(例如小于40.00nM、小于37.00nM、小于33.00nM、小于30.00nM、小于25.00nM、小于10.00nM、小于5.00nM、小于2.00nM、小于1.00nM、小于0.50nM或更小)，所述IC 50值通过ELISA方法检测；在一些实施方案中，所述IC 50值通过本披露测试例4方法检测；
G.具有阻断BAFF与BCMA结合活性；优选地，阻断BAFF与BCMA结合的IC 50值小于2.00nM(例如小于2.00nM、小于1.50nM、小于1.00nM、小于0.90nM、小于0.80nM、小于0.70nM、小于0.60nM、小于0.50nM、小于0.40nM或更小)，所述IC 50值通过ELISA方法检测；在一些实施方案中，所述IC 50值通过本披露测试例4方法检测；
H.具有阻断BAFF与BAFF-R结合活性；优选地，阻断BAFF与BAFF-R结合的IC 50值小于2.00nM(例如小于2.00nM、小于1.70nM、小于1.60nM、小于1.00nM、小于0.80nM、小于0.70nM或更小)，所述IC 50值通过ELISA方法检测；在一些实施方案中，所述IC 50值通过本披露测试例4方法检测；
I.具有阻断BAFF与TACI结合活性；优选地，阻断BAFF与TACI结合的IC 50值小于1.00nM(例如小于1.00nM、小于0.50nM、小于0.30nM、小于0.20nM、小于0.15nM或更小)，所述IC 50值通过ELISA方法检测；在一些实施方案中，所述IC 50值通过本披露测试例4方法检测；
J.具有抑制IFNγ、IL-10和/或IgA细胞因子分泌活性；在一些实施方案中，所述IC 50值通过本披露测试例9方法检测；
K.具有抑制B细胞增殖和/或T细胞增殖的活性；在一些实施方案中，所述抗ICOSL抗体或抗ICOSL抗体融合蛋白抑制ICOSL诱导T细胞增殖的活性；在一些实施方案中，所述抑制T细胞增殖活性通过本披露测试例6方法检测；在一些实施方案中，所述抗ICOSL抗体融合蛋白抑制BAFF或APRIL诱导的B细胞增殖的活性；在一些实施方案中，所述抑制B细胞增殖活性通过本披露测试例7方法检测。
示例性的抗ICOSL抗体融合蛋白
在一些实施方案中，所述抗ICOSL抗体融合蛋白，其中，所述TACI多肽相比野生型的TACI多肽(序列如SEQ ID NO：51所示)不容易发生断裂。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中，所述TACI多肽如SEQ ID NO：58所示或是SEQ ID NO：58的截短片段或是SEQ ID NO：58的变体或是SEQ ID NO：58的截短片段的变体；其中所述截短片段包含SEQ ID NO：58的第48位至第85位氨基酸残基，所述变体为在SEQ ID NO：58或其截 短片段上具有选自由第49、52、53、57、65、82和83位组成的组中的一个或更多个氨基酸替换，其中所述氨基酸替换的位点为相对于序列SEQ ID NO：58的自然顺序编号的氨基酸残基位点。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中，所述TACI多肽，其中所述TACI多肽的截短片段包含：SEQ ID NO：58的第48位至第86位氨基酸残基；SEQ ID NO：58的第48位至第87位氨基酸残基；或SEQ ID NO：58的第48位至第88位氨基酸残基。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中，所述TACI多肽，序列如SEQ ID NO：60-63任一项所示。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中，所述TACI多肽，其序列为SEQ ID NO：58变体或者SEQ ID NO：58的截短片段(例如SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63)的变体，所述变体为在SEQ ID NO：58或其截短片段序列(例如SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63)上具有选自第49、52、53、57、65、82和83位中的任意1个、2个、3个、4个、5个、6个或7个氨基酸的替换，所述氨基酸替换的位点为相对于序列SEQ ID NO：58的自然顺序编号的氨基酸残基位点。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中所述TACI多肽为在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有选自由49T或49R、52S、53E或53Q、57E、65T或65A、82A或82R、和83Y组成的组中的一个或更多个氨基酸替换(例如1个、2个、3个、4个、5个、6个或7个氨基酸的替换)，其中所述氨基酸替换的位点为相对于序列SEQ ID NO：58的自然顺序编号的氨基酸残基位点。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中所述TACI多肽为：在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有选自49T、52S、53E、53Q、57E和82A中的任一个氨基酸替换。在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有49R和65T氨基酸替换。在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有49R和65A氨基酸替换。在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有49R、65T和82R氨基酸替换。在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有53E和57E氨基酸替换。在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有52S、53E和57E氨基酸替换。在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有49T和82A氨基酸替换。在SEQ ID NO： 58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有49T和83Y氨基酸替换。在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有49T、82A和83Y氨基酸替换。或者在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有49T、53E、57E和82A氨基酸替换。其中前述氨基酸替换的位点为相对于序列SEQ ID NO：58的自然顺序编号的氨基酸残基位点。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中所述TACI多肽的序列如SEQ ID NO：60-63、SEQ ID NO：66-83中的任一所示。在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中所述TACI多肽序列如SEQ ID NO：83所示。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其由2条相同的第一链和2条相同的第二链组成，其中，
第一链：其从N端到C端依次为：[抗ICOSL抗体的重链]-[连接子1]-[TACI多肽1]，和
第二链，其从N端到C端依次为：[TACI多肽2]-[连接子2]-[抗ICOSL抗体的轻链]；或第二链，其从N端到C端依次为[抗ICOSL抗体的轻链]-[连接子2]-[TACI多肽2]；或第二链，其为抗ICOSL抗体的轻链；
其中，所述TACI多肽1和TACI多肽2可以相同或不相同，所述连接子1和连接子2可以相同或不相同。在一些实施方案中，所述TACI多肽1和TACI多肽2相同。所述连接子1和连接子2独立地为(G xS) y连接子，其中，x选自1-5的整数，y选自0-6的整数。更优选地，所述连接子1和连接子2独立地为如SEQ ID NO：89或90所示的连接子。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中所述抗ICOSL抗体为前面任一项所述的抗ICOSL抗体。
在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中，所述的抗ICOSL抗体融合蛋白的第一链如SEQ ID NO：86所示，且第二链如SEQ ID NO：88、87或48所示。在一些实施方案中，如上任一项所述的抗ICOSL抗体融合蛋白，其中，所述的抗ICOSL抗体融合蛋白的第一链如SEQ ID NO：84所示，且第二链如SEQ ID NO：85或46所示。
抗ICOSL抗体或其融合蛋白的变体
在某些实施方案中，涵盖本文中提供的抗ICOSL抗体或其融合蛋白的氨基酸序列变体。例如，可以期望改善抗体的结合亲和力和/或其它生物学特性。可以通过将合适的修饰引入编码抗体的核苷酸序列中，或者通过肽合成来制备抗体的氨基酸序列变体。此类修饰包括例如对抗ICOSL抗体或其融合蛋白的氨基酸序列内的残基的删除、和/或插入、和/或取代。可以进行删除、插入、和取代的任何组合以得到最终的构建体，只要最终的构建体拥有期望的特征，例如抗原结合特性。
取代、插入、和删除变体
在某些实施方案中，提供了具有一处或多处氨基酸取代的抗体变体。取代诱变感兴趣的位点包括CDR和FR。保守取代在表3中在“优选的取代”的标题下显示。更实质的变化在表3中在“示例性取代”的标题下提供，并且如下文参照氨基酸侧链类别进一步描述的。可以将氨基酸取代引入感兴趣的抗体中，并且对产物筛选期望的活性，例如保留/改善的抗原结合，降低的免疫原性，或改善的ADCC或CDC。
表3.氨基酸的取代
原始残基 示例性取代 优选的取代
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；正亮氨酸 Leu
Leu(L) 正亮氨酸；Ile；Val；Met；Ala；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；正亮氨酸 Leu
依照常见的侧链特性，氨基酸可以如下分组:
