TW202334225A

TW202334225A - Anti-icosl antibody fusion protein and the use thereof

Info

Publication number: TW202334225A
Application number: TW111143243A
Authority: TW
Inventors: 毛浪勇; 應華; 李玲玲; 黃旋; 金薪盛; 陶維康
Original assignee: 大陸商江蘇恆瑞醫藥股份有限公司; 大陸商上海恆瑞醫藥有限公司
Priority date: 2021-11-11
Filing date: 2022-11-11
Publication date: 2023-09-01
Also published as: CN118176216A; WO2023083298A1

Abstract

The present disclosure relates to anti-ICOSL antibody fusion protein and the use thereof. In particular, the present disclosure relates to anti-ICOSL antibody, fusion protein and the use thereof.

Description

抗ICOSL抗體融合蛋白及用途 Anti-ICOSL antibody fusion protein and its uses

本申請要求2021年11月11日提交的專利申請(申請號CN202111330908.5)的優先權。 This application claims priority from the patent application (application number CN202111330908.5) submitted on November 11, 2021.

本揭露屬於生物技術領域，更具體地，本揭露關於一種抗ICOSL抗體及其融合蛋白和應用。 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.

這裡的陳述僅是提供與本揭露有關的背景資訊，而不必然地構成現有技術。 The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

系統性紅斑狼瘡(systemic lupus erythematosus，SLE)是一種影響多種器官的自身免疫性疾病。在遺傳和環境等因素的作用下，導致人體失去免疫耐受，產生自身抗體和核苷酸免疫複合物進入血液循環系統，免疫複合物啟動TLR受體，刺激pDC細胞分泌大量的干擾素α。干擾素進一步刺激髓系細胞分泌多種細胞因子，分別促進B細胞和T細胞的啟動(Curr Opin Rheumatol.2017 Mar；29(2)：178-186.)。已知BAFF和APRIL是兩種B細胞的啟動因子，它們藉由與B細胞表面的受體作用促進B細胞的分化成熟和產生抗體(Nat Rev Rheumatol.2016 Nov 22；12(12)：716-730.)。而ICOSL和ICOS的信號通路在T細胞啟動和T-B細胞相互作用的過程中發揮了非常重要的作用。T細胞啟動後釋放促炎細胞因子導致促炎T細胞的分化並破壞Treg細胞在體內的平衡。此外，T-B細胞之間的相互作用會進一步促進T細胞和B細胞的活化並產生促炎細胞因子進而形成持續的免疫反應迴圈(Nat Rev Rheumatol.2014 Jan；10(1)：23-34.)。在SLE的發病過程中，T細胞和B細胞這2條免疫途徑都參與其中，同時抑制T細胞和B細胞啟動以及T-B細胞相互作用的信號通路，對SLE會有比較好的作用效果。其它研發還發現，ICOS/ICOSL信號途徑在其它機體炎症反應、過敏反應、自身免疫性疾病、移植排斥等免疫應答以及B細胞或T細胞障礙(例如癌症)發生過程中也發揮了重要的作用。 Systemic lupus erythematosus (SLE) is an autoimmune disease that affects multiple organs. Under the influence of genetic and environmental factors, the human body loses immune tolerance and produces autoantibodies and nucleotide immune complexes that enter the blood circulation system. The immune complexes activate TLR receptors and stimulate pDC cells to secrete a large amount of interferon α. Interferon further stimulates myeloid cells to secrete a variety of cytokines, promoting the initiation of B cells and T cells respectively (Curr Opin Rheumatol. 2017 Mar; 29(2): 178-186.). BAFF and APRIL are known to be two B cell initiating factors. They 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. 2016 Nov 22;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 cells are activated leads to the differentiation of pro-inflammatory T cells and destroys the balance of Treg cells in the body. In addition, the interaction between T-B cells will further promote the activation of T cells and B cells and produce pro-inflammatory cytokines to form a sustained immune response cycle (Nat Rev Rheumatol. 2014 Jan; 10(1): 23-34. ). In the pathogenesis of SLE, both immune pathways, T cells and B cells, are involved. At the same time, inhibiting the signaling pathways of T cell and B cell activation and T-B cell interaction will have a better effect on SLE. Other research and development has also found that the ICOS/ICOSL signaling pathway also plays an important role in the occurrence of 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，也稱為B7RP1、B7H2、CD275)，是屬於B7家族的一個免疫共刺激分子。ICOSL主要在抗原遞呈細胞表面表達，包括B細胞，巨噬細胞，單核細胞和樹突狀細胞(BioDrugs.2013 Feb；27(1)：1-13.)。目前已知ICOSL的受體是ICOS，ICOS主要在T細胞表面表達，ICOSL和ICOS介導的信號通路主要在T細胞的分化和T細胞依賴的B細胞活化中發揮作用(Nat Rev Cancer.2012 Mar 22；12(4)：252-64.)。ICOSL-ICOS信號通路的啟動可以分別促進Th2、Th1、Th17和Tfh細胞分泌表達IL-4、IFNγ、IL-23R和IL-21，這些細胞因子和受體參與維持多種T細胞亞型的活性並且促進他們進一步擴增(Curr Opin Immunol.2010 Jun；22(3)：326-32.)。此外，ICOSL在Tfh細胞遷移和T-B細胞相互作用的過程中也發揮了非常重要的功能。 ICOSL (Inducible costimulator-ligand, also known as B7RP1, B7H2, CD275) is an immune costimulatory 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.). It is currently known that 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 T cell-dependent B cell activation (Nat Rev Cancer. 2012 Mar 22;12(4):252-64.). The initiation of the ICOSL-ICOS signaling pathway can promote the secretion and expression of IL-4, IFNγ, IL-23R and IL-21 by Th2, Th1, Th17 and Tfh cells respectively. These cytokines and receptors are involved in maintaining the activity of multiple T cell subtypes and Promote their further expansion (Curr Opin Immunol. 2010 Jun; 22(3): 326-32.). In addition, 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，也稱為TNFRSF13B)是一種膜結合受體，具有包含二個富含半胱胺酸的假性重複片斷 (cysteine-rich pseudo-repeats)的胞外區、一個跨膜區和與CAML(鈣調節劑和親環蛋白配體)相互作用的胞質區。TACI與B細胞和T細胞的一種亞型相關。TACI受體與腫瘤壞死因子配體家族的BAFF(B-cell activating factor，也稱為TNFSF13B)相結合。BAFF是屬於TNF家族的一種B細胞啟動因子。BAFF主要在骨髓細胞膜表面表達，以三聚體的形式存在。在細胞膜表面的BAFF會被蛋白酶水解形成可溶性的BAFF進入血液循環系統，可溶性的BAFF具有多聚化的特徵，最多可以形成60聚體。另外BAFF也可以跟同家族的另外一個蛋白APRIL作用形成異源的三聚體。目前已知在B細胞表面有三個BAFF的受體，分別為BAFF-R、BCMA和TACI。BAFF與這三個受體作用，參與B細胞的分化成熟、存活和調節。APRIL與BAFF有兩個共同的受體，分別為BCMA和TACI。APRIL與這兩個受體作用參與B細胞的存活和調節(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對維持B細胞的體內平衡非常重要，BAFF信號通路的過度啟動會導致自反應B細胞的存活並產生自身抗體促進自身免疫反應(Cancro,M.P.,D.P.D'Cruz,and M.A.Khamashta,J Clin Invest,2009.119：p.1066-73)。 TACI (Transmembrane activator and CAML interactor, also known as TNFRSF13B) is a membrane-bound receptor containing two cysteine-rich pseudorepeat segments (cysteine-rich pseudo-repeats) extracellular domain, a transmembrane domain and a cytoplasmic domain that interacts with CAML (calcium modulator and cyclophilin ligand). TACI is associated with a subtype 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 priming factor belonging to the TNF family. BAFF is mainly expressed on the surface of bone marrow cell membranes and exists in the form of trimers. BAFF on the cell membrane surface will be hydrolyzed by proteases to form soluble BAFF, which enters the blood circulation system. Soluble BAFF has multimerization characteristics and can form up to 60-mers. In addition, BAFF can also interact with another protein in the same family, APRIL, to form a heterologous trimer. It is currently known that there are three 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 share 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 very important for maintaining B cell homeostasis. Excessive activation of the BAFF signaling pathway will lead 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).

本揭露構建了一種抗ICOSL抗體融合蛋白，其包含抗ICOSL抗體和TACI多肽。 The present disclosure constructs an anti-ICOSL antibody fusion protein, which contains an anti-ICOSL antibody and a TACI polypeptide.

在一些實施方案中，該抗ICOSL抗體融合蛋白，其中該抗ICOSL抗體包含重鏈可變區和輕鏈可變區，其中，該重鏈可變區包含HCDR1、HCDR2和HCDR3，該輕鏈可變區包含LCDR1、LCDR2和LCDR3，其中， In some embodiments, 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, and the light chain may The variable area includes LCDR1, LCDR2 and LCDR3, where,

(i)該重鏈可變區的HCDR1、HCDR2和HCDR3分別包含SEQ ID NO：42、3、39、40或41中的HCDR1、HCDR2和HCDR3的胺基酸序列，和該輕鏈可變區的LCDR1、LCDR2和LCDR3分別包含SEQ ID NO：38、4、32、33、34、35、36或37中的LCDR1、LCDR2和LCDR3的胺基酸序列；或 (i) HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 42, 3, 39, 40 or 41, and the light chain variable region LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequence of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 38, 4, 32, 33, 34, 35, 36 or 37; or

(ii)該重鏈可變區的HCDR1、HCDR2和HCDR3分別包含SEQ ID NO：30、1、28或29中的HCDR1、HCDR2和HCDR3的胺基酸序列，和該輕鏈可變區的LCDR1、LCDR2和LCDR3分別包含SEQ ID NO：22、2、20、21、23、24、25、26或27中的LCDR1、LCDR2和LCDR3的胺基酸序列。 (ii) HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 30, 1, 28 or 29, and LCDR1 of the light chain variable region , LCDR2 and LCDR3 respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 22, 2, 20, 21, 23, 24, 25, 26 or 27.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中該重鏈可變區的HCDR1、HCDR2和HCDR3以及輕鏈可變區的LCDR1、LCDR2和LCDR3是根據選自Kabat、IMGT、Chothia、AbM和Contact的編號規則定義的。在一些實施方案中，該重鏈可變區的HCDR1、HCDR2和HCDR3以及輕鏈可變區的LCDR1、LCDR2和LCDR3是根據Kabat編號規則定義的。在一些實施方案中，該重鏈可變區的HCDR1、HCDR2和HCDR3以及輕鏈可變區的LCDR1、LCDR2和LCDR3是根據IMGT編號規則定義的。在一些實施方案中，該重鏈可變區的HCDR1、HCDR2和HCDR3以及輕鏈可變區的LCDR1、LCDR2和LCDR3是根據Chothia編號規則定義的。在一些實施方案中，該重鏈可變區的HCDR1、HCDR2和HCDR3以及輕鏈可變區的LCDR1、LCDR2和LCDR3是根據AbM編號規則定義的。在一些實施方案中，該重鏈可變區的 HCDR1、HCDR2和HCDR3以及輕鏈可變區的LCDR1、LCDR2和LCDR3是根據Contact編號規則定義的。 In some embodiments, the anti-ICOSL antibody fusion protein as described in 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, The numbering rules of IMGT, Chothia, AbM and Contact are defined. In some embodiments, 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 rule. In some embodiments, 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 IMGT numbering convention. In some embodiments, 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 rule. In some embodiments, 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 heavy chain variable region HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to the Contact numbering rule.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中， In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, wherein,

(i)該重鏈可變區的HCDR1包含SEQ ID NO：11的胺基酸序列，HCDR2包含SEQ ID NO：31或12的胺基酸序列，和HCDR3包含SEQ ID NO：13的胺基酸序列，和該輕鏈可變區的LCDR1包含SEQ ID NO：14的胺基酸序列，LCDR2包含SEQ ID NO：15的胺基酸序列，和LCDR3包含SEQ ID NO：16的胺基酸序列；或 (i) HCDR1 of the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 11, HCDR2 includes the amino acid sequence of SEQ ID NO: 31 or 12, and HCDR3 includes the amino acid sequence of SEQ ID NO: 13 sequence, and LCDR1 of the light chain variable region includes the amino acid sequence of SEQ ID NO: 14, LCDR2 includes the amino acid sequence of SEQ ID NO: 15, and LCDR3 includes the amino acid sequence of SEQ ID NO: 16; or

(ii)該重鏈可變區的HCDR1包含SEQ ID NO：5的胺基酸序列，HCDR2包含SEQ ID NO：19或6的胺基酸序列，和HCDR3包含SEQ ID NO：7的胺基酸序列，和該輕鏈可變區的LCDR1包含SEQ ID NO：8的胺基酸序列，LCDR2包含SEQ ID NO：17或9的胺基酸序列，和LCDR3包含SEQ ID NO：10或18的胺基酸序列。 (ii) HCDR1 of the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 5, HCDR2 includes the amino acid sequence of SEQ ID NO: 19 or 6, and HCDR3 includes the amino acid sequence of SEQ ID NO: 7 sequence, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8, LCDR2 comprises the amino acid sequence of SEQ ID NO: 17 or 9, and LCDR3 comprises the amine of SEQ ID NO: 10 or 18 amino acid sequence.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, wherein

(i)該重鏈可變區的HCDR1包含SEQ ID NO：11的胺基酸序列，HCDR2包含SEQ ID NO：31的胺基酸序列，和HCDR3包含SEQ ID NO：13的胺基酸序列，和該輕鏈可變區的LCDR1包含SEQ ID NO：14的胺基酸序列，LCDR2包含SEQ ID NO：15的胺基酸序列，和LCDR3包含SEQ ID NO：16的胺基酸序列；或 (i) HCDR1 of the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 11, HCDR2 includes the amino acid sequence of SEQ ID NO: 31, and HCDR3 includes the amino acid sequence of SEQ ID NO: 13, and LCDR1 of the light chain variable region includes the amino acid sequence of SEQ ID NO: 14, LCDR2 includes the amino acid sequence of SEQ ID NO: 15, and LCDR3 includes the amino acid sequence of SEQ ID NO: 16; or

(ii)該重鏈可變區的HCDR1包含SEQ ID NO：5的胺基酸序列，HCDR2包含SEQ ID NO：19的胺基酸序列，和HCDR3包含SEQ ID NO：7的胺基酸序列，和該輕鏈可變區的LCDR1包含SEQ ID NO：8的胺基酸序列，LCDR2包含SEQ ID NO：17的胺基酸序列，和LCDR3包含SEQ ID NO：10的胺基酸序列。 (ii) HCDR1 of the heavy chain variable region includes the amino acid sequence of SEQ ID NO:5, HCDR2 includes the amino acid sequence of SEQ ID NO:19, and HCDR3 includes the amino acid sequence of SEQ ID NO:7, and LCDR1 of the light chain variable region includes the amino acid sequence of SEQ ID NO:8, LCDR2 includes the amino acid sequence of SEQ ID NO:17, and LCDR3 includes the amino acid sequence of SEQ ID NO:10.

在一些實施方案中，如上任一項所述的抗ICQSL抗體融合蛋白，其中 In some embodiments, the anti-ICQSL antibody fusion protein as described in any one of the above, wherein

該重鏈可變區的HCDR1包含SEQ ID NO：11的胺基酸序列，HCDR2包含SEQ ID NO：12的胺基酸序列，和HCDR3包含SEQ ID NO：13的胺基酸序列，和該輕鏈可變區的LCDR1包含SEQ ID NO：14的胺基酸序列，LCDR2包含SEQ ID NO：15的胺基酸序列，和LCDR3包含SEQ ID NO：16的胺基酸序列。 HCDR1 of the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 11, HCDR2 includes the amino acid sequence of SEQ ID NO: 12, and HCDR3 includes the amino acid sequence of SEQ ID NO: 13, and the light chain variable region LCDR1 of the chain variable region includes the amino acid sequence of SEQ ID NO:14, LCDR2 includes the amino acid sequence of SEQ ID NO:15, and LCDR3 includes the amino acid sequence of SEQ ID NO:16.

該重鏈可變區的HCDR1包含SEQ ID NO：5的胺基酸序列，HCDR2包含SEQ ID NO：6的胺基酸序列，和HCDR3包含SEQ ID NO：7的胺基酸序列，和該輕鏈可變區的LCDR1包含SEQ ID NO：8的胺基酸序列，LCDR2包含SEQ ID NO：9的胺基酸序列，和LCDR3包含SEQ ID NO：10的胺基酸序列；或 HCDR1 of the heavy chain variable region includes the amino acid sequence of SEQ ID NO:5, HCDR2 includes the amino acid sequence of SEQ ID NO:6, and HCDR3 includes the amino acid sequence of SEQ ID NO:7, and the light chain variable region LCDR1 of the chain variable region includes the amino acid sequence of SEQ ID NO: 8, LCDR2 includes the amino acid sequence of SEQ ID NO: 9, and LCDR3 includes the amino acid sequence of SEQ ID NO: 10; or

該重鏈可變區的HCDR1包含SEQ ID NO：5的胺基酸序列，HCDR2包含SEQ ID NO：6的胺基酸序列，和HCDR3包含SEQ ID NO：7的胺基酸序列，和該輕鏈可變區的LCDR1包含SEQ ID NO：8的胺基酸序列，LCDR2包含SEQ ID NO：17的胺基酸序列，和LCDR3包含SEQ ID NO：10的胺基酸序列；或 HCDR1 of the heavy chain variable region includes the amino acid sequence of SEQ ID NO:5, HCDR2 includes the amino acid sequence of SEQ ID NO:6, and HCDR3 includes the amino acid sequence of SEQ ID NO:7, and the light chain variable region LCDR1 of the chain variable region includes the amino acid sequence of SEQ ID NO: 8, LCDR2 includes the amino acid sequence of SEQ ID NO: 17, and LCDR3 includes the amino acid sequence of SEQ ID NO: 10; or

該重鏈可變區的HCDR1包含SEQ ID NO：5的胺基酸序列，HCDR2包含SEQ ID NO：19的胺基酸序列，和HCDR3包含SEQ ID NO：7的胺基酸序列，和該輕鏈可變區的LCDR1包含SEQ ID NO：8的胺基酸序列，LCDR2包含SEQ ID NO：9的胺基酸序列，和LCDR3包含SEQ ID NO：10的胺基酸序列。 HCDR1 of the heavy chain variable region includes the amino acid sequence of SEQ ID NO:5, HCDR2 includes the amino acid sequence of SEQ ID NO:19, and HCDR3 includes the amino acid sequence of SEQ ID NO:7, and LCDR1 of the light chain variable region includes the amino acid sequence of SEQ ID NO:8, LCDR2 includes the amino acid sequence of SEQ ID NO:9, and LCDR3 includes the amino acid sequence of SEQ ID NO:10.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，該抗ICOSL抗體為鼠源抗體、嵌合抗體或人源化抗體。 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, the anti-ICOSL antibody is a murine antibody, a chimeric antibody or a humanized antibody.

在一些實施方案中，該抗體是人源化抗體。 In some embodiments, the antibody is a humanized antibody.