(1)疏水性的：正亮氨酸，Met，Ala，Val，Leu，Ile；
(2)中性，亲水性的：Cys，Ser，Thr，Asn，Gln；
(3)酸性的：Asp，Glu；
(4)碱性的：His，Lys，Arg；
(5)影响链取向的残基：Gly，Pro；
(6)芳香族的：Trp，Tyr，Phe。
非保守取代会需要用这些类别之一的成员替换另一个类别的成员。
一类取代变体涉及取代亲本抗体(例如人源化或人抗体)的一个或多个CDR残基。一般地，经选择用于进一步研究的所得变体相对于亲本抗体会具有某些生物学特性(例如升高的亲和力，降低的免疫原性)的改变(例如改善)，和/或会基本上保留亲本抗体的某些生物学特性。一种示例性的取代变体是亲和力成熟的 抗体，可以例如使用基于噬菌体展示的亲和力成熟技术(如本文所述的那些技术)，便利地产生所述抗体。简言之，将一个或多个CDR残基突变，并将变体抗体在噬菌体上展示，并对其筛选特定的生物学活性(例如结合亲和力)。可以对CDR做出改变(例如取代)，例如以改善抗体亲和力。可以对CDR“热点”，即在体细胞成熟过程期间高频率经历突变的密码子所编码的残基，和/或对接触抗原的残基做出此类改变，同时对所得的变体VH或VL测试结合亲和力。在亲和力成熟的一些实施方案中，通过多种方法(例如易错PCR、链改组、或寡核苷酸指导的诱变)的任一种，将多样性引入所选择用于成熟的可变基因中。然后，创建次级文库。然后，筛选文库以鉴定具有期望的亲和力的任何抗体变体。另一种引入多样性的方法涉及CDR定向的方法，其中将几个CDR残基(例如4-6个残基)随机化。可以例如使用丙氨酸扫描诱变或建模来特异性鉴定涉及抗原结合的CDR残基。特别地，经常靶向HCDR3和LCDR3。
在某些实施方案中，取代、插入或缺失可以在一个或多个CDR内进行，只要此类变化不实质性降低抗体结合抗原的能力。例如，可以对CDR做出保守变化(例如保守取代，如表3中提供的)，其不实质性降低结合亲和力。此类变化可以例如在CDR中的抗原接触残基外部。在上文提供的变体VH和VL序列的某些实施方案中，每个CDR是未改变的，或者含有不超过1、2或3处氨基酸取代。
一种可用于鉴定抗体中可以作为诱变靶位的残基或区域的方法称作“丙氨酸扫描诱变”。在这种方法中，鉴定一个残基或残基组(例如带电荷的残基，诸如Arg、Asp、His、Lys和Glu)，并且用中性或带负电荷的氨基酸(例如，Ala或聚丙氨酸)替换以确定该抗体与抗原的相互作用是否受影响。可以在对初始取代显示功能敏感性的氨基酸位置引入进一步的取代。此外，可通过研究抗原-抗体复合物的晶体结构来鉴定抗体与抗原间的接触点。这些接触残基及邻近残基可以作为取代候选物被打靶或消除。可以筛选变体以确定它们是否含有期望的特性。
氨基酸序列插入包括在多肽的氨基和/或羧基端融合长度范围为1个残基至100或更多个残基，和单个或多个氨基酸残基的序列内插入。末端插入的例子包括具有N端甲硫氨酰基残基的抗体。抗体分子的其它插入变体包括抗体的N或C端与酶(或延长抗体的半衰期的多肽)的融合物。
Fc区的修饰
在一个方面，本披露的抗ICOSL抗体或抗ICOSL抗体融合蛋白的Fc区包含一个或多个氨基酸取代，所述一个或多个氨基酸取代减少其与Fc受体的结合，例如其与Fcγ受体的结合，并且降低或消除效应子功能。天然IgG Fc区，具体地是IgG 1Fc区或IgG 4Fc区，可能导致本披露的融合蛋白靶向表达Fc受体的细胞，而不是表达抗原的细胞。在一些实施方案中，本披露改造的Fc区表现出降低的对Fc受体的结合亲和力和/或降低的效应子功能。在一些实施方案中，改造的Fc区与天然Fc区相比，对Fc受体的结合亲和力下降50％、80％、90％或95％以上。在一些 实施方案中，所述的Fc受体是Fcγ受体。在一些实施方案中，所述Fc受体是人Fcγ受体，例如FcγRI、FcγRIIa、FcγRIIB、FcγRIIIa。在一些实施方案中，改造的Fc区与天然Fc区相比，对补体，如C1q的结合亲和力也降低。在一些实施方案中，改造的Fc区与天然Fc区相比，对新生儿Fc受体(FcRn)的结合亲和力不降低。在一些实施例中，改造的Fc区具有降低的效应子功能，所述降低的效应子功能可以包括但不限于以下中的一个或多个：降低的补体依赖性细胞毒性(CDC)、降低的抗体依赖性细胞介导的细胞毒性(ADCC)、降低的抗体依赖性细胞吞噬(ADCP)、减少的细胞因子分泌、减少的免疫复合物介导的抗原呈递细胞的抗原摄取、减少的与NK细胞的结合、减少的与巨噬细胞的结合、减少的与单核细胞的结合、减少的与多形核细胞的结合、减少的直接信号传导诱导性细胞凋亡、降低的树突细胞成熟或减少的T细胞引发。对于IgG 1Fc区，在238、265、269、270、297、327和329等位置的氨基酸残基取代可降低的效应子功能。在一些实施方案中，所述Fc区是人IgG 1Fc区，并且在234和235位置的氨基酸残基为A，编号依据为EU索引。对于IgG 4Fc区，在228等位置的氨基酸残基取代可降低的效应子功能。
抗ICOSL抗体或抗ICOSL抗体融合蛋白可包含与Fc区的两个亚基融合的不同结合域，因此可能导致不期望的同源二聚化。为了提高产率和纯度，可以在本披露的融合蛋白的Fc区中引入促进异源二聚化的修饰将是有利的。在一些实施方案中，本披露的Fc区包含根据杵臼(knob-into-hole，KIH)技术的改造，该方法涉及在第一亚基的界面处引入凸起结构(knob)以及在第二亚基的界面处引入孔结构(hole)。使得所述凸起结构可以定位在孔结构中，促进异源二聚体的形成并抑制同源二聚体的产生。凸起结构是通过用较大侧链(例如酪氨酸或色氨酸)取代来自第一亚基的界面的小氨基酸侧链而构建的。而孔结构是通过用较小的氨基酸侧链(例如丙氨酸或苏氨酸)取代大氨基酸侧链而在第二亚基的界面中创建的。凸起结构和孔结构通过改变编码多肽的核酸来制备，可选的氨基酸取代如下表4所示：
表4.KIH突变组合
Figure PCTCN2022131362-appb-000011
除了杵臼技术外，用于修饰抗体的重链的CH3结构域以实现异源二聚化的其他技术也是本领域中已知的，例如WO96/27011、WO98/050431、EP1870459、WO2007/110205、WO 007/147901、WO2009/089004、WO2010/129304、WO2011/90754、WO2011/143545、WO2012/058768、WO2013/157954和WO 013/096291。
抗原结合分子还可包含二硫键改造，例如Fc区的第一亚基包含354C突变和 第二亚基包含349C突变，使Fc区的第一亚基和第二亚基之间产生工程化二硫键，促进Fc区第一亚基和第二亚基的异源二聚化。
抗原结合分子的Fc区还可进一步引入其它的氨基酸改造，例如降低免疫原性的同种异型氨基酸残基突变。在一些实施方案中，IgG1的Fc引入356E和358M突变。
Fc区的C末端可以是以氨基酸残基PGK结束的完整C末端；也可以是缩短的C末端，例如在所述缩短的C末端中已经去除了一个或两个C末端氨基酸残基。在一个优选的方面中，重链的C末端是以PG结束的缩短的C末端。因此，在一些实施方案中，完整抗体可以包括去除了所有K447残基和/或G446+K447残基的抗体混合物。在一些实施方案中，完整抗体可以包括没有去除K447残基和/或G446+K447残基的抗体混合物。在一些实施方案中，完整抗体具有带有和不带有K447残基和/或G446+K447残基的抗体混合物。
重组方法
抗ICOSL抗体或抗ICOSL抗体融合蛋白可以使用重组方法来产生。对于这些方法，提供编码抗体或融合蛋白的一个或更多个分离的核酸。
在一个实施方案中，本披露提供了编码如前所述的抗ICOSL抗体或抗ICOSL抗体融合蛋白的分离的核酸。此类核酸可以给自独立的编码前述的任一多肽链。在另一方面中，本披露提供了包含此类核酸的一种或多种载体(例如表达载体)。在另一方面中，本披露提供了包含此类核酸的宿主细胞。在一个实施方案中，提供制备抗体或融合蛋白的方法，其中所述方法包括，在适合表达的条件下，培养包含编码所述抗体或融合蛋白的核酸的宿主细胞，如上文所提供的，和任选地从宿主细胞(或宿主细胞培养基)回收所述抗ICOSL抗体或抗ICOSL抗体融合蛋白。
为了重组产生抗ICOSL抗体或抗ICOSL抗体融合蛋白，将编码蛋白的核酸分离并插入一个或更多个载体中，用于在宿主细胞中进一步克隆和/或表达。此类核酸可以使用常规程序容易地分离和测序，或者通过重组方法产生或通过化学合成获得。
用于克隆或表达编码抗ICOSL抗体或抗ICOSL抗体融合蛋白的载体的适当宿主细胞包括本文描述的原核或真核细胞。例如，可在细菌中产生，特别是当不需要糖基化和Fc效应子功能时。在表达后，可以在可溶级分中从细菌细胞糊状物分离，并且可进一步纯化。
除了原核生物以外，真核微生物诸如丝状真菌或酵母也是用于编码抗体或融合蛋白的载体的合适的克隆或表达宿主，包括真菌和酵母菌株。适于表达抗体或融合蛋白的合适的宿主细胞也可源自多细胞生物体(无脊椎动物和脊椎动物)；无脊椎动物细胞的例子包括植物和昆虫细胞。已经鉴定了许多杆状病毒株，其可与昆虫细胞联合使用，特别是用于草地贪夜蛾(Spodoptera frugiperda)细胞的转染；还可利用植物细胞培养物作为宿主，例如US5959177、US6040498、US6420548、 US7125978和US6417429；也可将脊椎动物细胞用作宿主，例如适应于在悬浮液中生长的哺乳动物细胞系。适宜的哺乳动物宿主细胞系的其它例子是经SV40转化的猴肾CVl系(COS-7)；人胚肾系(293或293T细胞)；幼仓鼠肾细胞(BHK)；小鼠塞托利(sertoli)细胞(TM4细胞)；猴肾细胞(CV1)；非洲绿猴肾细胞(VERO-76)；人宫颈癌细胞(HELA)；犬肾细胞(MDCK)；水牛鼠(buffalo rat)肝细胞(BRL3A)；人肺细胞(W138)；人肝细胞(Hep G2)；小鼠乳房肿瘤(MMT 060562)；TRI细胞；MRC 5细胞；和FS4细胞。其它适宜的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括DHFR-CHO细胞；以及骨髓瘤细胞系，如Y0、NS0和Sp2/0。关于适合产生抗体或融合蛋白的某些哺乳动物宿主细胞系的综述参见例如Yazaki，P.和Wu，A.M.，Methods in Molecular Biology，Vol.248，Lo，B.K.C.(编)，Humana Press，Totowa，NJ(2004)，第255-268页。
测定
本文提供的抗ICOSL抗体或抗ICOSL抗体融合蛋白可以通过本领域已知的多种测定法对其物理/化学特征和/或生物学活性进行鉴定、筛选或表征。