(i)該重鏈可變區包含與SEQ ID NO：42、39、40或41具有至少90%(例如至少90%、95%、96%、97%、98%或99%)序列同一性的胺基酸序列，和該輕鏈可變區包含與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%)序列同一性的胺基酸序列；或 (i) 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 an amino acid sequence, and the 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 the amino acid sequence; or

(ii)該重鏈可變區包含與SEQ ID NO：30、28或29具有至少90%(例如至少90%、95%、96%、97%、98%或99%)序列同一性的胺基酸序列，和該輕鏈可變區包含與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%)序列同一性的胺基酸序列；或 (ii) The heavy chain variable region comprises an amine having 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 the 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 of the amino acid sequence; or

(iii)該重鏈可變區包含與SEQ ID NO：3具有至少90%(例如至少90%、95%、96%、97%、98%或99%)序列同一性的胺基酸序列，和該輕鏈可變區包含與SEQ ID NO：4具有至少90%(例如至少90%、95%、96%、97%、98%或99%)序列同一性的胺基酸序列；或 (iii) the 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: 3, and the light chain variable region comprises an amino acid sequence that has at least 90% (eg, at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 4; or

(iv)該重鏈可變區包含與SEQ ID NO：1具有至少90%(例如至少90%、95%、96%、97%、98%或99%)序列同一性的胺基酸序列，和該輕鏈可變區包含與SEQ ID NO：2具有至少90%(例如至少90%、95%、96%、97%、98%或99%)序列同一性的胺基酸序列。 (iv) the 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, and 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.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，該抗ICOSL抗體的重鏈可變區的框架區上包含選自第37、43和49位元(根據Kabat編號系統編號)中的一個或更多個胺基酸突變，和/或輕鏈可變區的框架區上包含選自第1、2、3、42、43、60和85位元(根據Kabat編號系統編號)中的一個或更多個胺基酸突變。 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, the framework region of the heavy chain variable region of the anti-ICOSL antibody includes positions selected from the group consisting of positions 37, 43 and 49 (numbered according to the Kabat numbering system ), and/or the framework region of the light chain variable region contains a position selected from positions 1, 2, 3, 42, 43, 60 and 85 (numbered according to the Kabat numbering system ) one or more amino acid mutations.

在一些實施方案中，該抗ICOSL抗體融合蛋白，其中該重鏈可變區的HCDR1包含SEQ ID NO：11的胺基酸序列，HCDR2包含SEQ ID NO：31或12的胺基酸序列，和HCDR3包含SEQ ID NO：13的胺基酸序列，和該輕鏈可變區的LCDR1包含SEQ ID NO：14的胺基酸序列，LCDR2包含SEQ ID NO：15的胺基酸序列，和LCDR3包含SEQ ID NO：16的胺基酸序列；該抗體的重鏈可變區的框架區上包含選自：37I、43E和49A(根據Kabat編號系統編號)中的一個或更多個胺基酸突變，和/或該輕鏈可變區的框架區上包含選自：1N、2T、3V、42Q、43S、60D、85V(根據Kabat編號系統編號)中的一個或更多個胺基酸突變。 In some embodiments, the anti-ICOSL antibody fusion protein, wherein HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11, HCDR2 comprises the amino acid sequence of SEQ ID NO: 31 or 12, and HCDR3 includes the amino acid sequence of SEQ ID NO: 13, and LCDR1 of the light chain variable region includes the amino acid sequence of SEQ ID NO: 14, LCDR2 includes the amino acid sequence of SEQ ID NO: 15, and LCDR3 includes The amino acid sequence of SEQ ID NO: 16; the framework region of the heavy chain variable region of the antibody contains one or more amino acid mutations selected from: 37I, 43E and 49A (numbered according to the Kabat numbering system) , and/or the framework region of the light chain variable region contains one or more amino acid mutations selected from: 1N, 2T, 3V, 42Q, 43S, 60D, 85V (numbered according to Kabat numbering system).

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，該抗ICOSL抗體的重鏈可變區的框架區上包含選自第1、24、69、71、73、78位元(根據Kabat編號系統編號)中的一個或更多個胺基酸突變，和/或輕鏈可變區的框架區上包含選自第1、2和4位元(根據Kabat編號系統編號)中的一個或更多個胺基酸突變。 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, the framework region of the heavy chain variable region of the anti-ICOSL antibody includes positions selected from positions 1, 24, 69, 71, 73, and 78 one or more amino acid mutations in (numbered according to the Kabat numbering system), and/or light chain variability The framework region of the region contains one or more amino acid mutations selected from positions 1, 2 and 4 (numbered according to the Kabat numbering system).

在一些實施方案中，該抗ICOSL抗體融合蛋白，其中抗ICOSL抗體的重鏈可變區的HCDR1包含SEQ ID NO：5的胺基酸序列，HCDR2包含SEQ ID NO：19或6的胺基酸序列，和HCDR3包含SEQ ID NO：7的胺基酸序列，和該輕鏈可變區的LCDR1包含SEQ ID NO：8的胺基酸序列，LCDR2包含SEQ ID NO：17或9的胺基酸序列，和LCDR3包含SEQ ID NO：10或18的胺基酸序列；該抗體的重鏈可變區的框架區上包含選自：1E、24T、69L、71V、73K和78A(根據Kabat編號系統編號)中的一個或更多個胺基酸突變，和/或該輕鏈可變區的框架區上包含選自：1A、2V和4L(根據Kabat編號系統編號)中的一個或更多個胺基酸突變。 In some embodiments, 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 sequence, and HCDR3 includes the amino acid sequence of SEQ ID NO: 7, and LCDR1 of the light chain variable region includes the amino acid sequence of SEQ ID NO: 8, and LCDR2 includes the amino acid sequence of SEQ ID NO: 17 or 9 sequence, and LCDR3 includes the amino acid sequence of SEQ ID NO: 10 or 18; the heavy chain variable region of the antibody includes a framework region selected from: 1E, 24T, 69L, 71V, 73K and 78A (according to the Kabat numbering system one or more amino acid mutations in the numbering), and/or the framework region of the light chain variable region contains one or more selected from: 1A, 2V and 4L (numbering according to the Kabat numbering system) Amino acid mutations.

(i)該重鏈可變區包含SEQ ID NO：42、39、40或41的胺基酸序列，和該輕鏈可變區包含SEQ ID NO：38、32、33、34、35、36或37的胺基酸序列；或 (i) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 42, 39, 40 or 41, and the light chain variable region comprises SEQ ID NO: 38, 32, 33, 34, 35, 36 or an amino acid sequence of 37; or

(ii)該重鏈可變區包含SEQ ID NO：30、28或29的胺基酸序列，和該輕鏈可變區包含SEQ ID NO：22、20、21、23、24、25、26或27的胺基酸序列；或 (ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 30, 28 or 29, and the light chain variable region comprises SEQ ID NO: 22, 20, 21, 23, 24, 25, 26 or an amino acid sequence of 27; or

(iii)該重鏈可變區包含SEQ ID NO：1的胺基酸序列，和該輕鏈可變區包含SEQ ID NO：2的胺基酸序列；或 (iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 2; or

(iv)該重鏈可變區包含SEQ ID NO：3的胺基酸序列，和該輕鏈可變區包含SEQ ID NO：4的胺基酸序列。 (iv) The heavy chain variable region includes the amino acid sequence of SEQ ID NO:3, and the light chain variable region includes the amino acid sequence of SEQ ID NO:4.

(i)該重鏈可變區包含SEQ ID NO：42的胺基酸序列，和該輕鏈可變區包含SEQ ID NO：38的胺基酸序列；或 (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 42, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 38; or

(ii)該重鏈可變區包含SEQ ID NO：30的胺基酸序列，和該輕鏈可變區包含SEQ ID NO：22的胺基酸序列。 (ii) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 30, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 22.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中該抗ICOSL抗體包含抗體重鏈恆定區和輕鏈恆定區。在一些實施方案中，該重鏈恆定區為人IgG重鏈恆定區。在一些實施方案中，該重鏈恆定區選自人IgG1、IgG2、IgG3和IgG4恆定區。在一些實施方案中，該輕鏈恆定區選自人抗體κ或λ鏈恆定區。在一些實施方案中，該重鏈恆定區為人IgG4重鏈恆定區，該輕鏈恆定區為人κ輕鏈恆定區。在一些實施方案中，該重鏈恆定區的Fc區具有一個或更多個能夠減少Fc區與Fc受體結合的胺基酸取代。在一些實施方案中，該Fc區具有L234A、L235A突變，和/或S228P突變，和/或YTE突變(M252Y、S254T和T256E)，該突變編號依據為EU索引。在一些實施方案中，該重鏈恆定區包含SEQ ID NO：43的胺基酸序列，該輕鏈恆定區包含SEQ ID NO：44的胺基酸序列。 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, wherein the anti-ICOSL antibody comprises an antibody heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is a human IgG heavy chain constant region. In some embodiments, the heavy chain constant region is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 constant regions. In some embodiments, the light chain constant region is selected from the group consisting of human antibody kappa or lambda chain constant regions. In some embodiments, the heavy chain constant region is a human IgG4 heavy chain constant region and the light chain constant region is a human kappa light chain constant region. In some embodiments, the Fc region of the heavy chain constant region has one or more amino acid substitutions that reduce binding of the Fc region to Fc receptors. In some embodiments, the Fc region has L234A, L235A mutations, and/or S228P mutations, and/or YTE mutations (M252Y, S254T, and T256E), and the mutation numbering is based on the EU index. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:43 and the light chain constant region comprises the amino acid sequence of SEQ ID NO:44.

(i)該抗ICOSL抗體的重鏈包含與SEQ ID NO：47具有至少90%(例如至少90%、95%、96%、97%、98%或99%)序列同一性的胺基酸序列，和輕鏈包含與SEQ ID NO：48具有至少90%(例如至少90%、95%、96%、97%、98%或99%)序列同一性的胺基酸序列；或 (i) 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: 47 , and light chain Comprises an amino acid sequence that has at least 90% (eg, at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 48; or

(ii)該抗ICOSL抗體的重鏈包含與SEQ ID NO：45具有至少90%(例如至少90%、95%、96%、97%、98%或99%)序列同一性的胺基酸序列，和輕鏈包含與SEQ ID NO：46具有至少90%(例如至少90%、95%、96%、97%、98%或99%)序列同一性的胺基酸序列。 (ii) The heavy chain of the anti-ICOSL antibody comprises an amino acid sequence that has at least 90% (eg, at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 45 , and the light chain 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: 46.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白， In some embodiments, an anti-ICOSL antibody fusion protein as described in any one of the above,

(i)該抗ICOSL抗體的重鏈包含SEQ ID NO：47的胺基酸序列，和該抗ICOSL抗體的輕鏈包含SEQ ID NO：48的胺基酸序列；或 (i) the heavy chain of the anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 47, and the light chain of the anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 48; or

(ii)該抗ICOSL抗體的重鏈包含SEQ ID NO：45的胺基酸序列，和該抗ICOSL抗體的輕鏈包含SEQ ID NO：46的胺基酸序列。 (ii) The heavy chain of the anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 45, and the light chain of the anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 46.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中，該TACI多肽具有更好的防止斷裂功能。 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, wherein the TACI polypeptide has a better function of preventing fragmentation.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中該TACI多肽為包含SEQ ID NO：58的第48位至第85位胺基酸殘基的多肽或其變體；其中，該變體為選自第49、52、53、57、65、82和83位中的一個或更多個位點上具有胺基酸替換，該胺基酸替換的位點為相對於序列SEQ ID NO：58的自然順序編號的胺基酸殘基位點。在一些實施方案中，該TACI多肽的變體為在選自由49T或49R、52S、53E或53Q、57E、65T或65A、82A或82R、和83Y組成的組中的一個或更多個胺基酸替換，該胺基酸替換的位點為相對於序列SEQ ID NO：58的自然順序編號的胺基酸殘基位點。 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, wherein the TACI polypeptide is a polypeptide comprising amino acid residues 48 to 85 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 Natural sequence numbered amino acid residue positions of the sequence SEQ ID NO: 58. In some embodiments, the variant of the TACI polypeptide is one or more amine groups selected from the group consisting of 49T or 49R, 52S, 53E or 53Q, 57E, 65T or 65A, 82A or 82R, and 83Y Acid substitution, 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.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中，該TACI多肽如SEQ ID NO：58所示或是SEQ ID NO：58的截短片段或其變體。該截短片段包含SEQ ID NO：58的第48位至第85位胺基酸殘基。該變體為在SEQ ID NO：58或其截短片段上具有選自第49、52、53、57、65、82和83位中的一個或更多個胺基酸替換，其中該胺基酸替換的位點為相對於序列SEQ ID NO：58的自然順序編號的胺基酸殘基位點。 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, wherein the TACI polypeptide is as shown in SEQ ID NO: 58 or is a truncated fragment of SEQ ID NO: 58 or a variant thereof. The truncated fragment includes amino acid residues 48 to 85 of SEQ ID NO: 58. The variant is 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 The site of acid substitution is the amino acid residue site numbered relative to the natural sequence of sequence SEQ ID NO:58.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中該TACI多肽的截短片段包含：SEQ ID NO：58的第48位至第86位胺基酸殘基；SEQ ID NO：58的第48位至第87位胺基酸殘基；或SEQ ID NO：58的第48位至第88位胺基酸殘基。 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, wherein the truncated fragment of the TACI polypeptide comprises: amino acid residues 48 to 86 of SEQ ID NO: 58; SEQ ID Amino acid residues 48 to 87 of NO: 58; or amino acid residues 48 to 88 of SEQ ID NO: 58.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中，該TACI多肽，序列如SEQ ID NO：60-63任一項所示。 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, wherein the TACI polypeptide has a sequence as shown in any one of SEQ ID NO: 60-63.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中，該TACI多肽，其序列為SEQ ID NO：58變體或者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)的變體，該變體為在SEQ ID NO：58或其截短片段序列上具有選自第49、52、53、57、65、82和83位中的任意1個、2個、3個、4個、5個、6個或7個胺基酸的替換，該胺基酸替換的位點為相對於序列SEQ ID NO：58的自然順序編號的胺基酸殘基位點。 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, wherein the TACI polypeptide has a sequence of SEQ ID NO: 58 variant or a truncated fragment of SEQ ID NO: 58 (such as SEQ ID NO : 60, SEQ ID NO: 61, SEQ ID NO: 62 or a truncated fragment of the SEQ ID NO: 63 sequence), the variant has a sequence on SEQ ID NO: 58 or a truncated fragment thereof selected from Replacement of any 1, 2, 3, 4, 5, 6 or 7 amino acids at positions 49, 52, 53, 57, 65, 82 and 83, the amino acid substituted The positions are amino acid residue positions numbered relative to the natural sequence of the sequence SEQ ID NO:58.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中該TACI多肽為在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有選自由49T或49R、52S、53E或53Q、 57E、65T或65A、82A或82R、和83Y組成的組中的一個或更多個胺基酸替換(例如1個、2個、3個、4個、5個、6個或7個胺基酸替換)，其中該胺基酸替換的位點為相對於序列SEQ ID NO：58的自然順序編號的胺基酸殘基位點。 In some embodiments, the anti-ICOSL antibody fusion protein as described in 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 a sequence selected from 49T or 49R, 52S, 53E or 53Q, One or more amino acid substitutions from the group consisting of 57E, 65T or 65A, 82A or 82R, and 83Y (e.g., 1, 2, 3, 4, 5, 6, or 7 amine groups Acid substitution), wherein the position of the amino acid substitution is the amino acid residue position numbered relative to the natural sequence of sequence SEQ ID NO: 58.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中該TACI多肽為：在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有選自49T、52S、53E、53Q、57E和82A中的任一個胺基酸替換。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胺基酸替換。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胺基酸替換。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胺基酸替換。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胺基酸替換。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胺基酸替換。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胺基酸替換。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胺基酸替換。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胺基酸替換。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胺基酸替換。其中前述胺基酸替換的位點為相對於序列SEQ ID NO：58的自然順序編號的胺基酸殘基位點。 In some embodiments, the anti-ICOSL antibody fusion protein as described in 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 an amino acid substitution selected from any one of 49T, 52S, 53E, 53Q, 57E and 82A. There are 49R and 65T amino acid substitutions in the sequence SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63. There are 49R and 65A amino acid substitutions in the sequence SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63. There are 49R, 65T and 82R amino acid substitutions in the sequence SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63. There are 53E and 57E amino acid substitutions in the sequence SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63. There are 52S, 53E and 57E amino acid substitutions in the sequence SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63. There are 49T and 82A amino acid substitutions in the sequence SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63. There are 49T and 83Y amino acid substitutions in the sequence SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63. There are 49T, 82A and 83Y amino acid substitutions in the sequence SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63. or on the sequence SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 or SEQ ID NO:63 With 49T, 53E, 57E and 82A amino acid substitutions. The sites where the aforementioned amino acids are substituted are amino acid residue sites numbered relative to the natural sequence of sequence SEQ ID NO: 58.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中，該TACI多肽，其胺基酸序列如SEQ ID NO：51至83中任一所示。 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, wherein the TACI polypeptide has an amino acid sequence as shown in any one of SEQ ID NO: 51 to 83.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中該TACI多肽的胺基酸序列如SEQ ID NO：60-63、SEQ ID NO：66-83中的任一所示。 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, wherein the amino acid sequence of the TACI polypeptide is as shown in any one of SEQ ID NO: 60-63, SEQ ID NO: 66-83 .

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中該TACI多肽的胺基酸序列如SEQ ID NO：83所示。 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, wherein the amino acid sequence of the TACI polypeptide is shown in SEQ ID NO: 83.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其包括： In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, including:

(A)第一鏈：[抗ICOSL抗體的重鏈]-[連接子1]-[TACI多肽1]，和 (A) First chain: [heavy chain of anti-ICOSL antibody]-[linker 1]-[TACI polypeptide 1], and

第二鏈：[TACI多肽2]-[連接子2]-[抗ICOSL抗體的輕鏈](圖1)； Second chain: [TACI polypeptide 2]-[linker 2]-[light chain of anti-ICOSL antibody] (Figure 1);

(B)第一鏈：[抗ICOSL抗體的重鏈]-[連接子1]-[TACI多肽1]，和 (B) First chain: [heavy chain of anti-ICOSL antibody]-[linker 1]-[TACI polypeptide 1], and

第二鏈：抗ICOSL抗體的輕鏈(圖2)；或 Second chain: light chain of anti-ICOSL antibody (Figure 2); or

(C)第一鏈：[抗ICOSL抗體的重鏈]-[連接子1]-[TACI多肽1]，和 (C) First chain: [heavy chain of anti-ICOSL antibody]-[linker 1]-[TACI polypeptide 1], and

第二鏈：[抗ICOSL抗體的輕鏈]-[連接子2]-[TACI多肽2](圖3)； Second chain: [light chain of anti-ICOSL antibody]-[linker 2]-[TACI polypeptide 2] (Figure 3);

其中，該(A)、(B)或(C)中，該TACI多肽1和TACI多肽2是相同或不相同的，該連接子1和連接子2是相同或不相同的。 Wherein, in (A), (B) or (C), the TACI polypeptide 1 and TACI polypeptide 2 are the same or different, and the linker 1 and the linker 2 are the same or different.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中，該TACI多肽1或TACI多肽2，其胺基酸序列如SEQ ID NO：51至83中任一所示。 In some embodiments, the anti-ICOSL antibody fusion protein as described in 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.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中該TACI多肽1或TACI多肽2的胺基酸序列如SEQ ID NO：60-63、SEQ ID NO：66-83中的任一所示。 In some embodiments, the anti-ICOSL antibody fusion protein as described in 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 Any of the shown.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中該TACI多肽1或TACI多肽2的胺基酸序列如SEQ ID NO：83所示。 In some embodiments, the anti-ICOSL antibody fusion protein as described in 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.