在一个方面中，例如通过已知方法如ELISA、蛋白印迹法等，测试本披露的抗ICOSL抗体或抗ICOSL抗体融合蛋白活性。
治疗方法与施用途径
本文提供的任何抗ICOSL抗体或抗ICOSL抗体融合蛋白可用于治疗方法。
在又一个方面，本披露提供抗ICOSL抗体或抗ICOSL抗体融合蛋白在药物的制造或制备中的用途。在一些实施方案中，所述药物用于治疗自身免疫性疾病、炎性疾病、B细胞障碍或T细胞障碍，所述疾病是与ICOSL过表达相关的疾病。
在一些实施方案中，所述自身免疫性疾病或炎性疾病选自：系统性红斑狼疮、类风湿性关节炎、移植物抗宿主病、哮喘、免疫性血小板减少性紫癜、多发性硬化、糖尿病引起的炎性疾病、银屑病、炎性肠病、克罗恩病、溃疡性结肠炎、格雷夫斯病和桥本氏甲状腺炎等。在一些实施方案中，所述B细胞障碍或T细胞障碍为肿瘤疾病。在一些实施方案中，所述肿瘤选自：头颈癌、非小细胞肺癌、尿路上皮癌、白血病、肉瘤、黑色素瘤、腺癌、结直肠癌、前列腺肿瘤、乳腺癌和小细胞肺癌等。在一些实施方案中，所述自身免疫性疾病为系统性红斑狼疮。在一个此类实施方案中，所述用途进一步包括向受试者施用有效量的至少一种另外的治疗剂(例如一种、两种、三种、四种、五种或六种另外的治疗剂)。根据任意以上实施方案的“受试者”可以是人。
在又一个的方面，提供包含所述抗ICOSL抗体或抗ICOSL抗体融合蛋白的药物组合物，例如，其用于以上任何制药用途或治疗方法。在一个实施方案中，药物组合物包含本文提供的任何抗体或融合蛋白和药学上可接受的载体。在另一个实施方案中，药物组合物还包含至少一种另外的治疗剂。
本披露的抗ICOSL抗体或抗ICOSL抗体融合蛋白可单独使用或与其他试剂联 合用于治疗。例如，本披露的抗ICOSL抗体或抗ICOSL抗体融合蛋白可与至少一种另外的治疗剂联合施用。
“联合施用”是指为了达到治疗目的，而将两种或两种以上活性化合物同时或先后提供给受试者的方法。当涉及“联合施用”时，每次施用之间的时间间隔，足以实现施用的各活性化合物之间的协同作用。两种或两种以上活性化合物在相同的不同容器中。
本披露的抗ICOSL抗体或抗ICOSL抗体融合蛋白(和任何另外的治疗剂)可通过任何合适的手段施用，包括肠胃外、肺内和鼻内，并且如果需要局部治疗，则病灶内施用。肠胃外输注包括肌肉内、静脉内、动脉内、腹膜内或皮下施用。给药可以通过任何适当的途径，例如，通过注射，诸如静脉内或皮下注射，这部分取决于施用是短期的还是长期的。本文考虑多种给药时间方案，包括但不限于，单次或在多个时间点多次施用，推注施用和脉冲输注。
本披露的抗ICOSL抗体或抗ICOSL抗体融合蛋白将以符合良好医疗实践(Good Manufacturing Practice)的方式配制、给药和施用。在此背景下考虑的因素包括所治疗的具体病症、所治疗的具体哺乳动物、个体患者的临床状况、病症的起因、试剂的递送部位、施用方法、施用时间安排以及医学从业者已知的其他因素。多肽或融合蛋白可以与或不与目前用于预防或治疗所述病症的一种或更多种试剂一起配制。此类其它试剂的有效量取决于药物组合物中存在的量、病症或治疗的类型以及其它因素。这些通常以与本文所述相同的剂量和施用路径使用，或以本文所述剂量的约1至99％使用，或以其它剂量使用，并通过经验/临床确定为合适的任何途径使用。
具体实施方案中的有效量，可以从来源于动物模型测试系统的剂量-应答曲线而得到，并允许根据医生的判断和每位患者的情况来决定。受试者一次施用所需的药物的量可以方便地通过计算受试者体重和一次用药所需单位体重剂量得到。可以通过实验动物与人的单位体重剂量之间的等效剂量换算关系来确定用药量(Freireich等人1966，Cancer Chemother Rep 50：219)。例如，可以根据FDA、SFDA等药品管理机构提出的指导意见。在一些实施方式中，可以使用按照人和鼠的体表面积折算系数，来换算人和鼠的剂量(如参照测试例9-11中的剂量)。
为了预防或治疗疾病，本披露的抗ICOSL抗体或抗ICOSL抗体融合蛋白(当单独使用或与一种或更多种其他另外的治疗剂组合使用时)的适当的剂量将取决于待治疗的疾病的类型，治疗分子的类型，疾病的严重性和病程，是为预防还是治疗目的施用，之前的治疗，患者的临床病史和对治疗分子的响应，和主治医师的判断。治疗分子恰当地以一次或经过一系列治疗施用于患者。
制品
在本披露的另一方面中，提供一种制品(如药盒或试剂盒)，所述制品包含可用于治疗、预防和/或诊断上述病症的材料。该制品包含容器和在容器上或与容 器联合的标签或包装插页(package insert)。合适的容器包括，例如，瓶子、管形瓶、注射器、IV溶液袋等。容器可以自各种材料诸如玻璃或塑料形成。容器装有单独的抗体或其融合蛋白、或与另一种成分组合，并且可具有无菌的存取口(例如，容器可以是具有塞子的静脉内溶液袋或管形瓶)。容器中的至少一种活性试剂是本披露的抗ICOSL抗体或抗ICOSL抗体融合蛋白。标签或包装插页指示使用该抗体或其融合蛋白是来治疗选择的病况。
此外，制品可以包含：(a)其中装有组合物的第一容器，其中所述组合物包含本披露的抗ICOSL抗体或抗ICOSL抗体融合蛋白；和(b)其中装有组合物的第二容器，其中所述组合物包含另外的细胞毒性剂或其他方面的治疗剂。
备选地，制品可进一步包含第二(或第三)容器，所述第二(或第三)容器包含药学上可接受的缓冲液。从商业和用户立场，它可进一步包括所需的其他材料，包括其他缓冲剂、稀释剂、滤器、针头和注射器。
应当理解，对于数值范围的表述，例如“10至100”或“至少90％”是一种简洁的书写方式。尽管没有给出该范围内的每一个点值(包括整数和分数)，但视为已经在文中明确得以披露。
实施例与测试例
以下结合实施例和测试例进一步描述本披露，但这些实施例和测试例并非限制着本披露的范围。本披露实施例和测试例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1：小鼠抗人ICOSL单克隆抗体的制备
使用人ICOSL蛋白(Sino Biological，11559-H08H)免疫SJL小鼠。免疫3次后取血测定血清中抗体的效价，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合，将融合好的杂交瘤细胞铺在96孔细胞培养板中，置于37℃，5％CO 2培养箱中进行培养。取细胞培养上清液通过酶联免疫吸附分析法(ELISA)进行检测。将筛选出的阳性克隆进行扩增冻存保种和二到三次亚克隆直至获得单细胞克隆。选择的杂交瘤克隆用无血清细胞培养法进一步制备和纯化抗体。得到的杂交瘤抗体用FACS检测抗体与细胞表面人ICOSL蛋白的结合和对受体的阻断情况(方法见本公开测试例1和测试例3)，挑选出结合活性和阻断活性好的杂交瘤细胞株。
获得单克隆杂交瘤细胞株mAb201和mAb259克隆单克隆抗体的序列。过程如下：收集对数生长期杂交瘤细胞，用Trizol(Invitrogen，Cat#15596-018)提取RNA，反转录为cDNA。用cDNA为模板进行PCR扩增后送测序公司测序，得到的DNA序列对应的抗体氨基酸序列如下表5、表6所示：
表5.鼠源抗ICOSL抗体的可变区序列
Figure PCTCN2022131362-appb-000012
备注：表中下划线部分表示CDR序列(根据Kabat编号系统确认)。
表6.抗体CDR序列
Figure PCTCN2022131362-appb-000013
备注：表中CDR是根据Kabat编号系统确认的。
将上述mAb201和mAb259候选分子可变区序列通过PCR扩增VH/VL序列，再与表达载体pHr(带信号肽及hIgG4/hkappa恒定区基因(CH1-Fc/CL)片段)进行同源重组。示范性地，人重链IgG4恒定区序列如SEQ ID NO：43所示，人轻链κ恒定区序列如SEQ ID NO：44所示，构建重组嵌合抗体全长表达质粒VH-CH1-Fc-pHr/VL-CL-pHr，进而获得其嵌合抗体Ch201和Ch259。
实施例2：鼠源抗人ICOSL单克隆抗体的人源化
通过比对IMGT人类抗体重轻链可变区种系基因数据库，分别挑选同源性高的重轻链可变区种系基因作为模板，将鼠源抗体的CDR分别Grafted(嫁接)到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列，然后将可变区序列与人恒定区序列融合，获得人源化抗体。以下示范性地描述mAb201和mAb259鼠源抗体的人源化，范例中抗体的CDR氨基酸残基由Kabat编号系统确定并注释。
1、鼠源抗体mAb201的人源化
挑选同源性高的重轻链可变区种系基因作为模板。鼠源抗体mAb201的人源化轻链模板选自IGKV2-28*01和IGKJ4*01，即选择人种系轻链IGKV2-28*01的FR1，FR2，FR3，和IGKJ4*01的JK4区(作为FR4)作为人源化抗体轻链框架区；人源化重链模板选自IGHV1-46*01和IGHJ6*01，即选择人种系重链IGHV1-46*01的FR1，FR2，FR3，和IGHJ6*01的JH6区(作为FR4)作为人源化抗体重链框架区。
首先，将鼠源抗体mAb201的CDR分别移植到相应的人源模板中，替换人源模板的CDR区；然后，对人源化抗体的轻链可变区的第1、2和/或4位(根据Kabat编号系统编号)氨基酸残基进行突变，重链可变区的第1、24、69、71、73和/或78位(根据Kabat编号系统编号)氨基酸残基进行突变；此外，还对轻链可变区的LCDR2：LVSNRFS(SEQ ID NO：9)中的第1位氨基酸残基由L突变为K，获得新的LCDR2：KVSNRFS(SEQ ID NO：17)；对轻链可变区的LCDR3：FQSNYLPLT(SEQ ID NO：10)中的第8位氨基酸残基由L突变为P，获得新的LCDR3：FQSNYLPPT(SEQ ID NO：18)；对重链可变区的HCDR2：MIHPNGGSTNYNEKFKS(SEQ ID NO：6)中的第5位氨基酸残基由N突变为H，获得新的HCDR2：MIHPHGGSTNYNEKFKS(SEQ ID NO：19)。鼠源抗体mAb201人源化序列如下所示：
>hAb201VL1(Graft)的氨基酸序列(SEQ ID NO：20)
Figure PCTCN2022131362-appb-000014
>hAb201VL2(Graft+D1A，I2V，M4L)的氨基酸序列(SEQ ID NO：21)
Figure PCTCN2022131362-appb-000015
>hAb201VL3(Graft+L50K)的氨基酸序列(SEQ ID NO：22)