本揭露中的序數詞“第一”、“第二”、“1”、“2”(如“連接子2”中的“2”)僅用於區分不同的特徵、要素、元件或步驟，不意圖限制數量、順序、水準。 The ordinal words "first", "second", "1", and "2" (such as "2" in "connector 2") in this disclosure are only used to distinguish different features, elements, components or steps. There is no intention to limit the quantity, order, or level.

在一些實施方案中，該連接子1和連接子2各自獨立地具有(G_xS)_y的結構，其中，x選自1-5的整數，y選自0-6的整數。在一些實施方案中，該連接子1和連接子2各自獨立地具有(G_xS)_y的結構，其中，x選自1-5的整數，y選自1-6的整數。在一些實施方案中，該連接子1和連接子2各自獨立地為GGGS(如SEQ ID NO：89所示)或GGGGSGGGGSGGGGS(如SEQ ID NO：90所示)。 In some embodiments, the linker 1 and the linker 2 each independently have the 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 the linker 2 each independently have the 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 linker 2 are each independently GGGS (as shown in SEQ ID NO: 89) or GGGGSGGGGSGGGGS (as shown in SEQ ID NO: 90).

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中，該抗ICOSL抗體融合蛋白具有：包含SEQ ID NO：86的胺基酸序列的第一鏈，和包含SEQ ID NO：88、87或48的胺基酸序列的第二鏈；或該抗ICOSL抗體融合蛋白具有：包含SEQ ID NO：84的胺基酸序列的第一鏈，和包含SEQ ID NO：85或46的胺基酸序列的第二鏈。在一些實施方案中，該抗ICOSL抗體融合蛋白具有2條包含SEQ ID NO：86的胺基酸序列的第一鏈，和2條包含SEQ ID NO：88的胺基酸序列的第二鏈。在一些實施方案中，該抗ICOSL抗體融合蛋白具有2條包含SEQ ID NO：86的胺基酸序列的第一鏈，和2條包含SEQ ID NO：87的胺基酸序列的第二鏈。在一些實施方案中，該抗ICOSL抗體融合蛋白具有2條包含SEQ ID NO：86的胺基酸序列的第一鏈，和2條包含SEQ ID NO：48的胺基酸序列的第二鏈。在一些實施方案中，該抗ICOSL抗體融合蛋白具有2條包含SEQ ID NO：84的胺基酸序列的第一鏈，和2條包含SEQ ID NO：85的胺基酸序列的第二鏈。在一些實施方案中，該抗ICOSL抗體融合蛋白具有2條包含SEQ ID NO：84的胺基酸序列的第一鏈，和2條包含SEQ ID NO：46的胺基酸序列的第二鏈。 In some embodiments, the anti-ICOSL antibody fusion protein as described in 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 a first chain comprising the amino acid sequence of 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 of the amino acid sequence of SEQ ID NO: 84, and the first chain of the amino acid sequence of SEQ ID NO: 85 or 46 The amino acid sequence of the second strand. In some embodiments, the anti-ICOSL antibody fusion protein has 2 first strands comprising the amino acid sequence of SEQ ID NO:86, and 2 second strands comprising the amino acid sequence of SEQ ID NO:88. In some embodiments, the anti-ICOSL antibody fusion protein has 2 first strands comprising the amino acid sequence of SEQ ID NO: 86, and 2 first strands comprising the amino acid sequence of SEQ ID NO: 86 The second strand of the amino acid sequence of NO: 87. In some embodiments, the anti-ICOSL antibody fusion protein has 2 first strands comprising the amino acid sequence of SEQ ID NO: 86, and 2 second strands comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the anti-ICOSL antibody fusion protein has 2 first strands comprising the amino acid sequence of SEQ ID NO:84, and 2 second strands comprising the amino acid sequence of SEQ ID NO:85. In some embodiments, the anti-ICOSL antibody fusion protein has 2 first strands comprising the amino acid sequence of SEQ ID NO: 84, and 2 second strands comprising the amino acid sequence of SEQ ID NO: 46.

另一方面，本揭露提供一種抗ICOSL抗體，其中該抗ICOSL抗體包含重鏈可變區和輕鏈可變區，其中，該重鏈可變區包含HCDR1、HCDR2和HCDR3，該輕鏈可變區包含LCDR1、LCDR2和LCDR3，其中， In another aspect, the present disclosure provides an anti-ICOSL antibody, wherein the anti-ICOSL antibody includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, and the light chain variable region The area includes LCDR1, LCDR2 and LCDR3, where,

在一些實施方案中，如上任一項所述的抗ICOSL抗體，其中該重鏈可變區的HCDR1、HCDR2和HCDR3以及輕鏈可變區的LCDR1、LCDR2和LCDR3是根據選自Kabat、IMGT、Chothia、AbM和Contact的編號規則定義的。在一些實施方案中，該重鏈可變區的HCDR1、HCDR2和HCDR3以及輕鏈可變區的LCDR1、LCDR2和LCDR3是根據Kabat編號規則定義的。在一些實施方案中，該重鏈可變區的HCDR1、HCDR2和HCDR3以及輕鏈可變區的LCDR1、LCDR2和LCDR3是根據IMGT編號規則定義的。在一些實施方案中，該重鏈可變區的HCDR1、HCDR2和HCDR3以及輕鏈可變區的LCDR1、LCDR2和LCDR3是根據Chothia編號規則定義的。在一些實施方案中，該重鏈可變區的HCDR1、HCDR2和HCDR3以及輕鏈可變區的LCDR1、LCDR2和LCDR3是根據AbM編號規則定義的。在一些實施方案中，該重鏈可變區的HCDR1、HCDR2和HCDR3以及輕鏈可變區的LCDR1、LCDR2和LCDR3是根據Contact編號規則定義的。 In some embodiments, the anti-ICOSL antibody as described in 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, The numbering rules of Chothia, AbM and Contact are defined. In some embodiments, the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and the light chain variable LCDR1, LCDR2 and LCDR3 of the area are defined according to the Kabat numbering rule. In some embodiments, 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 IMGT numbering convention. In some embodiments, 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 rule. In some embodiments, 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, 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 Contact numbering rule.

在一些實施方案中，如上任一項所述的抗ICOSL抗體，其中， In some embodiments, an anti-ICOSL antibody as described in any one of the above, wherein,

在一些實施方案中，如上任一項所述的抗ICOSL抗體，其中 In some embodiments, an anti-ICOSL antibody as described in any one of the above, wherein

在一些實施方案中，如上任一項所述的抗ICOSL抗體，該抗ICOSL抗體為鼠源抗體、嵌合抗體或人源化抗體。在一些實施方案中，該抗體是人源化抗體。 In some embodiments, the anti-ICOSL antibody as described in any one of the above, the anti-ICOSL antibody is a murine antibody, a chimeric antibody or a humanized antibody. In some embodiments, the antibody is a humanized antibody.

(ii)該重鏈可變區包含與SEQ ID NO：30、28或29具有至少90%(例如至少90%、95%、96%、97%、98%或99%)序列同一性的胺基酸序列，和該輕鏈可變區包含與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%)序列同一性的胺基酸序列；或 (ii) The heavy chain variable region comprises an amine having 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 the light chain variable region comprises at least 90% identical to SEQ ID NO: 22, 20, 21, 23, 24, 25, 26 or 27 (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; or

在一些實施方案中，如上任一項所述的抗ICOSL抗體，該抗ICOSL抗體的重鏈可變區的框架區上包含選自第37、43和49位元(根據Kabat編號系統編號)中的一個或更多個胺基酸突變，和/或輕鏈可變區的框架區上包含選自第1、2、3、42、43、60和85位元(根據Kabat編號系統編號)中的一個或更多個胺基酸突變。在一些實施方案中，該抗ICOSL抗體融合蛋白，其中該重鏈可變區的HCDR1包含SEQ ID NO：11的胺基酸序列，HCDR2包含SEQ ID NO：31或12的胺基酸序列，和HCDR3包含SEQ ID NO：13的胺基酸序列，和該輕鏈可變區的LCDR1包含SEQ ID NO：14的胺基酸序列，LCDR2包含SEQ ID NO：15的胺基酸序列，和LCDR3包含SEQ ID NO：16的胺基酸序列；該抗體的重鏈可變區的框架區上包含選自：37I、43E和49A(根據Kabat編號系統編號)中的一個或更多個胺基酸突變，和/或該輕鏈可變區的框架區上包含選自： 1N、2T、3V、42Q、43S、60D、85V(根據Kabat編號系統編號)中的一個或更多個胺基酸突變。 In some embodiments, the anti-ICOSL antibody as described in any one of the above, the framework region of the heavy chain variable region of the anti-ICOSL antibody includes positions selected from the group consisting of positions 37, 43 and 49 (numbered according to the Kabat numbering system) One or more amino acid mutations, and/or the framework region of the light chain variable region contains one or more amino acid mutations selected from positions 1, 2, 3, 42, 43, 60 and 85 (numbered according to the Kabat numbering system) one or more amino acid mutations. In some embodiments, the anti-ICOSL antibody fusion protein, wherein HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11, HCDR2 comprises the amino acid sequence of SEQ ID NO: 31 or 12, and HCDR3 includes the amino acid sequence of SEQ ID NO: 13, and LCDR1 of the light chain variable region includes the amino acid sequence of SEQ ID NO: 14, LCDR2 includes the amino acid sequence of SEQ ID NO: 15, and LCDR3 includes The amino acid sequence of SEQ ID NO: 16; the framework region of the heavy chain variable region of the antibody contains one or more amino acid mutations selected from: 37I, 43E and 49A (numbered according to the Kabat numbering system) , and/or the framework region of the light chain variable region contains a component selected from: One or more amino acid mutations in 1N, 2T, 3V, 42Q, 43S, 60D, 85V (numbered according to Kabat numbering system).

在一些實施方案中，如上任一項所述的抗ICOSL抗體，該抗ICOSL抗體的重鏈可變區的框架區上包含選自第1、24、69、71、73、78位元(根據Kabat編號系統編號)中的一個或更多個胺基酸突變，和/或輕鏈可變區的框架區上包含選自第1、2和4位元(根據Kabat編號系統編號)中的一個或更多個胺基酸突變。在一些實施方案中，該抗ICOSL抗體融合蛋白，其中抗ICOSL抗體的重鏈可變區的HCDR1包含SEQ ID NO：5的胺基酸序列，HCDR2包含SEQ ID NO：19或6的胺基酸序列，和HCDR3包含SEQ ID NO：7的胺基酸序列，和該輕鏈可變區的LCDR1包含SEQ ID NO：8的胺基酸序列，LCDR2包含SEQ ID NO：17或9的胺基酸序列，和LCDR3包含SEQ ID NO：10或18的胺基酸序列；該抗體的重鏈可變區的框架區上包含選自：1E、24T、69L、71V、73K和78A(根據Kabat編號系統編號)中的一個或更多個胺基酸突變，和/或該輕鏈可變區的框架區上包含選自：1A、2V和4L(根據Kabat編號系統編號)中的一個或更多個胺基酸突變。 In some embodiments, the anti-ICOSL antibody as described in any one of the above, the framework region of the heavy chain variable region of the anti-ICOSL antibody includes positions selected from positions 1, 24, 69, 71, 73, and 78 (according to one or more amino acid mutations in the Kabat numbering system), and/or the framework region of the light chain variable region contains one selected from positions 1, 2, and 4 (according to the Kabat numbering system) or more amino acid mutations. In some embodiments, 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 sequence, and HCDR3 includes the amino acid sequence of SEQ ID NO: 7, and LCDR1 of the light chain variable region includes the amino acid sequence of SEQ ID NO: 8, and LCDR2 includes the amino acid sequence of SEQ ID NO: 17 or 9 sequence, and LCDR3 includes the amino acid sequence of SEQ ID NO: 10 or 18; the heavy chain variable region of the antibody includes a framework region selected from: 1E, 24T, 69L, 71V, 73K and 78A (according to the Kabat numbering system one or more amino acid mutations in the numbering), and/or the framework region of the light chain variable region contains one or more selected from: 1A, 2V and 4L (numbering according to the Kabat numbering system) Amino acid mutations.

在一些實施方案中，如上任一項所述的抗ICOSL抗體，其中該抗ICOSL抗體包含抗體重鏈恆定區和輕鏈恆定區。在一些實施方案中，該重鏈恆定區為人IgG重鏈恆定區。在一些實施方案中，該重鏈恆定區選自人IgG1、IgG2、IgG3和IgG4恆定區；在一些實施方案中，該輕鏈恆定區選自人抗體κ或λ鏈恆定區。在一些實施方案中，該重鏈恆定區為人IgG4重鏈恆定區，該輕鏈恆定區為人κ輕鏈恆定區。在一些實施方案中，該重鏈恆定區的Fc區具有一個或更多個能夠減少Fc區與Fc受體結合的胺基酸取代。在一些實施方案中，該Fc區具有L234A、L235A突變，和/或S228P突變，和/或YTE突變(M252Y、S254T和T256E)，該突變編號依據為EU索引。在一些實施方案中，該重鏈恆定區包含SEQ ID NO：43的胺基酸序列，該輕鏈恆定區包含SEQ ID NO：44的胺基酸序列。 In some embodiments, the anti-ICOSL antibody as described in any one of the above, wherein the anti-ICOSL antibody comprises an antibody heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is a human IgG heavy chain constant region. In some embodiments, the heavy chain constant region is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 constant regions; in some embodiments, the light chain constant region is selected from the group consisting of human antibody kappa or lambda chain constant regions. In some embodiments, the heavy chain constant region is a human IgG4 heavy chain constant region and the light chain constant region is a human kappa light chain constant region. In some embodiments, the Fc region of the heavy chain constant region has one or more amino acid substitutions that reduce binding of the Fc region to Fc receptors. In some embodiments, the Fc region has L234A, L235A mutations, and/or S228P mutations, and/or YTE mutations (M252Y, S254T, and T256E), and the mutation numbering is based on the EU index. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:43 and the light chain constant region comprises the amino acid sequence of SEQ ID NO:44.

(i)該抗ICOSL抗體的重鏈包含與SEQ ID NO：47具有至少90%(例如至少90%、95%、96%、97%、98%或99%)序列同一性的胺基酸序列，和輕鏈包含與SEQ ID NO：48具有至少90%(例如至少90%、95%、96%、97%、98%或99%)序列同一性的胺基酸序列；或 (i) 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: 47 , and the light chain 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: 48; or

在一些實施方案中，如上任一項所述的抗ICOSL抗體， In some embodiments, an anti-ICOSL antibody as described in any one of the above,

在一些實施方案中，本揭露還提供一種分離的抗ICOSL抗體或抗ICOSL抗體融合蛋白，其與前面任一項所述的抗ICOSL抗體或抗ICOSL抗體融合蛋白競爭性結合人ICOSL或其表位。 In some embodiments, the present disclosure also provides an isolated anti-ICOSL antibody or anti-ICOSL antibody fusion protein that competitively binds to human ICOSL or an epitope thereof with the anti-ICOSL antibody or anti-ICOSL antibody fusion protein described in any one of the preceding items. .

在一些實施方案中，前面任一項所述的抗ICOSL抗體或抗ICOSL抗體融合蛋白，其具有一種或更多種以下特性： In some embodiments, the anti-ICOSL antibody or anti-ICOSL antibody fusion protein of any of the preceding items has one or more of the following properties:

A.與人ICOSL(或其表位)和食蟹猴ICOSL(或其表位)特異性結合，不與鼠ICOSL特異性結合；較佳地，以小於5.00E-09M(例如小於5.00E-09M、小於4.00E-09M、小於3.00E-09M、小於2.00E-09M、小於1.50E-09M、9.00E-10M或更小)的KD值與人ICOSL結合，和/或以小於8.00E-09M(例如小於8.00E- 09M、小於7.00E-09M、小於6.00E-09M、小於5.00E-09M、小於4.00E-09M、小於3.00E-09M或更小)的KD值與食蟹猴ICOSL結合，該KD值藉由表面電漿共振測定法測量(例如BIACORE^®表面電漿共振測定法測量)；在一些實施方案中，該KD值藉由本揭露測試例8方法檢測； A. Specifically binds to human ICOSL (or its epitope) and cynomolgus monkey ICOSL (or its epitope), but does not specifically bind to mouse ICOSL; preferably, less than 5.00E-09M (for example, less than 5.00E-09M , less than 4.00E-09M, less than 3.00E-09M, less than 2.00E-09M, less than 1.50E-09M, 9.00E-10M or less) binds to human ICOSL, and/or binds to human ICOSL with a KD value less than 8.00E-09M (e.g., less than 8.00E-09M, less than 7.00E-09M, less than 6.00E-09M, less than 5.00E-09M, less than 4.00E-09M, less than 3.00E-09M or less) KD value combined with cynomolgus ICOSL , the KD value is measured by surface plasmon resonance assay (such as BIACORE ^® surface plasmon resonance assay); in some embodiments, the KD value is detected by the method of Test Example 8 of the present disclosure;

B.與人APRIL(或其表位)、食蟹猴APRIL(或其表位)和/或鼠APRIL(或其表位)特異性結合；較佳地，以小於2.00E-11M(例如小於2.00E-11M、小於1.80E-11M、小於1.70E-11M、小於1.60E-11M、小於1.20E-11M、小於4.60E-12M或更小)的KD值與人APRIL結合，以小於2.00E-10M(例如小於2.00E-10M、小於1.80E-10M、小於1.70E-10M、小於1.60E-10M、小於1.50E-10M、小於1.40E-10M、小於1.20E-10M或更小)的KD值與食蟹猴APRIL結合，和/或以小於2.00E-11M(例如小於2.00E-11M、小於1.80E-11M、小於1.60E-11M、小於1.40E-11M、小於1.20E-11M、小於1.00E-11M、小於8.00E-12M或更小)的KD值與鼠APRIL結合，該KD值藉由表面電漿共振測定法所測量(例如BIACORE^®表面電漿共振測定法測量)；在一些實施方案中，該KD值藉由本揭露測試例8方法檢測； B. Specifically binds to human APRIL (or its epitope), cynomolgus monkey APRIL (or its epitope) and/or mouse APRIL (or its epitope); preferably, less than 2.00E-11M (for example, less than 2.00E-11M, less than 1.80E-11M, less than 1.70E-11M, less than 1.60E-11M, less than 1.20E-11M, less than 4.60E-12M or less) KD value combined with human APRIL to less than 2.00E -10M (such as less than 2.00E-10M, less than 1.80E-10M, less than 1.70E-10M, less than 1.60E-10M, less than 1.50E-10M, less than 1.40E-10M, less than 1.20E-10M or less) Binds to cynomolgus monkey APRIL with a KD value of less than 2.00E-11M (e.g., less than 2.00E-11M, less than 1.80E-11M, less than 1.60E-11M, less than 1.40E-11M, less than 1.20E-11M, Binds to mouse APRIL with a KD value of less than 1.00E-11M, less than 8.00E-12M, or less) as measured by a surface plasmon resonance assay (such as the ^BIACORE® surface plasmon resonance assay); In some embodiments, the KD value is detected by the method of Test Example 8 of the present disclosure;