Figure PCTCN2022131362-appb-000016
>hAb201VL4(Graft+D1A，I2V，M4L+L50K)的氨基酸序列(SEQ ID NO：23)
Figure PCTCN2022131362-appb-000017
>hAb201VL5(Graft+L96P)的氨基酸序列(SEQ ID NO：24)
Figure PCTCN2022131362-appb-000018
Figure PCTCN2022131362-appb-000019
>hAb201VL6(Graft+D1A，I2V，M4L+L96P)的氨基酸序列(SEQ ID NO：25)
Figure PCTCN2022131362-appb-000020
>hAb201VL7(Graft+L50K，L96P)的氨基酸序列(SEQ ID NO：26)
Figure PCTCN2022131362-appb-000021
>hAb201VL8(Graft+D1A，I2V，M4L+L50K，L96P)的氨基酸序列(SEQ ID NO：27)
Figure PCTCN2022131362-appb-000022
>hAb201VH1(Graft+Q1E，A24T，M69L，R71V，T73K，V78A)的氨基酸序列(SEQ ID NO：28)
Figure PCTCN2022131362-appb-000023
>hAb201VH2(Graft+Q1E，R71V，T73K+N53H)的氨基酸序列(SEQ ID NO：29)
Figure PCTCN2022131362-appb-000024
>hAb201VH3(Graft+Q1E，A24T，M69L，R71V，T73K，V78A+N53H)的氨基酸序列(SEQ ID NO：30)
Figure PCTCN2022131362-appb-000025
注：上述序列依次为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中下划线部分为CDR序列(根据Kabat编号系统确认)，其余为FR序列，斜体加粗部分表示突变位点。
2、鼠源抗体mAb259的人源化
挑选同源性高的重轻链可变区种系基因作为模板，例如，鼠源抗体mAb259的人源化轻链模板选自IGKV4-1*01/IGKV1-39*01和IGKJ2*01，即选择人种系轻链IGKV4-1*01或IGKV1-39*01的FR1，FR2，FR3，和IGKJ2*01的JK4区(作为FR4)作为人源化抗体轻链框架区；人源化重链模板选自IGHV3-74*03和 IGHJ1*01，即选择人种系重链IGHV3-74*03的FR1，FR2，FR3，和IGHJ1*01的JH1区(作为FR4)作为人源化抗体重链框架区。首先，将鼠源抗体mAb259的CDR分别移植到相应的人源模板中，替换人源模板的CDR区；然后，对人源化抗体的轻链可变区的第1、2、3、42、43、60和/或85位(根据Kabat编号系统编号)氨基酸残基进行突变，重链可变区的的第37、43和/或49(根据Kabat编号系统编号)氨基酸残基进行突变。此外，对重链可变区的HCDR2：YISSSSGKVYADAVKG(SEQ ID NO：12)中的第3位氨基酸残基由S突变为D，获得新的HCDR2：YIDSSSGKVYADAVKG(SEQ ID NO：31)。鼠源抗体mAb259人源化序列如下所示：
>hAb259VL1(Graft(IGKV4-1*01-IGKJ2*01))的氨基酸序列(SEQ ID NO：32)
Figure PCTCN2022131362-appb-000026
>hAb259VL2(Graft(IGKV4-1*01-IGKJ2*01)+D1N，I2T，P43S)的氨基酸序列(SEQ ID NO：33)
Figure PCTCN2022131362-appb-000027
>hAb259VL3(Graft(IGKV1-39*01-IGKJ2*01)的氨基酸序列(SEQ ID NO：34)
Figure PCTCN2022131362-appb-000028
>hAb259VL4(Graft(IGKV1-39*01-IGKJ2*01+D1N，I2T，A43S，S60D，T85V)的氨基酸序列(SEQ ID NO：35)
Figure PCTCN2022131362-appb-000029
>hAb259VL5(Graft(IGKV1-39*01-IGKJ2*01+I2T)的氨基酸序列(SEQ ID NO：36)
Figure PCTCN2022131362-appb-000030
>hAb259VL6(Graft(IGKV1-39*01-IGKJ2*01+D1N，I2T，Q3V)的氨基酸序列(SEQ ID NO：37)
Figure PCTCN2022131362-appb-000031
Figure PCTCN2022131362-appb-000032
>hAb259VL7(Graft(IGKV1-39*01-IGKJ2*01+D1N，I2T，Q3V，K42Q)的氨基酸序列(SEQ ID NO：38)
Figure PCTCN2022131362-appb-000033
>hAb259VH1(Graft)的氨基酸序列(SEQ ID NO：39)
Figure PCTCN2022131362-appb-000034
>hAb259VH2(Graft+V37I，S49A)的氨基酸序列(SEQ ID NO：40)
Figure PCTCN2022131362-appb-000035
>hAb259VH3(Graft+V37I，K43E，S49A)的氨基酸序列(SEQ ID NO：41)
Figure PCTCN2022131362-appb-000036
>hAb259VH4(Graft+V37I，K43E，S49A+S52D)的氨基酸序列(SEQ ID NO：42)
Figure PCTCN2022131362-appb-000037
注：序列顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，其中下划线部分为CDR序列(根据Kabat编号系统确认)，其余为FR序列，斜体加粗部分表示突变位点。
3、人源化抗体的制备
分别构建抗体轻链和重链的表达载体，将人源化的抗体轻/重链分别交叉配对组合，转染293E细胞后收集培养上清纯化即得到人源化的全长抗体。人源化抗体重链恒定区可选自IgG1、IgG2、IgG3、IgG4及其变体的恒定区，示例性的，使用人重链IgG4恒定区(如SEQ ID NO：43所示)与前述人源化重链可变区融合形成抗体全长重链；人源化抗体轻链恒定区可选自人源κ、λ链或其变体的恒定区，示例性的，使用人轻链恒定区κ链(如SEQ ID NO：44所示)与前述人源化轻链可变区融合形成抗体全长轻链。
示例性的抗体的恒定区序列如下：
人IgG4的重链恒定区的氨基酸序列(SEQ ID NO：43)：
Figure PCTCN2022131362-appb-000038
Figure PCTCN2022131362-appb-000039
人κ链的轻链恒定区的氨基酸序列(SEQ ID NO：44)：
Figure PCTCN2022131362-appb-000040
示例性的，将前述mAb201的人源化抗体重链可变区羧基端与人重链IgG4恒定区(如SEQ ID NO：43)氨基端连接形成抗体全长重链，同时将人源化抗体轻链可变区羧基端与人轻链κ恒定区(如SEQ ID NO：44)氨基端连接形成抗体全长轻链，得到如下表7-1所示的mAb201的人源化抗体：
表7-1.mAb201人源化抗体
Figure PCTCN2022131362-appb-000041
备注：表中，例如201-H1L1表示重链可变区为hAb201VH1(如SEQ ID NO：28所示)，轻链可变区为hAb201VL1(如SEQ ID NO：20所示)，重链恒定区为IgG4恒定区(如SEQ ID NO：43所示)，轻链恒定区为κ恒定区(如SEQ ID NO：44)的人源化抗体。
另外，将前述来自mAb259的人源化抗体重链可变区羧基端与人重链IgG4恒定区氨基端连接形成抗体全长重链，同时将人源化抗体轻链可变区羧基端与人轻链κ恒定区氨基端连接形成抗体全长轻链，得到如下表7-2所示的mAb259人源化抗体：
表7-2.mAb259人源化抗体
Figure PCTCN2022131362-appb-000042
备注：表中，例如259-H1L1表示重链可变区为hAb259VH1(如SEQ ID NO：39所示)，轻链可变区为hAb259VL1(如SEQ ID NO：32所示)，重链恒定区为IgG4恒定区(如SEQ ID NO：43所示)，轻链恒定区为κ恒定区(如SEQ ID NO：44)的人源化抗体。
示例性的，人源化抗体201-H3L3和259-H4L7的轻链/重链序列如下表8所示：
表8.人源化抗体的轻/重链序列
Figure PCTCN2022131362-appb-000043
备注：表中斜体字为恒定区序列，下划线标记为可变区序列。
通过本披露测试例1方法检测抗ICOSL抗体对hICOSL-CHOK1的结合，通过本披露测试例3方法检测抗ICOSL抗体对ICOSL与ICOS结合的阻断活性的结合实验结果见下表9、表10：
表9.抗ICOSL抗体与hICOSL-CHOK1结合实验结果
Figure PCTCN2022131362-appb-000044
表10.抗ICOSL抗体阻断ICOSL与ICOS结合实验结果
Figure PCTCN2022131362-appb-000045
实施例3：抗ICOSL抗体融合蛋白的构建
将前述抗ICOSL抗体与TACI多肽融合，构建抗ICOSL抗体融合蛋白。示例性地，抗ICOSL抗体为人源化抗体259-H4L7或201-H3L3。TACI多肽可以是任 意适宜的TACI多肽，示例性地，TACI多肽为来源于专利申请PCT/CN2022/084256及其优先权202110348497.6(发明名称：一种新型TACI多肽，其融合蛋白及用途；申请日：2021/3/31)(通过援引完整收入本文)中所述的TACI多肽，包括例如下表11中所示的TACI多肽：
表11.TACI多肽的氨基酸序列
Figure PCTCN2022131362-appb-000046
Figure PCTCN2022131362-appb-000047