C.與人BAFF(或其表位)、食蟹猴BAFF(或其表位)和/或鼠BAFF(或其表位)特異性結合；較佳地，以小於7.00E-11M(例如小於7.00E-11M、小於5.00E-11M、小於4.00E-11M、小於3.00E-11M、小於2.00E-11M、小於1.00E-11M或更小)的KD值與人BAFF結合，以小於4.00E-10M(例如小於4.00E-10M、小於3.00E-10M、小於2.00E-10M、小於1.30E-10M或更小)的KD值與食蟹猴BAFF結合，和/或以小於6.00E-11M(例如小於6.00E-11M、小於5.00E-11M、小於4.00E-11M、小於3.20E-11M或更小)的KD值與鼠BAFF結合，該 KD值藉由表面電漿共振測定法所測量(例如BIACORE^®表面電漿共振測定法測量)；在一些實施方案中，該KD值藉由本揭露測試例8方法檢測； C. Specifically binds to human BAFF (or its epitope), cynomolgus BAFF (or its epitope) and/or mouse BAFF (or its epitope); preferably, less than 7.00E-11M (for example, less than 7.00E-11M, less than 5.00E-11M, less than 4.00E-11M, less than 3.00E-11M, less than 2.00E-11M, less than 1.00E-11M or less) KD value combined with human BAFF to less than 4.00E - Binding to cynomolgus BAFF with a KD value of less than 4.00E-10M, less than 3.00E-10M, less than 2.00E-10M, less than 1.30E-10M, or less, and/or 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 as measured by surface plasmon resonance assay (For example, measured by ^BIACORE® surface plasmon resonance assay); in some embodiments, the KD value is detected by the method of Test Example 8 of the present disclosure;

D.具有阻斷ICOSL與ICOS結合的活性；較佳地，阻斷人ICOSL與ICOS結合的IC₅₀值小於0.16nM(例如小於0.16nM、小於0.14nM、小於0.12nM、小於0.10nM、小於0.09nM、小於0.08nM、小於0.07nM、小於0.06nM、小於0.05nM、小於0.04nM或更小)，該IC₅₀值藉由FACS方法檢測；在一些實施方案中，該IC₅₀值藉由本揭露測試例3方法檢測； D. Has the activity of blocking the binding of ICOSL to ICOS; preferably, the IC ₅₀ value of blocking the binding of human ICOSL to ICOS is less than 0.16nM (for example, less than 0.16nM, less than 0.14nM, less than 0.12nM, less than 0.10nM, less than 0.09 nM, less than 0.08nM, less than 0.07nM, less than 0.06nM, less than 0.05nM, less than 0.04nM or less), the IC ₅₀ value is detected by the FACS method; in some embodiments, the IC ₅₀ value is tested by the present disclosure Example 3 method detection;

E.具有阻斷APRIL與BCMA結合的活性；較佳地，阻斷APRIL與BCMA結合的IC₅₀值小於40.00nM(例如小於40.00nM、小於35.00nM、小於30.00nM、小於20.00nM、小於10.00nM、小於5.00nM、小於2.00nM、小於0.10nM、小於0.06nM、小於0.02nM或更小)，該IC₅₀值藉由ELISA方法檢測；在一些實施方案中，該IC₅₀值藉由本揭露測試例4方法檢測； E. Has the activity of blocking the binding of APRIL to BCMA; preferably, the IC ₅₀ value of blocking the binding of APRIL to BCMA is less than 40.00nM (for example, 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 IC ₅₀ value is detected by the ELISA method; in some embodiments, the IC ₅₀ value is determined by the test example of the present disclosure 4 method detection;

F.具有阻斷APRIL與TACI結合的活性；較佳地，阻斷APRIL與TACI結合的IC₅₀值小於40.00nM(例如小於40.00nM、小於37.00nM、小於33.00nM、小於30.00nM、小於25.00nM、小於10.00nM、小於5.00nM、小於2.00nM、小於1.00nM、小於0.50nM或更小)，該IC₅₀值藉由ELISA方法檢測；在一些實施方案中，該IC₅₀值藉由本揭露測試例4方法檢測； F. Has the activity of blocking the binding of APRIL to TACI; preferably, the IC ₅₀ value of blocking the binding of APRIL to TACI is less than 40.00nM (for example, less than 40.00nM, less than 37.00nM, less than 33.00nM, less than 30.00nM, less than 25.00nM , less than 10.00nM, less than 5.00nM, less than 2.00nM, less than 1.00nM, less than 0.50nM or less), the IC ₅₀ value is detected by the ELISA method; in some embodiments, the IC ₅₀ value is determined by the test example of the present disclosure 4 method detection;

G.具有阻斷BAFF與BCMA結合的活性；較佳地，阻斷BAFF與BCMA結合的IC₅₀值小於2.00nM(例如小於2.00nM、小於1.50nM、小於1.00nM、小於0.90nM、小於0.80nM、小於0.70nM、小於0.60nM、小於0.50nM、小於0.40nM或更小)，該IC₅₀值藉由ELISA方法檢測；在一些實施方案中，該IC₅₀值藉由本揭露測試例4方法檢測； G. Has the activity of blocking the binding of BAFF to BCMA; preferably, the IC ₅₀ value of blocking the binding of BAFF to BCMA is less than 2.00nM (for example, 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 IC ₅₀ value is detected by the ELISA method; in some embodiments, the IC ₅₀ value is detected by the test example 4 method of the present disclosure;

H.具有阻斷BAFF與BAFF-R結合的活性；較佳地，阻斷BAFF與BAFF-R結合的IC₅₀值小於2.00nM(例如小於2.00nM、小於1.70nM、小於1.60nM、小於1.00nM、小於0.80nM、小於0.70nM或更小)，該IC₅₀值藉由ELISA方法檢測；在一些實施方案中，該IC₅₀值藉由本揭露測試例4方法檢測； H. Has the activity of blocking the binding of BAFF and BAFF-R; preferably, the IC ₅₀ value of blocking the binding of BAFF and BAFF-R is less than 2.00nM (for example, 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 the ELISA method; in some embodiments, the IC ₅₀ value is detected by the test example 4 method of the present disclosure;

I.具有阻斷BAFF與TACI結合的活性；較佳地，阻斷BAFF與TACI結合的IC₅₀值小於1.00nM(例如小於1.00nM、小於0.50nM、小於0.30nM、小於0.20nM、小於0.15nM或更小)，該IC₅₀值藉由ELISA方法檢測；在一些實施方案中，該IC₅₀值藉由本揭露測試例4方法檢測； I. Have the activity of blocking the binding of BAFF and TACI; preferably, the _IC50 value of blocking the binding of BAFF and TACI is less than 1.00nM (for example, 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 the ELISA method; in some embodiments, the IC ₅₀ value is detected by the test example 4 method of the present disclosure;

J.具有抑制細胞因子(如IFNγ、IL-10和/或IgA)分泌的活性；在一些實施方案中，該IC₅₀值藉由本揭露測試例9方法檢測； J. Have the activity of inhibiting the secretion of cytokines (such as IFNγ, IL-10 and/or IgA); in some embodiments, the IC ₅₀ value is detected by the method of Test Example 9 of the present disclosure;

K.具有抑制B細胞增殖和/或T細胞增殖的活性；在一些實施方案中，該抗ICOSL抗體或抗ICOSL抗體融合蛋白抑制ICOSL誘導T細胞增殖的活性；在一些實施方案中，該抑制T細胞增殖活性藉由本揭露測試例6方法檢測；在一些實施方案中，該抗ICOSL抗體融合蛋白抑制BAFF誘導的或APRIL誘導的B細胞增殖的活性；在一些實施方案中，該抑制B細胞增殖活性藉由本揭露測試例7方法檢測。 K. has the activity of inhibiting B cell proliferation and/or T cell proliferation; in some embodiments, the anti-ICOSL antibody or anti-ICOSL antibody fusion protein inhibits the activity of ICOSL-induced T cell proliferation; in some embodiments, the inhibiting T cell proliferation Cell proliferation activity is detected by the method of Test Example 6 of the present disclosure; in some embodiments, the anti-ICOSL antibody fusion protein inhibits BAFF-induced or APRIL-induced B cell proliferation activity; in some embodiments, the anti-ICOSL antibody fusion protein inhibits B cell proliferation activity Detected by the method of test example 7 of this disclosure.

本揭露還提供一種醫藥組成物，其包含前面任一項所述的抗ICOSL抗體融合蛋白或抗ICOSL抗體，以及一種或多種藥學上可接受的載體、稀釋劑或賦形劑。 The present disclosure also provides a pharmaceutical composition comprising the anti-ICOSL antibody fusion protein or anti-ICOSL antibody as described in any one of the preceding items, and one or more pharmaceutically acceptable carriers, diluents or excipients.

本揭露還提供一種醫藥組成物，其包含前面任一項所述的抗ICOSL抗體，和如SEQ ID NO：51至83中任一所示的TACI多肽，以及一種或多種藥學上可接受的載體、稀釋劑或賦形劑； The present disclosure also provides a pharmaceutical composition comprising the anti-ICOSL antibody as described in any one of the preceding items, a TACI polypeptide as shown in any one of SEQ ID NO: 51 to 83, and one or more pharmaceutically acceptable carriers , diluent or excipient;

較佳地，該TACI多肽序列如SEQ ID NO：60-63、SEQ ID NO：66-83中的任一所示； Preferably, the TACI polypeptide sequence is as shown in any one of SEQ ID NO: 60-63 and SEQ ID NO: 66-83;

更佳地，該TACI多肽序列如如SEQ ID NO：83所示。 More preferably, the TACI polypeptide sequence is as shown in SEQ ID NO: 83.

在一些實施方案中，本揭露提供一種核酸分子，其編碼前面任一項所述的抗ICOSL抗體融合蛋白或抗ICOSL抗體。 In some embodiments, the present disclosure provides a nucleic acid molecule encoding an anti-ICOSL antibody fusion protein or an anti-ICOSL antibody as described in any one of the preceding items.

在一些實施方案中，本揭露提供一種表達載體，其包含前述的核酸分子(DNA或RNA)。 In some embodiments, the present disclosure provides an expression vector comprising the aforementioned nucleic acid molecule (DNA or RNA).

在一些實施方案中，本揭露提供一種宿主細胞，其含前述的核酸分子。 In some embodiments, the present disclosure provides a host cell containing the aforementioned nucleic acid molecule.

在一些實施方案中，本揭露提供一種宿主細胞，其含前述的表達載體。 In some embodiments, the present disclosure provides a host cell containing the aforementioned expression vector.

本揭露提供的宿主細胞，不能發育成動物或植物個體。 The host cells provided by the present disclosure cannot develop into animal or plant individuals.

在一些實施方案中，本揭露提供一種治療自體免疫性疾病、炎性疾病、B細胞障礙或T細胞障礙的方法，該方法包括向有需要的受試者施用治療有效量的前面任一項所述的抗ICOSL抗體融合蛋白或抗ICOSL抗體或醫藥組成物的步驟。在一些實施方案中，本揭露方法用於治療自體免疫性疾病或炎性疾病。在一些實施方案中，本揭露方法用於治療B細胞障礙或T細胞障礙。 In some embodiments, the present disclosure provides a method of treating an autoimmune disease, an inflammatory disease, a B cell disorder, or a T cell disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of any of the foregoing. The steps of anti-ICOSL antibody fusion protein or anti-ICOSL antibody or pharmaceutical composition. In some embodiments, the disclosed methods are used to treat autoimmune or inflammatory diseases. In some embodiments, methods of the present disclosure are used to treat B cell disorders or T cell disorders.

在一些實施方案中，本揭露提供前述任一項所述的抗ICOSL抗體融合蛋白或抗ICOSL抗體、核酸分子或醫藥組成物在製備用於治療疾病的藥物中的用途。 In some embodiments, the present disclosure provides the use of any of the aforementioned anti-ICOSL antibody fusion proteins or anti-ICOSL antibodies, nucleic acid molecules or pharmaceutical compositions in the preparation of medicaments for treating diseases.

在另一個方面，本揭露還提供用作藥物的前述任一項所述的抗ICOSL抗體融合蛋白或抗ICOSL抗體、核酸分子或組成物。在一些實施方案中，藥物用於治療自體免疫性疾病、炎性疾病、B細胞障礙或T細胞障礙。在一些實施方案中，藥物用於治療自體免疫性疾病或炎性疾病。在一些實施方案中，藥物用於治療B細胞障礙或T細胞障礙。 In another aspect, the present disclosure also provides the anti-ICOSL antibody fusion protein or anti-ICOSL antibody, nucleic acid molecule or composition of any of the foregoing for use as a medicament. In some embodiments, Medications are used to treat autoimmune diseases, inflammatory diseases, B-cell disorders, or T-cell disorders. In some embodiments, the drug is used to treat autoimmune or inflammatory diseases. In some embodiments, the drug is used to treat B cell disorders or T cell disorders.

在一些實施方案中，前面任一項所述的疾病是與ICOSL高表達相關的疾病或病症。在一些實施方案中，前面任一項所述的疾病或病症是自體免疫性疾病、炎性疾病、B細胞障礙或T細胞障礙。在一些實施方案中，該自體免疫性疾病或炎性疾病選自：系統性紅斑狼瘡、類風濕性關節炎、移植物抗宿主病、哮喘、免疫性血小板減少性紫癜、多發性硬化、糖尿病引起的炎性疾病、銀屑病、炎性腸病、克羅恩病、潰瘍性結腸炎、格雷夫斯病和橋本氏甲狀腺炎。在一些實施方案中，該B細胞障礙或T細胞障礙為腫瘤。在一些實施方案中，該腫瘤選自：頭頸癌、非小細胞肺癌、尿路上皮癌、白血病、肉瘤、黑色素瘤、腺癌、結直腸癌、***腫瘤、乳腺癌和小細胞肺癌。在一些實施方案中，該自體免疫性疾病為系統性紅斑狼瘡。 In some embodiments, the disease of any of the preceding is a disease or disorder associated with high expression of ICOSL. In some embodiments, the disease or disorder of any of the preceding is an autoimmune disease, an inflammatory disease, a B cell disorder, or a T cell disorder. In some embodiments, the autoimmune or inflammatory disease is selected from: systemic lupus erythematosus, rheumatoid arthritis, graft versus host disease, asthma, immune thrombocytopenic purpura, multiple sclerosis, diabetes Caused by inflammatory diseases, psoriasis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Graves' disease and Hashimoto's thyroiditis. In some embodiments, the B cell disorder or T cell disorder is a tumor. In some embodiments, 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 tumors, breast cancer, and small cell lung cancer. In some embodiments, the autoimmune disease is systemic lupus erythematosus.

在一些實施方案中，前面任一項所述的治療，進一步包括向受試者施用另外的治療藥物。 In some embodiments, the treatment of any of the preceding items further includes administering to the subject an additional therapeutic agent.

圖1為抗ICOSL抗體融合蛋白如259H4L7-T11和201H3L3-T11的結構示意圖。 Figure 1 is a schematic structural diagram of anti-ICOSL antibody fusion proteins such as 259H4L7-T11 and 201H3L3-T11.

圖2為抗ICOSL抗體融合蛋白如201H3L3-T7和259H4L7-T7的結構示意圖。 Figure 2 is a schematic structural diagram of anti-ICOSL antibody fusion proteins such as 201H3L3-T7 and 259H4L7-T7.

圖3為抗ICOSL抗體融合蛋白如259H4L7-T9的結構示意圖。 Figure 3 is a schematic structural diagram of an anti-ICOSL antibody fusion protein such as 259H4L7-T9.

圖4為抗ICOSL抗體融合蛋白抑制IL-10的分泌實驗結果。 Figure 4 shows the experimental results of anti-ICOSL antibody fusion protein inhibiting the secretion of IL-10.

圖5為抗ICOSL抗體融合蛋白抑制IFNγ的分泌實驗結果。 Figure 5 shows the experimental results of anti-ICOSL antibody fusion protein inhibiting the secretion of IFNγ.

圖6為抗ICOSL抗體融合蛋白抑制IgA的分泌實驗結果。 Figure 6 shows the experimental results of anti-ICOSL antibody fusion protein inhibiting IgA secretion.

圖7為抗ICOSL抗體融合蛋白抑制KLH特異性IgG的產生實驗結果。 Figure 7 shows the experimental results of anti-ICOSL antibody fusion protein inhibiting the production of KLH-specific IgG.

圖8為抗ICOSL抗體融合蛋白抑制KLH特異性IgM的產生實驗結果。 Figure 8 shows the experimental results of anti-ICOSL antibody fusion protein inhibiting the production of KLH-specific IgM.

術語Terminology

為了更容易理解本揭露，以下對某些技術和科學術語進行了描述。除非在本文中另有明確定義，本文使用的全部技術和科學術語具有與所屬技術領域具有通常知識者通常所理解的相同含義。 To make this disclosure easier to understand, certain technical and scientific terms are described below. Unless otherwise expressly defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which they belong.

說明書和申請專利範圍中所用的單數形式“一個”、“一種”和“該”包括複數指代，除非上下文清楚表明並非如此。 As used in the specification and claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

除非上下文另外清楚要求，否則在專利說明書和申請專利範圍中，應將詞語“包含”、“具有”、“包括”等理解為“包括但不僅限於”的意義，而不是排他性或窮舉性意義。 Unless the context clearly requires otherwise, in the patent specification and the scope of the patent application, the words "include", "have", "including", etc. should be understood to mean "including but not limited to" and not in the exclusive or exhaustive sense. .

術語“細胞因子”是由一個細胞群體釋放的、作為細胞間介質作用於其它細胞的蛋白質的統稱。這樣的細胞因子的例子包括淋巴因子、單核因子、趨化因子和傳統的多肽激素。示例性的細胞因子包括：IL-10、IFN-γ、IL-6、TNFα、IL-17和IL-5。 The term "cytokine" is a general term for proteins released by one cell population that act as intercellular mediators on other cells. Examples of such cytokines include lymphokines, monokines, chemokines, and traditional peptide hormones. Exemplary cytokines include: IL-10, IFN-γ, IL-6, TNFα, IL-17, and IL-5.

本揭露所述的TACI是一種膜結合受體。野生型人TACI胞外區(第1-165位)參見本揭露的SEQ ID NO：51。本揭露中的“TACI胞外結構域”與“TACI胞外區”可相互替換。 TACI of the present disclosure is a membrane-bound receptor. The wild-type human TACI extracellular region (positions 1-165) is shown in SEQ ID NO: 51 of the present disclosure. "TACI extracellular domain" and "TACI extracellular region" in this disclosure are interchangeable.

ICOSL(Inducible costimulator-ligand，也稱為B7RP1，B7H2，CD275)，是屬於B7家族的一個免疫共刺激分子。目前已知ICOSL的受體是ICOS，ICOS主要在T細胞表面表達，ICOSL和ICOS介導的信號通路主要在T細胞的分化和T細胞依賴的B細胞活化中發揮作用(Nat Rev Cancer.2012 Mar 22；12(4)：252-64.)。 ICOSL (Inducible costimulator-ligand, also known as B7RP1, B7H2, CD275) is an immune costimulatory molecule belonging to the B7 family. It is currently known that 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 T cell-dependent B cell activation (Nat Rev Cancer. 2012 Mar 22;12(4):252-64.).

“ICOSL”作為抗體或其融合蛋白的靶點時，應作廣泛的理解，旨在涵蓋ICOSL在哺乳動物體內各階段中的各種形式的分子，例如但不限於ICOSL基因在擴增、複製、轉錄、剪接、加工、轉譯、修飾過程中所產生的分子(例如前體BCMA、成熟ICOSL、膜表達的ICOSL、ICOSL剪接變體、修飾的ICOSL、或其片段)；該術語也涵蓋人工製備的或體外表達的ICOSL。 When "ICOSL" is used as the target of an antibody or its fusion protein, it should be understood broadly and is intended to cover various forms of molecules in various stages of ICOSL in the mammalian body, such as but not limited to the amplification, replication, and transcription of the ICOSL gene. , molecules produced during splicing, processing, translation, modification (such as precursor BCMA, mature ICOSL, membrane-expressed ICOSL, ICOSL splice variants, modified ICOSL, or fragments thereof); this term also covers artificially prepared or In vitro expression of ICOSL.