示例性的，将TACI多肽(例如TACI-9-15c)融合到抗ICOSL抗体(例如201-H3L3或259-H4L7)重链的C端、轻链的N端或C端，并在融合抗体重链或轻链的C端的TACI多肽的C末端添加QK作为保护氨基酸，以防止TACI末端的氨基酸被羧肽酶切除。构建获得多种抗ICOSL抗体融合蛋白，其中259H4L7-T11和201H3L3-T11的结构示意图见图1、201H3L3-T7和259H4L7-T7的结构示意图见图2、259H4L7-T9的结构示意图见图3，所述抗ICOSL抗体融合蛋白包括2条相同的第一链和2条相同的第二链，具体如下所示：
>201H3L3-T7第一链的氨基酸序列(SEQ ID NO：84)
Figure PCTCN2022131362-appb-000048
>201H3L3-T7第二链的氨基酸序列同201-H3L3的轻链(SEQ ID NO：46)。
>201H3L3-T11第一链的氨基酸序列同201H3L3-T7第一链的氨基酸序列(SEQ ID NO：84)；
>201H3L3-T11第二链的氨基酸序列(SEQ ID NO：85)
Figure PCTCN2022131362-appb-000049
>259H4L7-T7第一链的氨基酸序列(SEQ ID NO：86)
Figure PCTCN2022131362-appb-000050
>259H4L7-T7第二链的氨基酸序列同259-H4L7的轻链(SEQ ID NO：48)。
>259H4L7-T9第一链的氨基酸序列同259H4L7-T7第一链的氨基酸序列(SEQ ID NO：86)；
>259H4L7-T9第二链的氨基酸序列(SEQ ID NO：87)
Figure PCTCN2022131362-appb-000051
>259H4L7-T11第一链的氨基酸序列同259H4L7-T7第一链的氨基酸序列(SEQ ID NO：86)；
>259H4L7-T11第二链的氨基酸序列(SEQ ID NO：88)
Figure PCTCN2022131362-appb-000052
注：上述序列中，单下划线为TACI序列，双下划线为抗体可变区序列，斜体为抗体恒定区序列，粗体为连接子序列。
本披露测试例中所用对照分子为AMG-570(抗ICOSL抗体与结合BAFF多肽融合而成抗体融合蛋白)的序列如下：
>AMG-570的重链的氨基酸序列(SEQ ID NO：49)
Figure PCTCN2022131362-appb-000053
Figure PCTCN2022131362-appb-000054
>AMG-570的轻链的氨基酸序列(SEQ ID NO：50)
Figure PCTCN2022131362-appb-000055
备注：序列中单下划线为可变区，双下划线为BAFF结合多肽，粗体为连接氨基酸，其余为恒定区。
测试例
测试例1：FACS结合实验
为了检测待测分子对ICOSL的结合情况，通过FACS(流式细胞术)的方法检测其与过表达人ICOSL的CHOK1细胞(ATCC，CCL-61)hICOSL-CHOK1的结合活性。用0.25％胰酶消化hICOSL-CHOK1细胞，300g离心5分钟，用PBS(源培生物，B320)+2％FBS(Biosun，BS-0002-500)重悬细胞。取50μL细胞悬液加入到96孔U型板(Corning，3795)中，使每孔细胞数为10 5个。每孔加入50μL梯度稀释的待测样品溶液，于4℃避光孵育1小时。用PBS+2％FBS洗板2次，每孔加入100μL Alexa羊抗人IgG(H+L)FITC荧光二抗(Thermo，A-11013，1:500稀释)，4℃避光孵育1小时。洗板2次后，每孔加入100μL PBS重悬细胞，用流式细胞仪(BD Biosciences，FACS Canto II，338962)读取FITC通道的荧光值，用软件拟合出待测分子与细胞表面ICOSL的结合曲线，计算出EC 50值。实验结果见下表12所示。
表12.抗ICOSL抗体及其融合蛋白对hICOSL-CHOK1的结合实验结果
样品名称 与hICOSL-CHOK1结合的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
实验结果表明，本披露构建的抗ICOSL抗体及其融合蛋白对hICOSL-CHOK1具有很强的结合活性。
测试例2：ELISA结合实验
通过ELISA的方法(包被待测分子)检测待测分子对BAFF和APRIL蛋白的结合活性。具体方法如下：
用pH 7.4的PBS缓冲液将待测分子稀释至2μg/mL，以100μL/孔的体积加入96孔酶标板(Corning，3590)中，4℃过夜孵育。弃去液体后，每孔加入300μL用PBS稀释的1％Casein(37528，Thermo)进行封闭，37℃孵育2小时。封闭结束后，弃去封闭液，并用PBST缓冲液(pH 7.4PBS含0.1％tween-20)洗板3次后，每孔加入100μL梯度稀释的人BAFF(ACROBiosystems，BAF-H52D4)或人APRIL(R&D Systems，5860-AP-010/CF)溶液，于37℃孵育1小时，孵育结束后用PBST洗板3次。检测与BAFF的结合时，每孔加入100μL抗-his-HRP二抗(Sino biological，105327-MM02T-H，1:2000稀释)，检测与APRIL的结合时，每孔加入100μL抗-HA-HRP(Abcam，ab1190，1:4000稀释)，37℃孵育1小时。用PBST洗板3次后，每孔加入100μL TMB显色底物(KPL，5120-0077)，室温孵育10至15分钟，每孔加入50μl 1M H 2SO 4终止反应，用酶标仪读取在450nm处的吸收值，用软件拟合出待测分子与抗原的结合曲线，计算出EC 50值。实验结果见下表13、表14。
表13.抗ICOSL抗体融合蛋白对APRIL蛋白的结合实验结果
样品名称 与人APRIL结合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 不结合
表14.抗ICOSL抗体融合蛋白对BAFF蛋白的结合实验结果
样品名称 与人BAFF结合的EC 50(nM)
201H3L3-T7 16.14
201H3L3-T11 13.75
259H4L7-T7 18.31
259H4L7-T9 27.53
259H4L7-T11 14.92
实验结果表明，本披露构建的抗ICOSL抗体融合蛋白对BAFF和APRIL蛋白均具有较强的结合活性，而对照分子AMG-570对APRIL蛋白没有结合活性。
测试例3：阻断ICOSL与ICOS的结合实验
通过FACS的方法检测待测分子对ICOSL与ICOS结合的阻断活性。用0.25％胰酶消化hICOSL-CHOK1细胞(同测试例1)，300g离心5分钟，用PBS(源培生物，B320)+2％FBS(Biosun，BS-0002-500)重悬细胞。取50μL细胞悬液加入到96孔板
U型板中，使每孔细胞数为10 5个。将梯度稀释的待测分子加入96孔板中，轻轻混匀，4℃避光孵育40分钟。PBS+2％FBS洗板2次，每孔加入100μL 20nM的hICOS-兔Fc蛋白(Sino Biological，10344-H31H)，4℃避光孵育1小时。洗板2次，每孔加入100μL羊抗兔IgG Fc FITC荧光二抗(Invitrogen，A16125，1:500稀释)，4℃避光孵育40分钟。洗板2次后每孔加100μL PBS重悬细胞，用流式细胞仪读取荧光数值。用软件拟合出抑制配体和受体结合的曲线，计算出IC 50值。实验结果见下表15。
表15.阻断ICOSL与ICOS结合的实验结果
样品名称 阻断ICOSL与ICOS结合的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
实验结果表明，本披露构建的抗ICOSL抗体及其融合蛋白对ICOSL与ICOS的结合，相较于对照，具有更强的阻断活性。
测试例4：阻断BAFF、APRIL和受体的结合实验
通过ELISA方法检测抗ICOSL抗体融合蛋白对BAFF或APRIL与其受体(BAFF/BAFF-R、BAFF/BCMA、BAFF/TACI、APRIL/BCMA、APRIL/TACI)结合的阻断活性。具体方法如下：
用pH 7.4的PBS缓冲液将受体蛋白稀释至2μg/mL，以100μL/孔的体积加入96孔酶标板中，4℃过夜孵育。弃去液体后，每孔加入200μL 1％Casein封闭液(Thermo，37528)进行封闭，37℃孵育2小时。封闭结束后，弃去封闭液，并用PBST缓冲液(pH 7.4PBS含0.1％tween-20)洗板3次后备用。将固定浓度的生物素(Biotin)标记的配体蛋白与梯度稀释的融合蛋白混合后37℃预孵育30分钟后加入封闭好的酶标板中，37℃孵育1.5小时。孵育结束后用PBST洗板3次，每孔加入100μL链霉亲和素-HRP(Invitrogen，434323，1:4000稀释)，37℃孵育1 小时。去上清，用PBST洗板3次后每孔加入100μL TMB显色底物(KPL，5120-0077)，室温孵育10至15分钟，每孔加入50μL 1M H 2SO 4终止反应，用酶标仪读取在450nm处的吸收值，用软件拟合出抑制配体和受体结合的曲线，计算出IC 50值。本测试例中所用配体蛋白的来源信息如下：BAFF(Sino biological，10056-HNCH)，APRIL(R&D Systems，5860-AP-010/CF)。所用受体蛋白的来源信息如下：BAFF-R(Sino biological，16079-H02H)，BCMA(Sino biological，10620-H02H)，TACI(ACROBiosystems，TAI-H5256)。实验结果见下表16、表17、表18：
表16.抗ICOSL抗体融合蛋白阻断APRIL与其受体结合实验结果
Figure PCTCN2022131362-appb-000056
表17.抗ICOSL抗体融合蛋白阻断BAFF与BCMA结合实验结果
样品 阻断BAFF与BCMA结合的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
表18.抗ICOSL抗体融合蛋白阻断BAFF与BAFF-R或TACI结合实验结果
Figure PCTCN2022131362-appb-000057
实验结果表明，本披露构建的抗ICOSL抗体融合蛋白能有效阻断BAFF或APRIL与其受体的结合，而阳性对照AMG-570不能有效阻断APRIL与其受体结合。
测试例5：Jurkat-ICOS-NFAT报告基因实验
通过Jurkat-ICOS-NFAT报告基因实验检测待测分子对ICOSL-ICOS信号通路 的抑制活性。实验方法如下：
将稳定表达人ICOSL的CHOK1细胞hICOSL-CHOK1重悬，按每孔40μL12500个细胞铺在96孔细胞板中。将ICOS的全长序列与CD3zeta的胞内区融合，在Jurkat细胞(ATCC，TIB-152)表面稳定表达，获得Jurkat-NFAT-ICOS-CD3zeta细胞。将Jurkat-NFAT-ICOS-CD3zeta细胞重悬后按每孔40μL 2×10 5个细胞铺在含hICOSL-CHOK1的96孔细胞板中。将待测样品梯度稀释，每孔20μL加入细胞板中，37℃培养箱中孵育48小时。取出细胞培养板，每孔加入50μL One-glo荧光素酶检测液(Promega，E6120)，用酶标仪(PerkinElmer，EnVision 2105)检测生物发光信号。将荧光值用软件拟合出抑制曲线，计算出IC 50值。实验结果见下表19。
表19.抑制Jurkat-ICOS-NFAT报告基因激活的实验结果
样品名称 抑制Jurkat-ICOS-NFAT报告基因激活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
实验结果表明，本披露构建的抗ICOSL抗体及其融合蛋白抑制Jurkat-ICOS-NFAT报告基因激活的活性比对照分子AMG-570强。
测试例6：T细胞增殖实验
通过T细胞增殖实验检测抗ICOSL抗体融合蛋白抑制ICOSL诱导T细胞增殖的活性。实验方法如下：
在96孔板(Corning，3599)中每孔加入100μL 0.5μg/mL抗人CD3抗体(Ebioscience，16-0037-85)，4℃过夜孵育，用PBS(源培生物，B320)洗涤3次，每孔加入100μL 5μg/mL ICOSL蛋白(R&D，165-B7-100)溶液，37℃孵育4小时。用人Pan T细胞分离试剂盒(Pan T Cell Isolation Kit，human，Miltenyi Biotec，130-096-535)按试剂盒说明书的方法从人外周血单核细胞(PBMC)中分离T细胞，按每孔2×10 5个细胞铺在包被好的96孔板中。将待测样品梯度稀释，加入细胞板中，37℃孵育48小时后，每孔加入50μL CTG(Cell titer Glo)检测液(Promega，G7573)，在酶标仪(PerkinElmer，Victor3)上检测生物发光信号。用软件拟合出抑制T细胞增殖的曲线，计算出IC 50值。实验结果见下表20：
表20.抑制T细胞增殖的实验结果
样品名称 抑制T细胞增殖的IC 50(nM)
201H3L3-T7 0.6146
201H3L3-T11 0.6429
259H4L7-T7 0.6007
259H4L7-T9 0.3753
259H4L7-T11 0.5890
实验结果表明，本披露构建的抗ICOSL抗体融合蛋白能有效抑制T细胞增殖。
测试例7：B细胞增殖实验
通过B细胞增殖实验检测抗ICOSL抗体融合蛋白抑制BAFF和APRIL诱导的B细胞增殖的活性。实验方法如下：
取小鼠脾脏进行研磨，4℃离心5分钟收集下层细胞，用洗涤溶液(PBS+2％FBS+2mM EDTA)清洗一次并离心，去上清后加入红细胞裂解液(RBC Lysis Buffer，Invitrogen，00-4333-57)，室温静置5分钟至红细胞完全裂解。再次离心并重悬细胞进行计数。细胞悬液用B细胞分离试剂盒(B Cell Isolation Kit，Miltenyi Biotec，130-090-862)进行分选，将分离的B细胞用RPMI 1640培养基(Gibco，11875119)+10％FBS(Gibco，10099-141)+50μM 2-巯基乙醇(Sigma-Aldrich，M6250)重悬并计数，细胞铺在96孔细胞板中备用。将BAFF(R&D Systems，7537-BF)或APRIL(R&D Systems，5860-AP)蛋白稀释至固定浓度，并加入梯度稀释的抗ICOSL抗体融合蛋白混匀，37℃预孵育30分钟后加入96孔细胞板中，37℃细胞培养箱中培养48小时。取出细胞培养板，每孔加入50μL Celltiter Glo检测液(Promega，G7573)，室温孵育10分钟，用酶标仪检测生物发光信号，将检测结果用软件拟合出抑制曲线，计算出IC 50值。实验结果见下表21、表22：
表21.抗ICOSL抗体融合蛋白抑制BAFF诱导的B细胞增殖的实验结果
样品 抑制BAFF诱导的B细胞增殖的活性(IC 50，pM)
201H3L3-T7 43.87
201H3L3-T11 24.96
259H4L7-T7 41.42
259H4L7-T9 20.33
259H4L7-T11 17.55
表22.抗ICOSL抗体融合蛋白抑制APRIL诱导的B细胞增殖的实验结果
样品 抑制APRIL诱导的B细胞增殖的活性(IC 50，pM)
201H3L3-T11 20.70
259H4L7-T9 29.60
259H4L7-T11 34.86
AMG-570 无抑制活性
实验结果表明，本披露构建的抗ICOSL抗体融合蛋白能抑制BAFF或APRIL诱导的B细胞增殖，而阳性对照分子AMG-570对APRIL没有抑制活性。
测试例8：亲和力测试
用生物传感芯片Protein A(GE，29127556)亲和捕获一定量的待测样品，然后于芯片表面流经一系列浓度梯度的抗原，利用Biacore(GE，8K)实时检测反应信号从而获得结合和解离曲线。在每个循环解离完成后，用10mM甘氨酸-盐酸溶液pH 1.5(GE，BR-1003-54)将生物芯片洗净再生。实验数据用BIAevaluation version4.1软件以1:1模型进行拟合，从而得出亲和力数值。
本测试中用到的相关抗原蛋白如下：人ICOSL(Sino biological，11559-H08H)，人BAFF(Sino biological，10056-HNCH)，人APRIL(R&D Systems，5860-AP-010/CF)，食蟹猴ICOSL(Sino biological，90800-C08H)，食蟹猴BAFF(Kactus，BAF-CM412)，食蟹猴APRIL(Kactus，APR-CM410B)，鼠ICOSL(Sino biological，50190-M08H)，鼠BAFF(Acro Biosystems，BAF-M521y)，鼠APRIL(R&D Systems，7907-AP/CF)。亲和力的测试结果见下表23至表30：
表23.抗ICOSL抗体融合蛋白与人APRIL结合的亲和力实验结果
Figure PCTCN2022131362-appb-000058
表24.抗ICOSL抗体融合蛋白与猴APRIL结合的亲和力实验结果
Figure PCTCN2022131362-appb-000059
表25.抗ICOSL抗体融合蛋白与鼠APRIL结合的亲和力实验结果
Figure PCTCN2022131362-appb-000060
表26.抗ICOSL抗体融合蛋白与人BAFF结合的亲和力实验结果
样品 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
表27.抗ICOSL抗体融合蛋白与猴BAFF结合的亲和力实验结果
样品 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
表28.抗ICOSL抗体融合蛋白与鼠BAFF结合的亲和力实验结果
样品 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
表29.抗ICOSL抗体及其融合蛋白与人ICOSL结合的亲和力实验结果
样品 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
表30.抗ICOSL抗体融合蛋白与猴ICOSL/鼠ICOSL结合的亲和力实验结果
Figure PCTCN2022131362-appb-000061
实验结果表明，本披露构建的抗ICOSL抗体融合蛋白对人、猴和鼠的APRIL均具有有很强的亲和力，而阳性对照分子AMG-570与人、猴和鼠的APRIL均不结合。本披露构建的抗ICOSL抗体融合蛋白与人、猴和鼠的BAFF的亲和力优于阳性对照分子AMG-570。本披露构建的抗ICOSL抗体及其融合蛋白与人ICOSL、和猴ICOSL均具有良好的结合活性，但对鼠ICOSL没有交叉结合活性。
测试例9：体内抑制细胞因子分泌活性评价
用人hICOSL-CHOK1细胞和人BAFF蛋白混合刺激小鼠，诱导小鼠体内产生IFNγ、IL-10和IgA等细胞因子，通过检测这些细胞因子的水平来评价抗ICOSL抗体融合蛋白的体内活性。实验过程如下：
SPF级雌性C57BL/6小鼠(浙江维通利华实验动物技术有限公司)，8周龄，将小鼠进行随机分4组，每组8只，通过腹腔混合注射hICOSL-CHOK1细胞(10 6个细胞/小鼠)和1mg/kg人BAFF蛋白(Sino biological，10056-HNCH)，每天注射一次，持续四天。各组(阳性对照(AMG-570 12mpk)组，259H4L7-T7 11.7mpk组，259H4L7-T11 12.4mpk组，阴性对照(PBS)组)分别在第一天和第三天在注射hICOSL-CHOK1细胞和BAFF蛋白前1小时腹腔注射待测样品。第五天收集各组小鼠血浆样品，分别检测IFNγ、IL-10和IgA的水平。其中AMG-570作为阳性对照，PBS作为阴性对照，其中AMG-570 12mpk(mpk也即给药剂量为mg/kg)，259H4L7-T7 11.7mpk和259H4L7-T11 12.4mpk的药物摩尔浓度相同。本测试例中所用的检测试剂盒来源：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)。实验结果如下表31及图4、图5、图6。
表31.抗ICOSL抗体融合蛋白抑制细胞因子分泌实验结果
样品 IFNγ下降百分比 IL-10下降百分比 IgA下降(％)
阴性对照(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％
备注：表中百分比为相比阴性对照，各试验组血浆中IFNγ、IL-10、IgA浓度下降的百分比。
实验结果表明，本披露构建的抗ICOSL抗体融合蛋白能显著抑制IFNγ、IL-10、IgA的分泌，抑制活性强于阳性对照分子AMG-570。
测试例10：小鼠体内免疫抑制实验