術語“和/或”，意指包含“和”與“或”兩種含義。例如短語“A、B和/或C”旨在涵蓋以下方面中的任一個：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(單獨)；B(單獨)；和C(單獨)。 The term "and/or" is meant to include both "and" and "or". For example the phrase "A, B and/or C" is intended to cover any of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

本揭露所用胺基酸三字母代碼和單字母代碼如J.biol.chem，243，p3558(1968)中所述。 The amino acid three-letter codes and single-letter codes used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

術語“胺基酸”是指天然存在的和合成的胺基酸，以及以與天然存在的胺基酸類似的方式起作用的胺基酸類似物和胺基酸模擬物。天然存在的胺基酸是由遺傳密碼編碼的那些胺基酸，以及後來修飾的那些胺基酸，例如羥脯胺酸、γ-羧基谷胺酸和O-磷酸絲胺酸。胺基酸類似物是指與天然存在的胺基酸具有相同基本化學結構(即與氫、羧基、胺基和R基團結合的α碳)的化合物，例如高絲胺酸、正亮胺酸、甲硫胺酸亞碸、甲硫胺酸甲基鋶。此類類似物具有修飾的R基團(例如，正亮胺酸)或修飾的肽骨架，但保留與天然存在的胺基酸相同的基本化學結構。胺基酸模擬物是指具有與胺基酸的一般化學結構不同的結構，但是以與天然存在的胺基酸類似的方式起作用的化學化合物。 The term "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 that are later modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogues are amino acids that are similar to naturally occurring amino acids. Compounds that have the same basic chemical structure (i.e., alpha carbon bonded to hydrogen, carboxyl, amine, and R groups), such as homoserine, norleucine, methionine teresine, and methionine methylthionine . Such analogs have modified R groups (eg, norleucine) or modified peptide backbones but retain the same basic chemical structure as the naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but act in a manner similar to naturally occurring amino acids.

術語“胺基酸突變”包括胺基酸取代(也稱胺基酸替換)、缺失、***和修飾。可以進行取代、缺失、***和修飾的任意組合來實現最終構建體，只要最終構建體擁有期望的特性，例如降低對Fc受體的結合。胺基酸序列缺失和***可以位於多肽鏈的胺基端和/或羧基端。在一個實施方案中，胺基酸突變是非保守性的胺基酸取代，即將一個胺基酸用具有不同結構和/或化學特性的另一種胺基酸替換。胺基酸取代包括由非天然存在的胺基酸或由20種天然胺基酸的衍生物(例如4-羥脯胺酸、3-甲基組胺酸、鳥胺酸、高絲胺酸、5-羥賴胺酸)替換。可以使用本領域中公知的遺傳或化學方法進行胺基酸突變。遺傳方法可以包括定點誘變、PCR，基因合成等。預計基因工程以外的改變胺基酸側鏈基團的方法，如化學修飾也可能是可用的。本文中可使用各種表述來指示胺基酸突變。本文中，可採用位置+胺基酸殘基的方式表示特定位元點的胺基酸殘基，例如366W，表示在366位點上的胺基酸殘基為W。T366W則表示第366位點上的胺基酸殘基由原來的T突變為了W。作為一個示例，當在請求項中以366W或T366W來描述序列時，不能理解為366位點處的原始胺基酸殘基T對保護範圍構成任何限制。 The term "amino acid mutation" includes amino acid substitutions (also known as amino acid substitutions), deletions, insertions and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to achieve the final construct, as long as the final construct possesses the desired properties, such as reduced binding to Fc receptors. Amino acid sequence deletions and insertions can be located at the amino terminus and/or carboxyl terminus of the polypeptide chain. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, ie, one amino acid is replaced by another amino acid with different structural and/or chemical properties. Amino acid substitutions include non-naturally occurring amino acids or derivatives of 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5 -Hydroxylysine) replacement. 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, etc. 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. Herein, the amino acid residue at a specific position can be represented by position + amino acid residue, for example, 366W, which means that the amino acid residue at position 366 is W. T366W means that the amino acid residue at position 366 has been mutated from the original T to W. As an example, when the sequence is described as 366W or T366W in the claim, it cannot be understood that the original amino acid residue T at position 366 constitutes any limitation on the scope of protection.

術語“抗體”以最廣義使用，並且涵蓋各種抗體結構，包括但不限於單株抗體、多株抗體；單特異性抗體，多特異性抗體(例如雙特異性抗體)；全長抗體和抗體片段(或抗原結合片段，或抗原結合部分)，只要它們展現出期望的抗原結合活性。“天然抗體”指天然存在的免疫球蛋白分子。例如，天然IgG抗體是約150,000道爾頓的異四聚糖蛋白，由二硫鍵結合的2條相同輕鏈和2條相同重鏈構成。從N至C端，每條重鏈具有一個可變區(VH)，又稱作可變重域、重鏈可變區，接著是重鏈恆定區，天然IgG重鏈恆定區通常含三個恆定域(CH1、CH2和CH3)。類似地，從N至C端，每條輕鏈具有一個可變區(VL)，又稱作可變輕域，或輕鏈可變域，接著是一個恆定輕域(輕鏈恆定區、CL)。術語“全長抗體”、“完整抗體”和“全抗體”在本文可互換使用，指具有與天然抗體結構基本類似的結構或具有如本文所限定的Fc區的重鏈的抗體。天然完整抗體輕鏈包括輕鏈可變區VL及恆定區CL，VL處於輕鏈的胺基末端，輕鏈恆定區包括κ鏈及λ鏈；重鏈包括可變區VH及恆定區(CH1、CH2及CH3)，VH處於重鏈的胺基末端，恆定區處於羧基末端，其中CH3最接近多肽的羧基末端，重鏈可屬於任何同種型，包括IgG(包括IgG1、IgG2、IgG3及IgG4亞型)、IgA(包括IgA1及IgA2亞型)、IgM及IgE。 The term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies); Full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), provided they exhibit the desired antigen-binding activity. "Native antibodies" refer to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetrameric proteins of approximately 150,000 Daltons, composed of 2 identical light chains and 2 identical heavy chains bonded by disulfide bonds. From N to C-terminus, 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. Natural IgG heavy chain constant regions usually contain three Constant domains (CH1, CH2 and CH3). Similarly, from N to C terminus, each light chain has a variable region (VL), also called a variable light domain, or light chain variable domain, followed by a constant light domain (light chain constant region, CL ). The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy chain with an Fc region as defined herein. The natural intact antibody light chain includes the light chain variable region VL and the constant region CL. The VL is at the amino end of the light chain. The light chain constant region includes the kappa chain and the lambda chain. The heavy chain includes the variable region VH and the constant region (CH1, CH2 and CH3), VH is at the amine terminus of the heavy chain, and the constant region is at the carboxyl terminus, with CH3 closest to the carboxyl terminus of the polypeptide. 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.

術語抗體“可變區”或“可變域”指抗體重鏈或輕鏈中參與抗體結合抗原的域。本文中，抗體重鏈可變區(VH)和輕鏈可變區(VL)各包含四個保守的框架區(FR)和三個互補決定區(CDR)。其中，術語“互補決定區”或“CDR”指可變結構域內主要促成與抗原結合的區域；“框架”或“FR”是指除CDR殘基之外的可變結構域殘基。VH包含3個CDR區：HCDR1、HCDR2和HCDR3；VL包含3個CDR區：LCDR1、LCDR2和LCDR3。每個VH和VL由從胺基末端(也稱N末端)排到羧基末端(也稱C末端)按以下順序排列的三個CDR和四個FR構成：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。 The term antibody "variable region" or "variable domain" refers to the domain of the antibody heavy or light chain that participates in the antibody's binding to antigen. Herein, the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved framework regions (FR) and three complementarity determining regions (CDR). Among them, the term "complementarity determining region" or "CDR" refers to the region within the variable domain that mainly contributes to binding to the antigen; "framework" or "FR" refers to the variable domain residues other than CDR residues. VH contains 3 CDR areas: HCDR1, HCDR2 and HCDR3; VL contains 3 CDR areas: LCDR1, LCDR2 and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amine terminus (also called N-terminus) to the carboxyl terminus (also called C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

可以藉由各種公知方案來確定CDR的胺基酸序列邊界，例如：“Kabat”編號規則(參見Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”編號規則、“ABM”編號規則、“contact”編號規則(參見Martin,ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001)和ImMunoGenTics(IMGT)編號規則(Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)；Front Immunol.2018 Oct 16；9：2278)等；各種編號系統之間的對應關係是所屬技術領域具有通常知識者熟知的，示例性的，如下表1中所示。 The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, such as: "Kabat" numbering rule (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering convention, "ABM" numbering convention, "contact" numbering convention (see Martin, ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001) and ImMunoGenTics (IMGT ) Numbering rules (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278), etc.; the correspondence between various numbering systems is a technical field It is well known to those of ordinary skill and is exemplified as shown in Table 1 below.

表1. CDR編號系統之間的關係

Table 1. Relationship between CDR numbering systems

除非另有說明，本揭露實施例中的可變區和CDR均適用“Kabat”編號規則。 Unless otherwise stated, the variable regions and CDRs in the embodiments of the present disclosure are all subject to the "Kabat" numbering rule.

術語“抗體片段”指不同於完整抗體的分子，其包含完整抗體的部分，該部分與完整抗體所結合的抗原相結合。抗體片段的實例包括但不限於Fv、Fab、Fab'、Fab'-SH、F(ab')₂、單域抗體、單鏈Fab(scFab)、雙抗體、線性抗體、單鏈抗體分子(例如scFv)，以及由抗體片段形成的多特異性抗體。 The term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab ' , Fab' -SH, F(ab ' ) ₂ , single domain antibody, single chain Fab (scFab), diabody, linear antibody, single chain antibody molecules (e.g. scFv), and multispecific antibodies formed from antibody fragments.

術語“Fc區”或“片段可結晶區”用於定義抗體重鏈的C末端區域，包括天然Fc區和改造的Fc區。在一些實施方案中，Fc區包含了相同或不同的兩個亞基。在一些實施方案中，人IgG重鏈的Fc區定義為從Cys226位置處的胺基酸殘基或從Pro230延伸至其羧基末端。用於本文所述抗體的合適Fc區包括人IgG1、IgG2(IgG2A、IgG2B)、IgG3和IgG4的Fc區。在一些實施方案中，Fc區的邊界還可以變化，例如缺失Fc區的C末端賴胺酸(根據EU編號系統的殘基447)或缺失Fc區的C末端甘胺酸和賴胺酸(根據EU編號系統的殘基446和447)。除非另有說明，Fc區的編號規則為EU編號系統，又稱作EU索引。 The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including native Fc regions and engineered Fc regions. In some embodiments, the Fc region contains the same or different Two subunits. In some embodiments, the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus. Suitable Fc regions for use in the antibodies described herein include those of human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. In some embodiments, the boundaries of the Fc region may also vary, such as deletion of the C-terminal lysine of the Fc region (according to residue 447 of the EU numbering system) or deletion of the C-terminal glycine and lysine of the Fc region (according to residues 446 and 447 of the EU numbering system). Unless otherwise stated, the numbering rule for the Fc region is the EU numbering system, also known as the EU index.

術語“嵌合抗體”指抗體中的重鏈和/或輕鏈的一部分自特定的來源或物種衍生，而重和/或輕鏈的剩餘部分自另外的不同來源或物種衍生的抗體。 The term "chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chain is derived from a specific source or species and the remaining portion of the heavy and/or light chain is derived from a different source or species.

術語“人源化抗體”是保留非人抗體的反應性同時在人中具有較低免疫原性的抗體。例如，可以藉由保留非人CDR區並用其人對應物(即，恆定區以及可變區的框架區部分)替換抗體的其餘部分來實現。 The term "humanized antibody" is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. This can be accomplished, 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).

術語“親和力”是指分子(例如，抗體)的單個結合部位與其結合配體(例如，抗原)之間非共價相互作用的總體的強度。除非另外指明，如本文所用，結合“親和力”是指內部結合親和力，其反映出結合對(例如，抗體與抗原)成員之間的相互作用。分子X對其配體Y的親和力通常可以由解離常數(KD)表示。親和力可以藉由本領域已知的常規方法(包括本文所述的那些方法)測量。 The term "affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to the 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 often be expressed by the dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.

如本文所使用的，術語“kassoc”或“ka”指特定抗體-抗原相互作用的締合速率，術語“kdis”或“kd”指特定抗體-抗原相互作用的解離速率。術語“KD”指解離常數，其獲得自kd與ka的比率(即kd/ka)並且表示為莫耳濃度(M)。可以使用本領域公知的方法測定抗體的KD值。例如，使用生物傳感系統例如系統測量表面電漿共振(例如Biacore)，或藉由溶液平衡滴定法(SET)測量溶液中的親和力。 As used herein, the term "kassoc" or "ka" refers to the association rate of a particular antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a particular antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd/ka) and is expressed as molar concentration (M). The KD value of an antibody can be determined using methods well known in the art. For example, measuring surface plasmon resonance using biosensing systems such as systems such as Biacore, or measuring affinity in solution by solution equilibrium titration (SET).

術語“效應子功能”指那些可歸於抗體Fc區(天然序列Fc區或胺基酸序列突變的Fc區)且隨抗體同種型而變化的生物學活性。抗體效應子功能的例子包括但不限於：C1q結合和補體依賴性細胞毒性、Fc受體結合、抗體依賴性細胞介導的細胞毒性(ADCC)、吞噬作用、細胞表面受體(例如B細胞受體)下調；和B細胞活化。 The term "effector function" refers to those biological activities that are attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence mutated Fc region) and that vary with the antibody isotype. Examples of antibody effector functions include, but are not limited to: C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, cell surface receptors (e.g., B cell receptors body) downregulation; and B cell activation.

術語“單株抗體”指基本上均質的抗體的群，即在該群中包含的抗體分子的胺基酸序列是相同的，除了可能少量存在的天然突變以外。相比之下，多株抗體製劑通常包含在其可變結構域具有不同胺基酸序列的多種不同抗體，其通常特異性針對不同表位。“單株”表示從基本上均質的抗體群體獲得的抗體的特徵，並且不應解釋為要求藉由任何特定方法來生產抗體。在一些實施方案中，本揭露提供的抗體是單株抗體。 The term "monoclonal antibody" refers to a population of antibodies that are substantially homogeneous, ie, the antibody molecules contained in the population are identical in amino acid sequence, except for natural mutations that may be present in minor amounts. In contrast, polyclonal antibody preparations typically contain multiple different antibodies with different amino acid sequences in their variable domains, often specific for different epitopes. "Single strain" refers to 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. In some embodiments, the antibodies provided by the present disclosure are monoclonal antibodies.

術語“抗原”是指能夠由諸如抗原結合蛋白(包括例如抗體)選擇性結合的分子或分子部分。抗原可具有一個或多個能夠與不同的抗原結合蛋白(例如抗體)相互作用的表位。 The term "antigen" refers to a molecule or portion of a molecule capable of selective binding 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).

術語“表位”指能夠與抗體(包括其抗原結合片段)特異性結合的抗原上的區域(area或region)。表位元可以由連續胺基酸串(線性表位元)形成或包含非連續胺基酸(構象表位)，例如因抗原的折疊(即藉由三級折疊)而使得非連續胺基酸在空間上互相接近。構象表位元和線性表位元的差別在於：在變性溶劑的存在下，抗體對構象表位的結合喪失。表位包含處於獨特空間構象的至少3，至少4，至少5，至少6，至少7，或8-10個胺基酸。篩選結合特定表位的抗體(即那些結合相同表位的)可以使用本領域例行方法來進行，例如但不限於丙胺酸掃描、肽印跡、肽切割分析、表位切除、表位元提取、抗原的化學修飾(見Prot.Sci.9(2000)487-496)和交叉阻斷。 The term "epitope" refers to an area or region on an antigen that is capable of specifically binding to an antibody (including antigen-binding fragments thereof). Epitopes can be formed from contiguous strings of amino acids (linear epitopes) or contain discontinuous amino acids (conformational epitopes), e.g. due to folding of the antigen (i.e. by tertiary folding). close to each other in space. The difference between conformational epitopes and linear epitopes is that in the presence of denaturing solvents, the antibody's binding to the conformational epitope is lost. An epitope contains 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 specific epitope (i.e., those that bind the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, Chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496) and cross-blocking.

術語“能夠特異性結合”、“特異性結合”或“結合”是指相比其他抗原或表位，抗體能夠以更高的親和力結合至某個抗原或其表位。通常地，抗體以約1×10^-7M或更小(例如約1×10^-8M、1×10^-9M、1×10^-10M、1×10^-11M或更小)的平衡解離常數(KD)結合抗原或其表位。在一些實施方案中，抗體與抗原結合的KD為該抗體結合至非特異性抗原(例如BSA、酪蛋白)的KD的10%或更低(例如1%)。可使用已知的方法來測量KD，例如藉由BIACORE^®表面電漿共振測定法所測量的。然而，特異性結合至抗原或其表位的抗體可能對其它相關的抗原具有交叉反應性，例如，對來自其它物種(同源)(諸如人或猴，例如食蟹獼猴(Macaca fascicularis)(cynomolgus，cyno)、黑猩猩(Pan troglodytes)(chimpanzee，chimp)或狨猴(Callithrix jacchus)(commonmarmoset，marmoset))的相應抗原具有交叉反應性。 The terms "capable of specifically binding", "specifically binding" or "binding" refer to the ability of an antibody to bind to an antigen or epitope thereof with higher affinity than to other antigens or epitopes. Typically, the antibody is present in a concentration of about 1×10 ⁻⁷ M or less (eg, about 1×10 ⁻⁸ M, 1×10 ⁻⁹ M, 1×10 ⁻¹⁰ M, 1×10 ⁻¹¹ M or less). The equilibrium dissociation constant (KD) binds an antigen or its epitope. In some embodiments, the KD of the antibody binding to the antigen is 10% or less (eg, 1%) of the KD of the antibody binding to a non-specific antigen (eg, BSA, casein). KD can be measured using known methods, such as by ^BIACORE® surface plasmon resonance assay. However, antibodies that specifically bind to an antigen or its epitope may be cross-reactive to other related antigens, e.g., from other species (homologous) such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus) The corresponding antigens of the chimpanzee (Pan troglodytes ) (chimpanzee, chimp) or the marmoset ( Callithrix jacchus ) (commonmarmoset, marmoset)) are cross-reactive.

術語“抗ICOSL抗體”和“結合ICOSL的抗體”是指能夠以足夠的親和力結合ICOSL或其表位的抗體。在一個實施例中，與無關的、非ICOSL蛋白的抗體的結合程度小於該抗體與ICOSL結合的約10%，該結合藉由電漿共振測定法測量(例如BIACORE^®)。在某些實施例中，與ICOSL蛋白結合的抗體具有以下解離常數(KD)<約1μM、<約100nM、<約10nM、<約1nM、<約0.1nM、<約0.01nM或<約0.001nM。在某些實施例中，抗ICOSL抗體結合人或食蟹猴ICOSL抗原表位。 The terms "anti-ICOSL antibody" and "ICOSL-binding antibody" refer to antibodies capable of binding ICOSL or an epitope thereof with sufficient affinity. In one embodiment, the degree of binding of the antibody to an unrelated, non-ICOSL protein is less than about 10% of the antibody's binding to ICOSL, as measured by a plasmon resonance assay (eg, ^BIACORE® ). In certain embodiments, the antibody that binds to the ICOSL protein has a dissociation constant (KD) of < about 1 μM, < about 100 nM, < about 10 nM, < about 1 nM, < about 0.1 nM, < about 0.01 nM, or < about 0.001 nM. . In certain embodiments, anti-ICOSL antibodies bind human or cynomolgus monkey ICOSL epitopes.