通过免疫抑制实验评估了融合蛋白在体内抑制免疫反应的活性。实验过程如下：SPF级雌性human ICOSL/human ICOS C57BL/6转基因小鼠(百奥赛图江苏基因生物技术有限公司)，小鼠体重15-18g，6至8周龄。在第1天用1mg/kg KLH (Keyhole Limpet Hemocyanin，血蓝蛋白)(Sigma，H7017)加佐剂(Thermo，77161)乳化混匀免疫小鼠，在第21天用5mg/kg KLH单独免疫小鼠。KLH是一种具有强免疫原性的蛋白，可以引起小鼠产生很强的免疫反应并产生KLH特异性的抗体。在KLH免疫前一天开始给药，每5天给药一次，总共持续35天。给药剂量为AMG-570 15mpk，259H4L7-T7 14.6mpk，259H4L7-T11 15.6mpk，各组的药物摩尔浓度相同，AMG-570作为阳性对照，PBS作为阴性对照。每周取各组小鼠的血清样品，通过检测小鼠血清中KLH特异性的IgG和IgM含量来评估融合蛋白抑制免疫反应的强度。本测试例中所用检测试剂盒如下：Mouse anti-KLH IgG ELISA Kit(Life diagnostics，KLHG-1)，Mouse anti-KLH IgM ELISA Kit(Life Diagnostics，KLHM-1)。实验结果见图7、图8。
KLH特异性IgG的检测结果表明，所有的给药组在第28天和第35天这两个时间点都能够显著的抑制KLH特异性IgG的产生，其中259H4L7-T7和259H4L7-T11组完全没有出现免疫反应，KLH特异性IgG的水平与正常小鼠相同，免疫抑制活性要强于AMG-570。KLH特异性IgM的检测结果表明，AMG-570只在第7-14天具有显著的抑制活性，而259H4L7-T7和259H4L7-T11在所有的时间点都显示出了显著的抑制活性，抑制活性要显著的强于AMG-570。
测试例11.大鼠体内药代动力学实验
用SD大鼠进行体内药代动力学测试。雄性SD大鼠(浙江维通利华实验动物技术有限公司)随机分组，每组4只，静脉注射给药，给药组于给药前及给药后5分钟、8小时、24小时、48小时、84小时、9天、10天、14天、21天、28天采集全血0.2mL，不加抗凝，取血后在4℃放置30分钟，1000g离心15分钟，取上层血清置于EP管中，于-80℃保存。用ELISA法检测血清中的血药浓度，用Winnolin软件计算受试药物的药代动力学参数和体内半衰期。
大鼠体内药代动力学实验结果见下表32，实验结果表明，本披露获得的融合蛋白具备较好的稳定性。
表32.大鼠体内药代动力学实验结果
Figure PCTCN2022131362-appb-000062

Claims (19)

  1. 一种抗ICOSL抗体融合蛋白，其包含抗ICOSL抗体和TACI多肽；
    其中所述抗ICOSL抗体包含重链可变区和轻链可变区，其中，
    所述重链可变区包含HCDR1、HCDR2和HCDR3，
    所述轻链可变区包含LCDR1、LCDR2和LCDR3，
    其中，
    (i)所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：42、3、39、40或41中的HCDR1、HCDR2和HCDR3的氨基酸序列，和
    所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：38、4、32、33、34、35、36或37中的LCDR1、LCDR2和LCDR3的氨基酸序列；或
    (ii)所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：30、1、28或29中的HCDR1、HCDR2和HCDR3的氨基酸序列，和
    所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：22、2、20、21、23、24、25、26或27中的LCDR1、LCDR2和LCDR3的氨基酸序列。
  2. 根据权利要求1所述的抗ICOSL抗体融合蛋白，其中：
    (i)所述重链可变区的HCDR1包含SEQ ID NO：11的氨基酸序列，HCDR2包含SEQ ID NO：31或12的氨基酸序列，和HCDR3包含SEQ ID NO：13的氨基酸序列，和
    所述轻链可变区的LCDR1包含SEQ ID NO：14的氨基酸序列，LCDR2包含SEQ ID NO：15的氨基酸序列，和LCDR3包含SEQ ID NO：16的氨基酸序列；或
    (ii)所述重链可变区的HCDR1包含SEQ ID NO：5的氨基酸序列，HCDR2包含SEQ ID NO：19或6的氨基酸序列，和HCDR3包含SEQ ID NO：7的氨基酸序列，和
    所述轻链可变区的LCDR1包含SEQ ID NO：8的氨基酸序列，LCDR2包含SEQ ID NO：17或9的氨基酸序列，和LCDR3包含SEQ ID NO：10或18的氨基酸序列；
    优选地，
    (i)所述重链可变区的HCDR1包含SEQ ID NO：11的氨基酸序列，HCDR2包含SEQ ID NO：31的氨基酸序列，和HCDR3包含SEQ ID NO：13的氨基酸序列，和
    所述轻链可变区的LCDR1包含SEQ ID NO：14的氨基酸序列，LCDR2包含SEQ ID NO：15的氨基酸序列，和LCDR3包含SEQ ID NO：16的氨基酸序列；或
    (ii)所述重链可变区的HCDR1包含SEQ ID NO：5的氨基酸序列，HCDR2包含SEQ ID NO：19的氨基酸序列，和HCDR3包含SEQ ID NO：7的氨基酸序列，和
    所述轻链可变区的LCDR1包含SEQ ID NO：8的氨基酸序列，LCDR2包含SEQ ID NO：17的氨基酸序列，和LCDR3包含SEQ ID NO：10的氨基酸序列。
  3. 根据权利要求1或2所述的抗ICOSL抗体融合蛋白，其中：
    (i)所述重链可变区包含与SEQ ID NO：42、39、40或41具有至少90％序列同一性的氨基酸序列，和所述轻链可变区包含与SEQ ID NO：38、32、33、34、35、36或37具有至少90％序列同一性的氨基酸序列；或
    (ii)所述重链可变区包含与SEQ ID NO：30、28或29具有至少90％序列同一性的氨基酸序列，和所述轻链可变区包含与SEQ ID NO：22、20、21、23、24、25、26或27具有至少90％序列同一性的氨基酸序列；或
    (iii)所述重链可变区包含与SEQ ID NO：3具有至少90％序列同一性的氨基酸序列，和所述轻链可变区包含与SEQ ID NO：4具有至少90％序列同一性的氨基酸序列；或
    (iv)所述重链可变区包含与SEQ ID NO：1具有至少90％序列同一性的氨基酸序列，和所述轻链可变区包含与SEQ ID NO：2具有至少90％序列同一性的氨基酸序列；
    优选地，(i)所述重链可变区包含SEQ ID NO：42的氨基酸序列，和所述轻链可变区包含SEQ ID NO：38的氨基酸序列；或
    (ii)所述重链可变区包含SEQ ID NO：30的氨基酸序列，和所述轻链可变区包含SEQ ID NO：22的氨基酸序列。
  4. 根据权利要求1至3中任一项所述的抗ICOSL抗体融合蛋白，其中所述的抗ICOSL抗体还包含抗体重链恒定区和轻链恒定区；
    优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区，所述轻链恒定区选自人抗体κ或λ链恒定区；
    更优选地，所述重链恒定区包含SEQ ID NO：43的氨基酸序列，所述轻链恒定区包含SEQ ID NO：44的氨基酸序列。
  5. 根据权利要求1至4中任一项所述的抗ICOSL抗体融合蛋白，其中，
    (i)所述抗ICOSL抗体的重链包含与SEQ ID NO：47具有至少90％序列同一性的氨基酸序列，和所述抗ICOSL抗体的轻链包含与SEQ ID NO：48具有至少90％序列同一性的氨基酸序列；或
    (ii)所述抗ICOSL抗体的重链包含与SEQ ID NO：45具有至少90％序列同 一性的氨基酸序列，和所述抗ICOSL抗体的轻链包含与SEQ ID NO：46具有至少90％序列同一性的氨基酸序列；
    优选地，
    (i)所述抗ICOSL抗体的重链包含SEQ ID NO：47的氨基酸序列，和所述抗ICOSL抗体的轻链包含SEQ ID NO：48的氨基酸序列；或
    (ii)所述抗ICOSL抗体的重链包含SEQ ID NO：45的氨基酸序列，和所述抗ICOSL抗体的轻链包含SEQ ID NO：46的氨基酸序列。
  6. 根据权利要求1至5中任一项所述的抗ICOSL抗体融合蛋白，其中所述TACI多肽为包含SEQ ID NO：58的第48位至第85位氨基酸残基的多肽或其变体；其中，所述变体包含在选自第49、52、53、57、65、82和83位中的一个或更多个位点上具有氨基酸替换，所述氨基酸替换的位点为相对于序列SEQ ID NO：58的自然顺序编号的氨基酸残基位点；
    优选地，所述变体包含选自由49T或49R、52S、53E或53Q、65T或65A、82A或82R、57E和83Y组成的组中的一个或更多个氨基酸替换，所述氨基酸替换的位点为相对于序列SEQ ID NO：58的自然顺序编号的氨基酸残基位点。
  7. 根据权利要求1至6中任一项所述的抗ICOSL抗体融合蛋白，其中所述TACI多肽的氨基酸序列如SEQ ID NO：51至83中任一所示；
    优选地，所述TACI多肽的氨基酸序列如SEQ ID NO：83所示。
  8. 根据权利要求1至7中任一项所述的抗ICOSL抗体融合蛋白，其包括：
    (A)第一链：[抗ICOSL抗体的重链]-[连接子1]-[TACI多肽1]，和
    第二链：[TACI多肽2]-[连接子2]-[抗ICOSL抗体的轻链]；或
    (B)第一链：[抗ICOSL抗体的重链]-[连接子1]-[TACI多肽1]，和
    第二链：抗ICOSL抗体的轻链；或
    (C)第一链：[抗ICOSL抗体的重链]-[连接子1]-[TACI多肽1]，和
    第二链：[抗ICOSL抗体的轻链]-[连接子2]-[TACI多肽2]；
    其中，所述(A)、(B)或(C)中，其中所述TACI多肽1和TACI多肽2是相同或不相同的，所述连接子1和连接子2是相同或不相同的；
    优选地，所述连接子1和连接子2各自独立地具有(G xS) y的结构，其中，x选自1-5的整数，y选自0-6的整数，
    更优选地，所述连接子1和连接子2各自独立地如SEQ ID NO：89或90所示；