術語“連接子”、“Linker”或“接頭”指連接兩個多肽片段的連接單元，通常具有一定的柔性，接頭的使用不會使蛋白質結構域原有的功能喪失。在本文中，同一結構中出現的連接子可以是相同或不同的。連接子可以是肽連接子，其包含一個或多個胺基酸，典型的約1-30個、2-24個或3-15個胺基酸。應用於本文的連接子可以是相同或不同的。 The terms "linker", "Linker" or "linker" refer to the connecting unit that connects two polypeptide fragments, which usually has a certain degree of flexibility. The use of the linker will not cause the original function of the protein domain to be lost. exist Herein, linkers appearing in the same structure may be the same or different. The linker can be a peptide linker, which contains 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.

術語“抗體依賴性細胞的細胞毒性”、“抗體依賴性細胞介導的細胞毒性”或“ADCC”是誘導細胞死亡的機制，該機制依賴於抗體包被靶細胞與具有裂解活性的效應細胞(諸如自然殺傷細胞(NK)、單核細胞、巨噬細胞和中性粒細胞)經由效應細胞上表達的Fcγ受體(FcγR)發生的相互作用。例如，NK細胞表達FcγRIIIa，而單核細胞表達FcγRI、FcγRII和FcγRIIIa。本文提供的抗體的ADCC活性可使用體外測定，使用表達抗原的細胞作為靶細胞和NK細胞作為效應細胞進行評定。根據從裂解的細胞中釋放的標記物(例如放射性受質、螢光染料或天然胞內蛋白)來檢測細胞裂解。 The terms "antibody-dependent cellular cytotoxicity", "antibody-dependent cell-mediated cytotoxicity" or "ADCC" are mechanisms of induction of cell death that rely on antibody coating of target cells in conjunction with effector cells with lytic activity ( Such as natural killer cells (NK), monocytes, macrophages, and neutrophils) interact via Fcγ receptors (FcγR) expressed on effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcγRIIIa. The ADCC activity of the antibodies provided herein can be assessed using an in vitro assay using cells expressing the antigen as target cells and NK cells as effector cells. Cell lysis is detected based on markers released from lysed cells, such as radioactive receptors, fluorescent dyes, or native intracellular proteins.

術語“抗體依賴性細胞吞噬作用”(“ADCP”)是指藉由吞噬細胞(諸如巨噬細胞或樹突狀細胞)的內化作用消除抗體包被的靶細胞的機制。 The term "antibody-dependent cellular phagocytosis" ("ADCP") refers to the mechanism by which antibody-coated target cells are eliminated by internalization by phagocytes, such as macrophages or dendritic cells.

術語“補體依賴性細胞毒性”或“CDC”是指誘導細胞死亡的機制，其中靶結合抗體的Fc效應域結合並啟動補體成分C1q，C1q繼而啟動補體級聯，從而導致靶細胞死亡。補體的啟動也可導致補體成分沉積在靶細胞表面上，這些補體成分藉由結合白細胞上的補體受體(例如，CR3)來促進CDC。 The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death in which the Fc effector domain of a target-binding antibody binds and initiates the complement component C1q, which in turn initiates the complement cascade, resulting in target cell death. Initiation of complement can also lead to the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (eg, CR3) on leukocytes.

術語“核酸”在本文中可與術語“多核苷酸”互換使用，並且是指呈單鏈或雙鏈形式的去氧核糖核苷酸或核糖核苷酸及其聚合物。該術語涵蓋含有已知核苷酸類似物或修飾的骨架殘基或連接的核酸，該核酸是合成的、天然存在的和非天然存在的，具有與參考核酸相似的結合特性，並且以類似於參考核苷酸的方式代謝。此類類似物的實例包括但不限於硫代磷酸酯、胺基磷酸酯、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)。“分離的”核酸指已經與其天然環境的組分分開的核酸分子。分離的核酸包括在下述細胞中含有的核酸分子，該細胞通常含有該核酸分子，但該核酸分子存在於染色體外或存在於不同於其天然染色體位置的染色體位置處。編碼多肽或融合蛋白的分離的核酸指編碼多肽或融合蛋白的一個或更多個核酸分子，包括在單一載體或分開的載體中的這樣的一個或更多個核酸分子，和存在於宿主細胞中一個或更多個位置的這樣的一個或更多個核酸分子。除非另有說明，否則特定的核酸序列還隱含地涵蓋其保守修飾的變體(例如，簡併密碼子取代)和互補序列以及明確指明的序列。具體地，如下詳述，簡併密碼子取代可以藉由產生如下序列而獲得，在這些序列中，一個或多個所選的(或全部)密碼子的第三位元被混合鹼基和/或去氧肌苷殘基取代。 The term "nucleic acid" is used interchangeably herein 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 that are synthetic, naturally occurring and non-naturally occurring, have similar binding properties to the reference nucleic acid, and are produced in a manner similar to Metabolism in the manner of reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, aminophosphates, methylphosphine Acid ester, chiral-methylphosphonate, 2-O-methylribonucleotide, peptide-nucleic acid (PNA). An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain the nucleic acid molecule but which are present extrachromosomally or at a chromosomal location that is different from its native chromosomal location. Isolated nucleic acid encoding a polypeptide or fusion protein refers to one or more nucleic acid molecules encoding a polypeptide or fusion protein, including such one or more nucleic acid molecules in a single vector or separate vectors, and present in a host cell One or more such nucleic acid molecules at one or more positions. Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences thereof as well as sequences explicitly indicated. Specifically, as detailed below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced by a mixed base and/or Deoxyinosine residue substitution.

術語“多肽”和“蛋白質”在本文中可互換使用，指胺基酸殘基的聚合物。該術語適用於胺基酸聚合物，其中一個或多個胺基酸殘基是相應天然存在的胺基酸或其人工化學類比物，以及天然存在的胺基酸聚合物和非天然存在的胺基酸聚合物。除非另外說明，否則特定的多肽序列還隱含地涵蓋其保守修飾的變體。 The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are the corresponding naturally occurring amino acids or artificial chemical analogs thereof, as well as naturally occurring amino acid polymers and non-naturally occurring amines acid polymer. Unless stated otherwise, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

術語序列“同一性”指，當對2條序列進行最佳比對時，2條序列的胺基酸/核酸在等價位置相同的程度(百分比)；其中，必要時在最佳比對中引入間隙以獲取最大序列同一性百分比，且不將任何保守性取代視為序列同一性的一部分。為測定序列同一性百分比，比對可以藉由本領域技術已知的技術來實現，例如使用公開可得到的電腦軟體，諸如BLAST、BLAST-2、ALIGN、ALIGN- 2或Megalign(DNASTAR)軟體。所屬技術領域具有通常知識者可確定適用於測量比對的參數，包括在所比較的序列全長上達成最大比對所需的任何演算法。 The term sequence "identity" refers to the extent (percentage) that the amino acids/nucleic acids of the two sequences are identical at equivalent positions when the two sequences are optimally aligned; where necessary, in the optimal alignment Gaps were introduced to obtain maximum percent sequence identity, and any conservative substitutions were not considered part of the sequence identity. To determine percent sequence identity, alignment can be performed by techniques known to those skilled in the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN- 2 or Megalign (DNASTAR) software. One of ordinary skill in the art can determine parameters suitable for measuring alignment, including any algorithms required to achieve maximal alignment over the entire length of the sequences being compared.

本揭露中，例如“相對於序列SEQ ID NO：XX的自然順序編號的胺基酸殘基位元點”意指物件序列與SEQ ID NO：XX序列進行最佳比對，獲得最高百分比同一性，此時對象序列與SEQ ID NO：XX序列對應位置的位點為2條序列的相對位點。例如TACI的胞外區序列SEQ ID NO：51和SEQ ID NO：60上相對於SEQ ID NO：58的自然順序編號的胺基酸殘基位點如表2所示： In this disclosure, for example, "amino acid residue positions numbered relative to the natural sequence of the sequence SEQ ID NO: XX" means that the object sequence is optimally aligned with the sequence SEQ ID NO: XX to obtain the highest percent identity. , at this time, the position corresponding to the target sequence and the SEQ ID NO: XX sequence is the relative position of the two sequences. For example, 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 number of SEQ ID NO: 58 are shown in Table 2:

表2. SEQ ID NO：58位點在其他序列上的相應位點 Table 2. Corresponding positions of SEQ ID NO: 58 on other sequences

(以SEQ ID NO：51或/SEQ ID NO：60為例)

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

術語“融合”或“連接”是指部件(例如TACI多肽和抗體重/輕鏈)直接地或經由一個或多個連接子藉由共價鍵連接。當連接子是肽連接子時，該共價鍵是肽鍵。 The term "fused" or "linked" means that the components (eg, TACI polypeptide and antibody heavy/light chain) are joined by a covalent bond, either directly or via one or more linkers. When the linker is a peptide linker, the covalent bond is a peptide bond.

術語“抗ICOSL抗體融合蛋白”是指抗ICOSL抗體與活性蛋白融合的蛋白質。例如，TACI多肽融合到抗ICOSL抗體的重鏈或輕鏈的N末端或C末端而形成蛋白。 The term "anti-ICOSL antibody fusion protein" refers to a protein in which an anti-ICOSL antibody is fused to an active protein. For example, a TACI polypeptide is fused to the N-terminus or C-terminus of the heavy chain or light chain of an anti-ICOSL antibody to form a protein.

術語“載體”意指能夠轉運與其連接的另一多核苷酸的多核苷酸分子。一種類型的載體是“質粒”，其是指環狀雙鏈DNA環，其中可以連接附加的DNA區段。另一種類型的載體是病毒載體，例如腺相關病毒載體(AAV或AAV2)，其中另外的DNA區段可以連接到病毒基因組中。某些載體能夠在引入它們的宿主細胞中自主複製(例如，具有細菌複製起點的細菌載體和附加型哺乳動物載體)。其他載體(例如，非附加型哺乳動物載體)可以在引入宿主細胞中後整合到宿主細胞的基因組中，從而與宿主基因組一起複製。術語“表達載體”或“表達構建體”是指可對宿主細胞進行轉化，且含有指導和/或控制(連同宿主細胞一起)與其可操作地連接的一個或多個異源編碼區的表達的核酸序列的載體。表達構建體可以包括但不限於影響或控制轉錄、轉譯且在存在內含子時影響與其可操作地連接的編碼區的RNA剪接的序列。 The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA circle into which additional DNA segments can be ligated. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can be introduced into the host cell and integrated into the host cell's genome, thereby replicating with the host genome. The term "expression vector" or "expression construct" refers to a vector capable of transforming a host cell and containing a vector that directs and/or controls (together with the host cell) the expression of one or more heterologous coding regions operably linked thereto. Nucleic acid sequence vectors. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation and, in the presence of introns, RNA splicing of the coding region operably linked thereto.

術語“宿主細胞”、“宿主細胞系”和“宿主細胞培養物”可互換使用，並且指已經導入外源核酸的細胞，包括此類細胞的後代。宿主細胞包括“轉化體”和“經轉化的細胞”，其包括原代的經轉化的細胞及自其衍生的後代，而不考慮傳代的次數。後代在核酸內容物上可以與親本細胞不完全相同，而是可以含有突變。本文中包括與在初始轉化細胞中篩選或選擇的細胞具有相同功能或生物學活性的突變體後代。宿主細胞包括原核和真核宿主細胞，其中真核宿主細胞包括但不限於哺乳動物細胞、昆蟲細胞系、植物細胞和真菌細胞。哺乳動物宿主細胞包括人、小鼠、大鼠、犬、猴、豬、山羊、牛、馬和倉鼠細胞，包括但不限於中國倉鼠卵巢(CHO)細胞、NSO、SP2細胞、HeLa細胞、幼倉鼠腎(BHK)細胞、猴腎細胞(COS)、人肝細胞癌細胞(例如，Hep G2)、A549細胞、3T3細胞和HEK-293細胞。真菌細胞包括酵母和絲狀真菌細胞，包括例如巴氏畢赤酵母(Pichiapastoris)、芬蘭畢赤酵母(Pichia finlandica)、海藻畢赤酵母(Pichia trehalophila)、科克拉馬畢赤酵母(Pichia koclamae)、膜狀畢赤酵母(Pichia membranaefaciens)、小畢赤酵母(Pichia minuta)(Ogataea minuta、Pichia lindneri)、仙人掌畢赤酵母(Pichiaopuntiae)、耐熱畢赤酵母(Pichia thermotolerans)、柳畢赤酵母(Pichia salictaria)、Pichia guercuum、皮傑普畢赤酵母(Pichia pijperi)、具柄畢赤酵母(Pichia stiptis)、甲醇畢赤酵母(Pichia methanolica)、畢赤酵母屬、釀酒酵母(Saccharomycescerevisiae)、釀酒酵母屬、多形漢遜酵母(Hansenula polymorpha)、克魯維酵母屬、乳酸克魯維酵母(Kluyveromyces lactis)、白色念珠菌(Candida albicans)、構巢麯黴(Aspergillus nidulans)、黑麯黴(Aspergillus niger)、米麯黴(Aspergillus oryzae)、裡氏木黴(Trichoderma reesei)、勒克氏菌(Chrysosporium lucknowense)、鐮刀菌屬(Fusarium sp.)、禾穀鐮刀菌(Fusarium gramineum)、菜鐮刀菌(Fusarium venenatum)、小立碗蘚(Physcomitrella patens)和粗糙脈孢菌(Neurospora crassa)、麯黴屬、、解脂耶氏酵母(Yarrowia lipolytica)。 The terms "host cell,""host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parent cells, but may contain mutations. Mutant progeny that have the same function or biological activity as the cells screened or selected in the initially 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, canine, monkey, porcine, goat, bovine, equine, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (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 opuntiae, Pichia thermotolerans , Pichia salictaria ), Pichia guercuum , Pichia pijperi , Pichia stiptis , Pichia methanolica , Pichia genus, Saccharomycescerevisiae , Saccharomyces cerevisiae, Hansenula polymorpha , Kluyveromyces lactis, Kluyveromyces lactis, Candida albicans , Aspergillus nidulans , Aspergillus niger , oryzae Aspergillus oryzae , Trichoderma reesei , Chrysosporium lucknowense , Fusarium sp. , Fusarium gramineum , Fusarium venenatum , Physcomitrella patens and Neurospora crassa , Aspergillus, and Yarrowia lipolytica .

“視需要”或“視需要地”意味著隨後所描述地事件或環境可以但不必發生，該說明包括該事件或環境發生或不發生的場合。 "As appropriate" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance does or does not occur.

術語“醫藥組成物”表示含有一種或多種本文所述的抗ICOSL抗體融合蛋白與其他化學組分的混合物，該其他組分例如生理學/可藥用的載體和賦形劑。 The term "pharmaceutical composition" refers to a mixture containing one or more anti-ICOSL antibody fusion proteins described herein and other chemical components, such as physiological/pharmaceutically acceptable carriers and excipients.

術語“藥學上可接受的載體、稀釋劑或賦形劑”指藥學配製劑中與活性成分不同的，且對受試者無毒的成分；例如，但不限於緩衝劑、賦形劑、穩定劑或防腐劑。 The term "pharmaceutically acceptable carrier, diluent or excipient" refers to an ingredient in a pharmaceutical formulation that is different from the active ingredient and is not toxic to the subject; such as, but not limited to, buffers, excipients, stabilizers or preservatives.

術語“受試者”或“個體”包括人類和非人類動物。非人動物包括所有脊椎動物(例如哺乳動物和非哺乳動物)例如非人靈長類(例如，食蟹猴)、綿羊、狗、牛、雞、兩棲動物和爬行動物。除非指出時，否則該術語“患者”或“受試者”在本文中可互換地使用。如本文所使用的，術語“食蟹猴(cyno)”或“食蟹猴(cynomolgus)”是指食蟹猴(Macaca fascicularis)。在某些實施方案中，個體或受試者是人。 The term "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, cattle, chickens, amphibians, and reptiles. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein. As used herein, the term "cyno" or "cynomolgus" refers to the crab-eating monkey ( Macaca fascicularis ). In certain embodiments, the individual or subject is a human.

“施用”或“給予”，當其應用於動物、人、實驗受試者、細胞、組織、器官或生物流體時，是指外源性藥物、治療劑、診斷劑或組成物與動物、人、受試者、細胞、組織、器官或生物流體的接觸。 "Administration" or "administration", when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, means the administration of an exogenous drug, therapeutic, diagnostic or composition to an animal, human , contact with subjects, cells, tissues, organs or biological fluids.

術語“樣品”是指從受試者分離的流體、細胞、或組織的採集物，以及存在於受試者體內的流體、細胞或組織。示例性樣品為生物流體，諸如血液、血清、漿膜液、血漿、淋巴液、尿液、唾液、囊液、淚液、***物、痰、分泌組織和器官的黏膜分泌物、***分泌物、腹水、胸膜、心包、腹膜、腹腔和其它體腔的流體、由支氣管灌洗液收集的流體、滑液、與受試者或生物來源接觸的液體溶液，例如細胞和器官培養基(包括細胞或器官條件培養基)、灌洗液等、組織活檢樣品、細針穿刺、手術切除的組織、器官培養物或細胞培養物。 The term "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. Exemplary samples are biological fluids such as blood, serum, serosal fluid, plasma, lymph fluid, urine, saliva, cystic fluid, tears, excreta, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, Fluids from the pleura, pericardium, peritoneum, peritoneal cavity and other body cavities, fluid collected from bronchial lavage, synovial fluid, fluid solutions in contact with subjects or biological sources, such as cell and organ culture media (including cell or organ conditioned media) , lavage fluid, etc., tissue biopsy samples, fine needle aspiration, surgically removed tissue, organ culture or cell culture.

“治療(treatment或treat)”和“處理”(及其語法變型)指試圖改變所治療個體的過程的臨床干預，並且可以為了預防或者在臨床病理學的過程期間實施。治療的期望效果包括但不限於預防疾病的發生或再發生，減輕症狀，減輕/減少疾病的任何直接或間接病理後果，預防轉移，降低疾病進展速率，改善或減輕疾病狀態，和消退或改善的預後。在一些實施方案中，使用本揭露的抗體或融合蛋白來延遲疾病的形成或減緩疾病的進展。 "Treatment" and "treatment" (and their grammatical variations) refer to a clinical intervention that attempts to alter the course of the individual being treated, and may be performed for prevention or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, reducing Lighten/reduce any direct or indirect pathological consequences of the disease, prevent metastasis, reduce the rate of disease progression, improve or alleviate the disease state, and resolve or improve the prognosis. In some embodiments, the antibodies or fusion proteins of the present disclosure are used to delay the development of disease or slow the progression of disease.

“有效量”一般是足以降低症狀的嚴重程度及/或頻率、消除這些症狀及/或潛在病因、預防症狀及/或其潛在病因出現及/或改良或改善由疾病狀態引起或與其相關的損傷的量。在一些實施例中，有效量是治療有效量或預防有效量。“治療有效量”是足以治療疾病狀態或症狀、尤其與該疾病狀態相關的狀態或症狀，或者以其他方式預防、阻礙、延遲或逆轉該疾病狀態或以任何方式與該疾病相關的任何其他不理想症狀的進展的量。“預防有效量”是當給予受試者時將具有預定預防效應，例如預防或延遲該疾病狀態的發作(或復發)，或者降低該疾病狀態或相關症狀的發作(或復發)可能性的量。完全治療或預防效未必在給予一個劑量之後便發生，可能在給予一系列劑量之後發生。因而，治療或預防有效量可以一次或多次給予的方式給予。“治療有效量”和“預防有效量”可取決於多種因素變化：諸如個體的疾病狀態、年齡、性別和體重，以及治療劑或治療劑組合在個體中引發期望的應答的能力。有效治療劑或治療劑組合的示例性指標包括例如患者改善的健康狀況。 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 impairments caused by or associated with a disease state. amount. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is one sufficient to treat a disease state or symptom, particularly a condition or symptom associated with that disease state, or to otherwise prevent, hinder, delay or reverse the disease state or any other adverse effect in any way related to the disease. The ideal amount of symptomatic progression. A "prophylactically effective amount" is an amount that, when administered to a subject, will have a predetermined prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or related symptoms. . Complete therapeutic or prophylactic effect does not necessarily occur after administration of one dose but may occur after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations. "Therapeutically effective amount" and "prophylactically effective amount" may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient.

本揭露的抗ICOSL抗體The disclosed anti-ICOSL antibodies

在一個方面中，本揭露設計了一種新的抗ICOSL抗體。其具有以下一種或更多種特性： In one aspect, the present disclosure designs a new anti-ICOSL antibody. It has one or more of the following properties:

A.與人ICOSL和食蟹猴ICOSL或其表位特異性結合，不與鼠ICOSL特異性結合；較佳地，以小於5.00E-09M(例如小於5.00E-09M、小於4.00E-09M、小於3.00E-09M、小於2.00E-09M、小於1.50E-09M、9.00E-10M或更小)的KD 值與人ICOSL結合，和/或以小於8.00E-09M(例如小於8.00E-09M、小於7.00E-09M、小於6.00E-09M、小於5.00E-09M、小於4.00E-09M、小於3.00E-09M或更小)的KD值與食蟹猴ICOSL結合，該KD值藉由表面電漿共振測定法所測量(例如BIACORE^®)；在一些實施方案中，該KD值藉由本揭露測試例8方法檢測； A. Specifically binds to human ICOSL and cynomolgus ICOSL or its epitope, but does not specifically bind to mouse ICOSL; preferably, less than 5.00E-09M (for example, less than 5.00E-09M, less than 4.00E-09M, less than Binds to human ICOSL with a KD value of less than 3.00E-09M, less than 2.00E-09M, less than 1.50E-09M, 9.00E-10M or less), and/or with a KD value less than 8.00E-09M (e.g., less than 8.00E-09M, A KD value of 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) binds to cynomolgus monkey ICOSL, and the KD value is determined by surface electricity Measured by plasma resonance measurement method (such as BIACORE ^® ); in some embodiments, the KD value is detected by the method of Test Example 8 of the present disclosure;

B.具有阻斷ICOSL與ICOS結合活性；較佳地，阻斷人ICOSL與ICOS結合的IC₅₀值小於0.16nM(例如小於0.16nM、小於0.14nM、小於0.12nM、小於0.10nM、小於0.09nM、小於0.08nM、小於0.07nM、小於0.06nM、小於0.05nM、小於0.04nM或更小)，該IC₅₀值藉由FACS方法檢測；在一些實施方案中，該IC₅₀值藉由本揭露測試例3方法檢測； B. Has the activity of blocking the binding of ICOSL to ICOS; preferably, the IC ₅₀ value of blocking the binding of human ICOSL to ICOS is less than 0.16nM (for example, 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 IC ₅₀ value is detected by the FACS method; in some embodiments, the IC ₅₀ value is detected by the test example of the present disclosure 3 method detection;

C.具有抑制IFNγ、IL-10和/或IgA細胞因子分泌活性；在一些實施方案中，該IC50值藉由本揭露測試例9方法檢測； C. Has the activity of inhibiting IFNγ, IL-10 and/or IgA cytokine secretion; in some embodiments, the IC50 value is detected by the method of Test Example 9 of the present disclosure;

D.具有抑制T細胞增殖活性；在一些實施方案中，該抗ICOSL抗體或抗ICOSL抗體融合蛋白抑制ICOSL誘導T細胞增殖的活性；在一些實施方案中，該抑制T細胞增殖活性藉由本揭露測試例6方法檢測。 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 inhibitory activity of T cell proliferation is tested by the present disclosure Example 6 method detection.

示例性的抗ICOSL抗體Exemplary anti-ICOSL antibodies

一方面，本揭露提供一種抗ICOSL抗體，其中該抗ICOSL抗體包含重鏈可變區和輕鏈可變區，其中，該重鏈可變區包含HCDR1、HCDR2和HCDR3，該輕鏈可變區包含LCDR1、LCDR2和LCDR3，其中， In one aspect, the disclosure provides an anti-ICOSL antibody, wherein the anti-ICOSL antibody includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region Contains LCDR1, LCDR2 and LCDR3, among which,

在一些實施方案中，如上任一項所述的抗ICOSL抗體，其中該重鏈可變區的HCDR1、HCDR2和HCDR3以及輕鏈可變區的LCDR1、LCDR2和LCDR3是根據選自Kabat編號規則定義的。 In some embodiments, the anti-ICOSL antibody as described in any one of the above, wherein the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to the Kabat numbering rule. of.

(i)該重鏈可變區的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，和該輕鏈可變區的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 (i) The heavy chain variable region has HCDR1 as set forth in SEQ ID NO: 11, HCDR2 as set forth in SEQ ID NO: 31 or 12, and HCDR3 as set forth in SEQ ID NO: 13, and the light chain variable region LCDR1 as set forth in SEQ ID NO: 14, LCDR2 as set forth in SEQ ID NO: 15, and LCDR3 as set forth in SEQ ID NO: 16; or

(ii)該重鏈可變區的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，和該輕鏈可變區的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。 (ii) The heavy chain variable region has HCDR1 as set forth in SEQ ID NO: 5, HCDR2 as set forth in SEQ ID NO: 19 or 6, and HCDR3 as set forth in SEQ ID NO: 7, and the light chain variable region The LCDR1 is shown in SEQ ID NO: 8, the LCDR2 is shown in SEQ ID NO: 17 or 9, and the LCDR3 is shown in SEQ ID NO: 10 or 18.

(i)該重鏈可變區的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，和該輕鏈可變區的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 (i) HCDR1 of the heavy chain variable region is set forth in SEQ ID NO: 11, HCDR2 is set forth in SEQ ID NO: 31, and HCDR3 is set forth in SEQ ID NO: 13, and LCDR1 of the light chain variable region as set forth in SEQ ID NO: 14, LCDR2 as set forth in SEQ ID NO: 15, and LCDR3 as set forth in SEQ ID NO: 16; or

(ii)該重鏈可變區的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，和該輕鏈可變區的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。 (ii) HCDR1 of the heavy chain variable region is set forth in SEQ ID NO: 5, HCDR2 is set forth in SEQ ID NO: 19, and HCDR3 is set forth in SEQ ID NO: 7, and LCDR1 of the light chain variable region As shown in SEQ ID NO:8, LCDR2 is shown in SEQ ID NO:17, and LCDR3 is shown in SEQ ID NO:10.

在一些實施方案中，如前任一項所述的抗ICOSL抗體，該抗ICOSL抗體是鼠源、嵌合或人源化的抗體。在一些實施方案中，該抗ICOSL抗體是人源化的。 In some embodiments, the anti-ICOSL antibody as described in any one of the preceding items is a murine, chimeric or humanized antibody. In some embodiments, the anti-ICOSL antibody is humanized.

在一些實施方案中，如前任一項所述的抗ICOSL抗體是人源化抗體。在一些實施方案中，該抗ICOSL抗體的重鏈可變區具有來源於IGHV3-74*03的FR1、FR2、FR3和來源於IGHJ1*01的FR4，並且其是未被取代的或具有選自37I、43E和49A(根據Kabat編號系統編號)組成的組中的一個或更多個胺基酸取代；和/或該輕鏈可變區具有來源於IGKV4-1*01/IGKV1-39*01的FR1、FR2、FR3和來源於IGKJ2*01的FR4，並且其是未被取代的或具有選自1N、2T、3V、42Q、43S、60D、85V(根據Kabat編號系統編號)組成的組中的一個或更多個胺基酸取代。在一些實施方案中，該抗ICOSL抗體，其中該重鏈可變區的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，和該輕鏈可變區的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。上述可變區和CDR是根據Kabat編號規則定義的。 In some embodiments, the anti-ICOSL antibody of any one of the preceding items is a humanized antibody. In some embodiments, the heavy chain variable region of the anti-ICOSL antibody has FR1, FR2, FR3 derived from IGHV3-74*03 and FR4 derived from IGHJ1*01, and is unsubstituted or has FR4 derived from IGHJ1*01. One or more amino acid substitutions in the group consisting of 37I, 43E and 49A (numbered according to the Kabat numbering system); and/or the light chain variable region has an amino acid substitution derived from IGKV4-1*01/IGKV1-39*01 FR1, FR2, FR3 and FR4 derived from IGKJ2*01 and which are unsubstituted or have a component selected from the group consisting of 1N, 2T, 3V, 42Q, 43S, 60D, 85V (numbered according to the Kabat numbering system) One or more amino acid substitutions. In some embodiments, the anti-ICOSL antibody, wherein the HCDR1 of the heavy chain variable region is as set forth in SEQ ID NO: 11, the HCDR2 is as set forth in SEQ ID NO: 31 or 12, and the HCDR3 is as set forth in SEQ ID NO: 13 is shown, and LCDR1 of the light chain variable region is shown in SEQ ID NO: 14, LCDR2 is shown in SEQ ID NO: 15, and LCDR3 is shown in SEQ ID NO: 16. The above variable regions and CDRs are defined according to Kabat numbering rules.

在一些實施方案中，如前任一項所述的抗ICOSL抗體是人源化抗體，在一些實施方案中，該抗ICOSL抗體的重鏈可變區具有來源於IGHV1-46*01的FR1、FR2、FR3和來源於IGHJ6*01的FR4，並且其是未被取代的或具有選自1E、24T、69L、71V、73K和78A(根據Kabat編號系統編號)組成的組中的一個或更多個胺基酸取代；和/或該輕鏈可變區具有來源於IGKV2-28*01的FR1、FR2、FR3和來源於IGKJ4*01的FR4，並且其是未被取代的或具有選自1A、2V和4L(根據Kabat編號系統編號)組成的組中的一個或更多個胺基酸取代。在一些實施方案中，該抗ICOSL抗體，其中該重鏈可變區的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，和該輕鏈可變區的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。上述可變區和CDR是根據Kabat編號規則定義的。 In some embodiments, the anti-ICOSL antibody as described in any one of the preceding items is a humanized antibody. In some embodiments, the heavy chain variable region of the anti-ICOSL antibody has FR1, FR2 derived from IGHV1-46*01 , FR3 and FR4 derived from IGHJ6*01 and which is unsubstituted or has one or more selected from the group consisting of 1E, 24T, 69L, 71V, 73K and 78A (numbered according to the Kabat numbering system) Amino acid substitution; 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 component selected from 1A, Substitution of one or more amino acids in the group consisting of 2V and 4L (numbered according to the Kabat numbering system). In some embodiments, the anti-ICOSL antibody, wherein the HCDR1 of the heavy chain variable region is as set forth in SEQ ID NO: 5, the HCDR2 is as set forth in SEQ ID NO: 19 or 6, and the HCDR3 is as set forth in SEQ ID NO: 7 is shown, and the LCDR1 of the light chain variable region is shown in SEQ ID NO: 8, and the LCDR2 of the light chain variable region is shown in SEQ ID SEQ ID NO: 17 or 9, and LCDR3 as SEQ ID NO: 10 or 18. The above variable regions and CDRs are defined according to Kabat numbering rules.

(i)該重鏈可變區如SEQ ID NO：42、39、40或41所示，和該輕鏈可變區如SEQ ID NO：38、32、33、34、35、36或37所示；或 (i) The heavy chain variable region is as set forth in SEQ ID NO: 42, 39, 40 or 41, and the light chain variable region is as set forth in SEQ ID NO: 38, 32, 33, 34, 35, 36 or 37 indication; or

(ii)該重鏈可變區如SEQ ID NO：30、28或29所示，和該輕鏈可變區如SEQ ID NO：22、20、21、23、24、25、26或27所示；或 (ii) The heavy chain variable region is as set forth in SEQ ID NO: 30, 28 or 29, and the light chain variable region is as set forth in SEQ ID NO: 22, 20, 21, 23, 24, 25, 26 or 27 indication; or

(iii)該重鏈可變區如SEQ ID NO：1所示，和該輕鏈可變區如SEQ ID NO：2所示；或 (iii) the heavy chain variable region is represented by SEQ ID NO: 1, and the light chain variable region is represented by SEQ ID NO: 2; or

(iv)該重鏈可變區如SEQ ID NO：3所示，和該輕鏈可變區如SEQ ID NO：4所示。 (iv) The heavy chain variable region is represented by SEQ ID NO:3, and the light chain variable region is represented by SEQ ID NO:4.

(i)該重鏈可變區如SEQ ID NO：42所示，和該輕鏈可變區如SEQ ID NO：38所示；或 (i) the heavy chain variable region is set forth in SEQ ID NO: 42, and the light chain variable region is set forth in SEQ ID NO: 38; or

(ii)該重鏈可變區如SEQ ID NO：30所示，和該輕鏈可變區如SEQ ID NO：22所示。 (ii) The heavy chain variable region is set forth in SEQ ID NO:30, and the light chain variable region is set forth in SEQ ID NO:22.

在一些實施方案中，如上任一項所述的抗ICOSL抗體，其中該抗ICOSL抗體包含抗體重鏈恆定區和輕鏈恆定區。在一些實施方案中，該重鏈恆定區為人IgG重鏈恆定區。在一些實施方案中，該重鏈恆定區選自人IgG1、IgG2、IgG3和IgG4恆定區；在一些實施方案中，該輕鏈恆定區選自人抗體κ或λ鏈恆定區。在一些實施方案中，該重鏈恆定區為人IgG4重鏈恆定區，該輕鏈恆定區為人κ輕鏈恆定區。在一些實施方案中，該重鏈恆定區的Fc區具有一個或更多個能夠減少Fc區與Fc受體結合的胺基酸取代。在一些實施方案中，該Fc區具有L234A、L235A突變，和/或S228P突變，和/或YTE突變(M252Y、S254T和T256E)，該突變編號依據為EU索引。在一些實施方案中，該重鏈恆定區包含SEQ ID NO：43的胺基酸序列，該輕鏈恆定區包含SEQ ID NO：44的胺基酸序列。 In some embodiments, the anti-ICOSL antibody as described in any one of the above, wherein the anti-ICOSL antibody comprises an antibody heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is a human IgG heavy chain constant region. In some embodiments, the heavy chain constant region is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 constant regions; in some embodiments, the light chain constant region is selected from the group consisting of human antibody kappa or lambda chain constant regions. In some embodiments, the heavy chain constant region is a human IgG4 heavy chain constant region and the light chain constant region is a human kappa light chain constant region. In some embodiments, the Fc region of the heavy chain constant region has one or more An amino acid substitution that reduces the binding of the Fc region to Fc receptors. In some embodiments, the Fc region has L234A, L235A mutations, and/or S228P mutations, and/or YTE mutations (M252Y, S254T, and T256E), and the mutation numbering is based on the EU index. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:43 and the light chain constant region comprises the amino acid sequence of SEQ ID NO:44.

(i)該抗ICOSL抗體的重鏈與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，和輕鏈與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 (i) 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 has at least 90% sequence identity to SEQ ID NO: 47 ID NO: 48 has at least 90% (eg, at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity; or

(ii)該抗ICOSL抗體的重鏈與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，和輕鏈與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。 (ii) 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 has at least 90% sequence identity to SEQ ID NO: 45 ID NO: 46 has at least 90% (eg, at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

(i)該抗ICOSL抗體的重鏈如SEQ ID NO：47所示，和該抗ICOSL抗體的輕鏈如SEQ ID NO：48所示；或 (i) The heavy chain of the anti-ICOSL antibody is set forth in SEQ ID NO: 47, and the light chain of the anti-ICOSL antibody is set forth in SEQ ID NO: 48; or

(ii)該抗ICOSL抗體的重鏈如SEQ ID NO：45所示，和該抗ICOSL抗體的輕鏈如SEQ ID NO：46所示。 (ii) The heavy chain of the anti-ICOSL antibody is set forth in SEQ ID NO:45, and the light chain of the anti-ICOSL antibody is set forth in SEQ ID NO:46.

本揭露的抗ICOSL抗體融合蛋白Anti-ICOSL antibody fusion proteins of the present disclosure

本揭露構建了一種抗ICOSL抗體融合蛋白，其包含抗ICOSL抗體和TACI多肽，其中該抗ICOSL抗體特異性結合人ICOSL或其表位。 The present disclosure constructs an anti-ICOSL antibody fusion protein, which includes an anti-ICOSL antibody and a TACI polypeptide, wherein the anti-ICOSL antibody specifically binds human ICOSL or its epitope.