    任选地，在(A)、(B)或(C)中，在第一链的C端还包括保护氨基酸；
    任选地，在(C)中，在第二链的C端还包括保护氨基酸；
    优选地，所述保护氨基酸是QK。
  9. 根据权利要求1至8中任一项所述的抗ICOSL抗体融合蛋白，其中，
    所述的抗ICOSL抗体融合蛋白具有：包含SEQ ID NO：86的氨基酸序列的第一链，和包含SEQ ID NO：88、87或48的氨基酸序列的第二链；或
    所述的抗ICOSL抗体融合蛋白具有：包含SEQ ID NO：84的氨基酸序列的第一链，和包含SEQ ID NO：85或46的氨基酸序列的第二链；
    优选地，所述的抗ICOSL抗体融合蛋白具有2条包含SEQ ID NO：86的氨基酸序列的第一链，和2条包含SEQ ID NO：88的氨基酸序列的第二链。
  10. 一种抗ICOSL抗体，其包含重链可变区和轻链可变区，所述重链可变区包含HCDR1、HCDR2和HCDR3，所述轻链可变区包含LCDR1、LCDR2和LCDR3，其中，
    (i)所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：42、3、39、40或41中的HCDR1、HCDR2和HCDR3的氨基酸序列，和
    所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：38、4、32、33、34、35、36或37中的LCDR1、LCDR2和LCDR3的氨基酸序列；或
    (ii)所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：30、1、28或29中的HCDR1、HCDR2和HCDR3的氨基酸序列，和
    所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：22、2、20、21、23、24、25、26或27中的LCDR1、LCDR2和LCDR3的氨基酸序列。
  11. 根据权利要求10所述的抗ICOSL抗体，其中：
    (i)所述重链可变区的HCDR1包含SEQ ID NO：11的氨基酸序列，HCDR2包含SEQ ID NO：31或12的氨基酸序列，和HCDR3包含SEQ ID NO：13的氨基酸序列，和
    所述轻链可变区的LCDR1包含SEQ ID NO：14的氨基酸序列，LCDR2包含SEQ ID NO：15的氨基酸序列，和LCDR3包含SEQ ID NO：16的氨基酸序列；或
    (ii)所述重链可变区的HCDR1包含SEQ ID NO：5的氨基酸序列，HCDR2包含SEQ ID NO：19或6的氨基酸序列，和HCDR3包含SEQ ID NO：7的氨基酸序列，和
    所述轻链可变区的LCDR1包含SEQ ID NO：8的氨基酸序列，LCDR2包含SEQ ID NO：17或9的氨基酸序列，和LCDR3包含SEQ ID NO：10或18的氨基酸序列；
    优选地，
    (i)所述重链可变区的HCDR1包含SEQ ID NO：11的氨基酸序列，HCDR2 包含SEQ ID NO：31的氨基酸序列，和HCDR3包含SEQ ID NO：13的氨基酸序列，和
    所述轻链可变区的LCDR1包含SEQ ID NO：14的氨基酸序列，LCDR2包含SEQ ID NO：15的氨基酸序列，和LCDR3包含SEQ ID NO：16的氨基酸序列；或
    (ii)所述重链可变区的HCDR1包含SEQ ID NO：5的氨基酸序列，HCDR2包含SEQ ID NO：19的氨基酸序列，和HCDR3包含SEQ ID NO：7的氨基酸序列，和
    所述轻链可变区的LCDR1包含SEQ ID NO：8的氨基酸序列，LCDR2包含SEQ ID NO：17的氨基酸序列，和LCDR3包含SEQ ID NO：10的氨基酸序列。
  12. 根据权利要求10或11所述的抗ICOSL抗体，其中：
    (i)所述重链可变区包含与SEQ ID NO：42、39、40或41具有至少90％序列同一性的氨基酸序列，和所述轻链可变区包含与SEQ ID NO：38、32、33、34、35、36或37具有至少90％序列同一性的氨基酸序列；或
    (ii)所述重链可变区包含与SEQ ID NO：30、28或29具有至少90％序列同一性的氨基酸序列，和所述轻链可变区包含与SEQ ID NO：22、20、21、23、24、25、26或27具有至少90％序列同一性的氨基酸序列；或
    (iii)所述重链可变区包含与SEQ ID NO：3具有至少90％序列同一性的氨基酸序列，和所述轻链可变区包含与SEQ ID NO：4具有至少90％序列同一性的氨基酸序列；或
    (iv)所述重链可变区包含与SEQ ID NO：1具有至少90％序列同一性的氨基酸序列，和所述轻链可变区包含与SEQ ID NO：2具有至少90％序列同一性的氨基酸序列；
    优选地，(i)所述重链可变区包含SEQ ID NO：42的氨基酸序列，和所述轻链可变区包含SEQ ID NO：38的氨基酸序列；或
    (ii)所述重链可变区包含SEQ ID NO：30的氨基酸序列，和所述轻链可变区包含SEQ ID NO：22的氨基酸序列。
  13. 根据权利要求10至12中任一项所述的抗ICOSL抗体，其中所述的抗ICOSL抗体还包含抗体重链恒定区和轻链恒定区；
    优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区，所述轻链恒定区选自人抗体κ或λ链恒定区；
    更优选地，所述重链恒定区包含SEQ ID NO：43的氨基酸序列，所述轻链恒定区包含SEQ ID NO：44的氨基酸序列。
  14. 根据权利要求10至13中任一项所述的抗ICOSL抗体，其中，
    (i)所述抗ICOSL抗体的重链包含与SEQ ID NO：47具有至少90％序列同一性的氨基酸序列，和所述抗ICOSL抗体的轻链包含与SEQ ID NO：48具有至少90％序列同一性的氨基酸序列；或
    (ii)所述抗ICOSL抗体的重链包含与SEQ ID NO：45具有至少90％序列同一性的氨基酸序列，和所述抗ICOSL抗体的轻链包含与SEQ ID NO：46具有至少90％序列同一性的氨基酸序列；
    优选地，
    (i)所述抗ICOSL抗体的重链包含SEQ ID NO：47的氨基酸序列，和所述抗ICOSL抗体的轻链包含SEQ ID NO：48的氨基酸序列；或
    (ii)所述抗ICOSL抗体的重链包含SEQ ID NO：45的氨基酸序列，和所述抗ICOSL抗体的轻链包含SEQ ID NO：46的氨基酸序列。
  15. 根据权利要求1至9中任一项所述的抗ICOSL抗体融合蛋白或权利要求10至14中任一项所述的抗ICOSL抗体，其具有一种或更多种以下特性：
    A.与人ICOSL和食蟹猴ICOSL特异性结合，不与鼠ICOSL特异性结合；优选地，以小于5.00E-09M的KD值与人ICOSL结合，和/或以小于8.00E-09M的KD值与食蟹猴ICOSL结合，所述KD值通过表面等离子体共振测定法测量；
    B.与人APRIL、食蟹猴APRIL和/或鼠APRIL特异性结合；优选地，以小于2.00E-11M的KD值与人APRIL结合，以小于2.00E-10M的KD值与食蟹猴APRIL结合，和/或以小于2.00E-11M的KD值与鼠APRIL结合，所述KD值通过表面等离子体共振测定法所测量；
    C.与人BAFF、食蟹猴BAFF和/或鼠BAFF特异性结合；优选地，以小于7.00E-11M的KD值与人BAFF结合，以小于4.00E-10M的KD值与食蟹猴BAFF结合，和/或以小于6.00E-11M的KD值与鼠BAFF结合，所述KD值通过表面等离子体共振测定法所测量；
    D.具有阻断ICOSL与ICOS结合的活性；优选地，阻断人ICOSL与ICOS结合的IC 50值小于0.16nM，所述IC 50值通过FACS方法检测；
    E.具有阻断APRIL与BCMA结合的活性；优选地，阻断APRIL与BCMA结合的IC 50值小于40.00nM，所述IC 50值通过ELISA方法检测；
    F.具有阻断APRIL与TACI结合的活性；优选地，阻断APRIL与TACI结合的IC 50值小于40.00nM，所述IC 50值通过ELISA方法检测；
    G.具有阻断BAFF与BCMA结合的活性；优选地，阻断BAFF与BCMA结合的IC 50值小于2.00nM，所述IC 50值通过ELISA方法检测；
    H.具有阻断BAFF与BAFF-R结合的活性；优选地，阻断BAFF与BAFF-R结合的IC 50值小于2.00nM，所述IC 50值通过ELISA方法检测；
    I.具有阻断BAFF与TACI结合的活性；优选地，阻断BAFF与TACI结合的IC 50值小于1.00nM，所述IC 50值通过ELISA方法检测；
    J.抑制细胞因子的分泌，优选地，细胞因子选自以下任一项或其组合：IFNγ、IL-10和/或IgA；
    K.抑制B细胞增殖和/或T细胞增殖。
  16. 一种药物组合物，其包含：
    权利要求1至9或15中任一项所述的抗ICOSL抗体融合蛋白或权利要求10至15中任一项所述的抗ICOSL抗体，以及
    一种或多种药学上可接受的载体、稀释剂或赋形剂。
  17. 核酸分子，其编码权利要求1至9或15中任一项所述的抗ICOSL抗体融合蛋白或权利要求10至15中任一项所述的抗ICOSL抗体。
  18. 一种宿主细胞，其含有权利要求17所述的核酸分子。
  19. 一种治疗自身免疫性疾病、炎性疾病、B细胞障碍或T细胞障碍的方法，所述方法包括向有需要的受试者施用治疗有效量的权利要求1至9或15中任一项所述的抗ICOSL抗体融合蛋白或权利要求10至15中任一项所述的抗ICOSL抗体或权利要求16所述药物组合物的步骤；
    优选地，所述自身免疫性疾病或炎性疾病选自：系统性红斑狼疮、类风湿性关节炎、移植物抗宿主病、哮喘、免疫性血小板减少性紫癜、多发性硬化、糖尿病引起的炎性疾病、银屑病、炎性肠病、克罗恩病、溃疡性结肠炎、格雷夫斯病和桥本氏甲状腺炎；所述B细胞障碍或T细胞障碍为肿瘤；
    更优选地，所述肿瘤选自：头颈癌、非小细胞肺癌、尿路上皮癌、白血病、肉瘤、黑色素瘤、腺癌、结直肠癌、前列腺肿瘤、乳腺癌和小细胞肺癌；所述自身免疫性疾病为系统性红斑狼疮。
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