在一些實施方案中，前面任一項所述抗ICOSL抗體融合蛋白，其具有一種或更多種以下特性： In some embodiments, the anti-ICOSL antibody fusion protein of any of the preceding items has one or more of the following properties:

A.與人ICOSL和食蟹猴ICOSL特異性結合，不與鼠ICOSL特異性結合；較佳地，以小於5.00E-09M(例如小於5.00E-09M、小於4.00E-09M、小於3.00E-09M、小於2.00E-09M、小於1.50E-09M、9.00E-10M或更小)的KD值與人ICOSL結合，和/或以小於8.00E-09M(例如小於8.00E-09M、小於7.00E-09M、小於6.00E-09M、小於5.00E-09M、小於4.00E-09M、小於3.00E-09M或更小)的KD值與食蟹猴ICOSL結合，該KD值藉由表面電漿共振測定法所測量(例如BIACORE^®)；在一些實施方案中，該KD值藉由本揭露測試例8方法檢測； A. Specifically binds to human ICOSL and cynomolgus ICOSL, but does not specifically bind to mouse ICOSL; preferably, less than 5.00E-09M (for example, less than 5.00E-09M, less than 4.00E-09M, less than 3.00E-09M , less than 2.00E-09M, less than 1.50E-09M, 9.00E-10M or less) binds to human ICOSL, and/or binds to human ICOSL with a KD value less than 8.00E-09M (e.g., less than 8.00E-09M, less than 7.00E- 09M, less than 6.00E-09M, less than 5.00E-09M, less than 4.00E-09M, less than 3.00E-09M or less) KD value combined with cynomolgus monkey ICOSL, the KD value was measured by surface plasmon resonance Measured (such as BIACORE ^® ); in some embodiments, the KD value is detected by the method of Test Example 8 of the present disclosure;

B.與人APRIL、食蟹猴APRIL和/或鼠APRIL特異性結合；較佳地，以小於2.00E-11M(例如小於2.00E-11M、小於1.80E-11M、小於1.70E-11M、小於1.60E-11M、小於1.20E-11M、小於4.60E-12M或更小)的KD值與人APRIL結合，以小於2.00E-10M(例如小於2.00E-10M、小於1.80E-10M、小於1.70E-10M、小於1.60E-10M、小於1.50E-10M、小於1.40E-10M、小於1.20E-10M或更小)的KD值與食蟹猴APRIL結合，和/或以小於2.00E-11M(例如小於2.00E-11M、小於1.80E-11M、小於1.60E-11M、小於1.40E-11M、小於1.20E-11M、小於1.00E-11M、小於8.00E-12M或更小)的KD值與鼠APRIL結合，該KD值藉由表面電漿共振測定法所測量(例如BIACORE^®)；在一些實施方案中，該KD值藉由本揭露測試例8方法檢測； B. Specifically binds to human APRIL, cynomolgus APRIL and/or mouse APRIL; preferably, less than 2.00E-11M (for example, less than 2.00E-11M, less than 1.80E-11M, less than 1.70E-11M, less than 1.60E-11M, less than 1.20E-11M, less than 4.60E-12M or less) combined with human APRIL to a KD value less than 2.00E-10M (e.g., less than 2.00E-10M, less than 1.80E-10M, less than 1.70 E-10M, less than 1.60E-10M, less than 1.50E-10M, less than 1.40E-10M, less than 1.20E-10M or less) binds to cynomolgus monkey APRIL, and/or binds to cynomolgus monkey APRIL with a KD value less than 2.00E-11M (For example, KD value less than 2.00E-11M, less than 1.80E-11M, less than 1.60E-11M, less than 1.40E-11M, less than 1.20E-11M, less than 1.00E-11M, less than 8.00E-12M or less) Binding to mouse APRIL, the KD value is measured by a surface plasmon resonance assay (such as BIACORE ^® ); in some embodiments, the KD value is detected by the method of Test Example 8 of the present disclosure;

C.與人BAFF、食蟹猴BAFF和/或鼠BAFF特異性結合；較佳地，以小於7.00E-11M(例如小於7.00E-11M、小於5.00E-11M、小於4.00E-11M、小於3.00E-11M、小於2.00E-11M、小於1.00E-11M或更小)的KD值與人BAFF結合，以小於4.00E-10M(例如小於4.00E-10M、小於3.00E-10M、小於2.00E-10M、小於1.30E-10M或更小)的KD值與食蟹猴BAFF結合，和/或以小於6.00E-11M(例如小於6.00E-11M、小於5.00E-11M、小於4.00E-11M、小於3.20E-11M或更小)的KD值與鼠BAFF結合，該KD值藉由表面電漿共振測定法所測量(例如BIACORE^®)；在一些實施方案中，該KD值藉由本揭露測試例8方法檢測； C. Specifically binds to human BAFF, cynomolgus BAFF and/or mouse BAFF; preferably, less than 7.00E-11M (for example, less than 7.00E-11M, less than 5.00E-11M, less than 4.00E-11M, less than A KD value of less than 3.00E-11M, less than 2.00E-11M, less than 1.00E-11M or less) is combined with human BAFF to be less than 4.00E-10M (e.g., less than 4.00E-10M, less than 3.00E-10M, less than 2.00 Binds to cynomolgus BAFF with a KD value of E-10M, less than 1.30E-10M, or less), and/or binds to cynomolgus BAFF with a KD value 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) binds to murine BAFF, the KD value being measured by a surface plasmon resonance assay (e.g., ^BIACORE® ); in some embodiments, the KD value is determined by the present disclosure Test example 8 method detection;

D.具有阻斷ICOSL與ICOS結合活性；較佳地，阻斷人ICOSL與ICOS結合的IC₅₀值小於0.16nM(例如小於0.16nM、小於0.14nM、小於0.12nM、小於0.10nM、小於0.09nM、小於0.08nM、小於0.07nM、小於0.06nM、小於0.05nM、小於0.04nM或更小)，該IC₅₀值藉由FACS方法檢測；在一些實施方案中，該IC₅₀值藉由本揭露測試例3方法檢測； D. Has the activity of blocking the binding of ICOSL to ICOS; preferably, the IC ₅₀ value of blocking the binding of human ICOSL to ICOS is less than 0.16nM (for example, 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 IC ₅₀ value is detected by the FACS method; in some embodiments, the IC ₅₀ value is detected by the test example of the present disclosure 3 method detection;

E.具有阻斷APRIL與BCMA結合活性；較佳地，阻斷APRIL與BCMA結合的IC₅₀值小於40.00nM(例如小於40.00nM、小於35.00nM、小於30.00nM、小於20.00nM、小於10.00nM、小於5.00nM、小於2.00nM、小於0.10nM、小於0.06nM、小於0.02nM或更小)，該IC₅₀值藉由ELISA方法檢測；在一些實施方案中，該IC₅₀值藉由本揭露測試例4方法檢測； E. Has the activity of blocking the binding of APRIL to BCMA; preferably, the IC ₅₀ value of blocking the binding of APRIL to BCMA is less than 40.00nM (for example, 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 IC ₅₀ value is detected by an ELISA method; in some embodiments, the IC ₅₀ value is determined by Test Example 4 of the present disclosure Method testing;

F.具有阻斷APRIL與TACI結合活性；較佳地，阻斷APRIL與TACI結合的IC₅₀值小於40.00nM(例如小於40.00nM、小於37.00nM、小於33.00nM、小於30.00nM、小於25.00nM、小於10.00nM、小於5.00nM、小於2.00nM、小於1.00nM、小於0.50nM或更小)，該IC₅₀值藉由ELISA方法檢測；在一些實施方案中，該IC₅₀值藉由本揭露測試例4方法檢測； F. Has the activity of blocking the binding of APRIL to TACI; preferably, the IC ₅₀ value of blocking the binding of APRIL to TACI is less than 40.00nM (for example, less than 40.00nM, less than 37.00nM, less than 33.00nM, less than 30.00nM, less than 25.00nM, less than 10.00nM, less than 5.00nM, less than 2.00nM, less than 1.00nM, less than 0.50nM or less), the IC ₅₀ value is detected by an ELISA method; in some embodiments, the IC ₅₀ value is determined by Test Example 4 of the present disclosure method testing;

G.具有阻斷BAFF與BCMA結合活性；較佳地，阻斷BAFF與BCMA結合的IC₅₀值小於2.00nM(例如小於2.00nM、小於1.50nM、小於1.00nM、小於0.90nM、小於0.80nM、小於0.70nM、小於0.60nM、小於0.50nM、小於0.40nM或更小)，該IC₅₀值藉由ELISA方法檢測；在一些實施方案中，該IC₅₀值藉由本揭露測試例4方法檢測； G. Has the activity of blocking the binding of BAFF and BCMA; preferably, the IC ₅₀ value of blocking the binding of BAFF and BCMA is less than 2.00nM (for example, less than 2.00nM, less than 1.50nM, less than 1.00nM, less than 0.90nM, less than 0.80nM, less than 0.70 nM, less than 0.60 nM, less than 0.50 nM, less than 0.40 nM or less), the IC ₅₀ value is detected by the ELISA method; in some embodiments, the IC ₅₀ value is detected by the test example 4 method of the present disclosure;

H.具有阻斷BAFF與BAFF-R結合活性；較佳地，阻斷BAFF與BAFF-R結合的IC₅₀值小於2.00nM(例如小於2.00nM、小於1.70nM、小於1.60nM、小於1.00nM、小於0.80nM、小於0.70nM或更小)，該IC₅₀值藉由ELISA方法檢測；在一些實施方案中，該IC₅₀值藉由本揭露測試例4方法檢測； H. Has the activity of blocking the binding of BAFF and BAFF-R; preferably, the IC ₅₀ value of blocking the binding of BAFF and BAFF-R is less than 2.00nM (for example, less than 2.00nM, less than 1.70nM, less than 1.60nM, less than 1.00nM, less than 0.80 nM, less than 0.70 nM or less), the IC ₅₀ value is detected by the ELISA method; in some embodiments, the IC ₅₀ value is detected by the test example 4 method of the present disclosure;

I.具有阻斷BAFF與TACI結合活性；較佳地，阻斷BAFF與TACI結合的IC₅₀值小於1.00nM(例如小於1.00nM、小於0.50nM、小於0.30nM、小於0.20nM、小於0.15nM或更小)，該IC₅₀值藉由ELISA方法檢測；在一些實施方案中，該IC₅₀值藉由本揭露測試例4方法檢測； I. Has the activity of blocking the binding of BAFF and TACI; preferably, the _IC50 value of blocking the binding of BAFF and TACI is less than 1.00nM (for example, 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 the ELISA method; in some embodiments, the IC ₅₀ value is detected by the test example 4 method of the present disclosure;

J.具有抑制IFNγ、IL-10和/或IgA細胞因子分泌活性；在一些實施方案中，該IC₅₀值藉由本揭露測試例9方法檢測； J. Has the activity of inhibiting IFNγ, IL-10 and/or IgA cytokine secretion; in some embodiments, the IC ₅₀ value is detected by the method of Test Example 9 of the present disclosure;

K.具有抑制B細胞增殖和/或T細胞增殖的活性；在一些實施方案中，該抗ICOSL抗體或抗ICOSL抗體融合蛋白抑制ICOSL誘導T細胞增殖的活性；在一些實施方案中，該抑制T細胞增殖活性藉由本揭露測試例6方法檢測；在一些實施方案中，該抗ICOSL抗體融合蛋白抑制BAFF或APRIL誘導的B細胞增殖的活性；在一些實施方案中，該抑制B細胞增殖活性藉由本揭露測試例7方法檢測。 K. has the activity of inhibiting B cell proliferation and/or T cell proliferation; in some embodiments, the anti-ICOSL antibody or anti-ICOSL antibody fusion protein inhibits the activity of ICOSL-induced T cell proliferation; in some embodiments, the inhibiting T cell proliferation The cell proliferation activity 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 inhibitory B cell proliferation activity is determined by the present invention. Exposed test case 7 method detection.

示例性的抗ICOSL抗體融合蛋白Exemplary anti-ICOSL antibody fusion proteins

在一些實施方案中，該抗ICOSL抗體融合蛋白，其中，該TACI多肽相比野生型的TACI多肽(序列如SEQ ID NO：51所示)不容易發生斷裂。 In some embodiments, the anti-ICOSL antibody fusion protein, wherein the TACI polypeptide is less prone to fragmentation than the wild-type TACI polypeptide (sequence shown in SEQ ID NO: 51).

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中，該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的變體或是SEQ ID NO：58的截短片段的變體；其中該截短片段包含SEQ ID NO：58的第48位至第85位胺基酸殘基，該變體為在SEQ ID NO：58或其截短片段上具有選自由第49、52、53、57、65、82和83位組成的組中的一個或更多個胺基酸替換，其中該胺基酸替換的位點為相對於序列SEQ ID NO：58的自然順序編號的胺基酸殘基位點。 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, wherein the TACI polypeptide is as shown in SEQ ID NO: 58 or is a truncated fragment of SEQ ID NO: 58 or is SEQ ID NO: 58 or a variant of a truncated fragment of SEQ ID NO: 58; wherein the truncated fragment includes amino acid residues 48 to 85 of SEQ ID NO: 58, and the variant is in SEQ ID NO: 58 NO: 58 or a truncated fragment thereof 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 substitutions are Points are amino acid residue positions numbered relative to the natural sequence of the sequence SEQ ID NO:58.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中，該TACI多肽，其中該TACI多肽的截短片段包含：SEQ ID NO：58的第48位至第86位胺基酸殘基；SEQ ID NO：58的第48位至第87位胺基酸殘基；或SEQ ID NO：58的第48位至第88位胺基酸殘基。 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, wherein the TACI polypeptide, wherein the truncated fragment of the TACI polypeptide comprises: the 48th to 86th amino groups of SEQ ID NO: 58 Acid residue; amino acid residues 48 to 87 of SEQ ID NO: 58; or amino acid residues 48 to 88 of SEQ ID NO: 58.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中，該TACI多肽，其序列為SEQ ID NO：58變體或者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)的變體，該變體為在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)上具有選自第49、52、53、57、65、82和83位中的任意1個、2個、3個、4個、5個、6個或7個胺基酸的替換，該胺基酸替換的位點為相對於序列SEQ ID NO：58的自然順序編號的胺基酸殘基位點。 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, wherein the TACI polypeptide has a sequence of SEQ ID NO: 58 variant 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), the variant is in 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 1, 2, 3 or 4 selected from positions 49, 52, 53, 57, 65, 82 and 83 , the substitution of 5, 6 or 7 amino acids, the amino acid substitution sites are amino acid residue sites numbered relative to the natural sequence of sequence SEQ ID NO: 58.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中該TACI多肽為在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有選自由49T或49R、52S、53E或53Q、57E、65T或65A、82A或82R、和83Y組成的組中的一個或更多個胺基酸替換(例如1個、2個、3個、4個、5個、6個或7個胺基酸的替換)，其中該胺基酸替換的位點為相對於序列SEQ ID NO：58的自然順序編號的胺基酸殘基位點。 In some embodiments, the anti-ICOSL antibody fusion protein as described in 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 The ID NO: 63 sequence has one or more amino acid substitutions (eg 1, substitution of 2, 3, 4, 5, 6 or 7 amino acids), wherein the position of the amino acid substitution is an amino acid numbered relative to the natural sequence of the sequence SEQ ID NO: 58 residue position.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中該TACI多肽為：在SEQ ID NO：58、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62或SEQ ID NO：63序列上具有選自49T、52S、53E、53Q、57E和82A中的任一個胺基酸替換。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胺基酸替換。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胺基酸替換。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胺基酸替換。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胺基酸替換。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胺基酸替換。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胺基酸替換。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胺基酸替換。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胺基酸替換。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胺基酸替換。其中前述胺基酸替換的位點為相對於序列SEQ ID NO：58的自然順序編號的胺基酸殘基位點。 In some embodiments, the anti-ICOSL antibody fusion protein as described in 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 an amino acid substitution selected from any one of 49T, 52S, 53E, 53Q, 57E and 82A. There are 49R and 65T amino acid substitutions in the sequence SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63. There are 49R and 65A amino acid substitutions in the sequence SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63. There are 49R, 65T and 82R amino acid substitutions in the sequence SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63. There are 53E and 57E amino acid substitutions in the sequence SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63. There are 52S, 53E and 57E amino acid substitutions in the sequence SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63. There are 49T and 82A amino acid substitutions in the sequence SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63. There are 49T and 83Y amino acid substitutions in the sequence SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63. In SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 or SEQ ID The NO:63 sequence has 49T, 82A and 83Y amino acid substitutions. Or have 49T, 53E, 57E and 82A amino acid substitutions in the sequence SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63. The sites where the aforementioned amino acids are substituted are amino acid residue sites numbered relative to the natural sequence of sequence SEQ ID NO: 58.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中該TACI多肽的序列如SEQ ID NO：60-63、SEQ ID NO：66-83中的任一所示。在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中該TACI多肽序列如SEQ ID NO：83所示。 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, wherein the sequence of the TACI polypeptide is as shown in any one of SEQ ID NO: 60-63, SEQ ID NO: 66-83. In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, wherein the TACI polypeptide sequence is shown in SEQ ID NO: 83.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其由2條相同的第一鏈和2條相同的第二鏈組成，其中， In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above is composed of 2 identical first chains and 2 identical second chains, wherein,

第一鏈：其從N端到C端依次為：[抗ICOSL抗體的重鏈]-[連接子1]-[TACI多肽1]，和 First chain: from N-terminus to C-terminus: [heavy chain of anti-ICOSL antibody]-[linker 1]-[TACI polypeptide 1], and

第二鏈，其從N端到C端依次為：[TACI多肽2]-[連接子2]-[抗ICOSL抗體的輕鏈]；或第二鏈，其從N端到C端依次為[抗ICOSL抗體的輕鏈]-[連接子2]-[TACI多肽2]；或第二鏈，其為抗ICOSL抗體的輕鏈； The second chain, from N terminus to C terminus, is: [TACI polypeptide 2]-[linker 2]-[light chain of anti-ICOSL antibody]; or the second chain, from N terminus to C terminus, is [ light chain of an anti-ICOSL antibody]-[linker 2]-[TACI polypeptide 2]; or a second chain that is a light chain of an anti-ICOSL antibody;

其中，該TACI多肽1和TACI多肽2可以相同或不相同，該連接子1和連接子2可以相同或不相同。在一些實施方案中，該TACI多肽1和TACI多肽2相同。該連接子1和連接子2獨立地為(G_xS)_y連接子，其中，x選自1-5的整數，y選自0-6的整數。更佳地，該連接子1和連接子2獨立地為如SEQ ID NO：89或90所示的連接子。 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 linker 2 are independently (G _x S) _y linkers, where 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 the linker 2 are independently the linkers shown in SEQ ID NO: 89 or 90.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中該抗ICOSL抗體為前面任一項所述的抗ICOSL抗體。 In some embodiments, the anti-ICOSL antibody fusion protein as described in any one of the above, wherein the anti-ICOSL antibody is the anti-ICOSL antibody as described in any one of the preceding items.

在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中，該抗ICOSL抗體融合蛋白的第一鏈如SEQ ID NO：86所示，且第二鏈如SEQ ID NO：88、87或48所示。在一些實施方案中，如上任一項所述的抗ICOSL抗體融合蛋白，其中，該抗ICOSL抗體融合蛋白的第一鏈如SEQ ID NO：84所示，且第二鏈如SEQ ID NO：85或46所示。 In some embodiments, the anti-ICOSL antibody fusion protein as described in 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 as described in 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 as shown in 46.

抗ICOSL抗體或其融合蛋白的變體Anti-ICOSL antibodies or variants thereof

在某些實施方案中，涵蓋本文中提供的抗ICOSL抗體或其融合蛋白的胺基酸序列變體。例如，可以期望改善抗體的結合親和力和/或其它生物學特性。可以藉由將合適的修飾引入編碼抗體的核苷酸序列中，或者藉由肽合成來製備抗體的胺基酸序列變體。此類修飾包括例如對抗ICOSL抗體或其融合蛋白的胺基酸序列內的殘基的刪除、和/或***、和/或取代。可以進行刪除、***、和取代的任何組合以得到最終的構建體，只要最終的構建體擁有期望的特徵，例如抗原結合特性。 In certain embodiments, amino acid sequence variants of the anti-ICOSL antibodies or fusion proteins thereof provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. 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 fusion protein thereof. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, so long as the final construct possesses the desired characteristics, such as antigen-binding properties.

取代、***、和刪除變體Substitute, insert, and delete variants

在某些實施方案中，提供了具有一處或多處胺基酸取代的抗體變體。取代誘變感興趣的位點包括CDR和FR。保守取代在表3中在“較佳的取代”的標題下顯示。更實質的變化在表3中在“示例性取代”的標題下提供，並且如下文參照胺基酸側鏈類別進一步描述的。可以將胺基酸取代引入感興趣的抗體中，並且對產物篩選期望的活性，例如保留/改善的抗原結合，降低的免疫原性，或改善的ADCC或CDC。 In certain embodiments, antibody variants with 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 the amino acid side chain class. Amino acid substitutions can be introduced into the antibody of interest and the product screened for desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

表3. 胺基酸的取代

Table 3. Substitution of amino acids

依照常見的側鏈特性，胺基酸可以如下分組： According to common side chain characteristics, amino acids can be grouped as follows:

(1)疏水性的：正亮胺酸，Met，Ala，Val，Leu，Ile； (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2)中性，親水性的：Cys，Ser，Thr，Asn，Gln； (2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3)酸性的：Asp，Glu； (3) Acidic: Asp, Glu;

(4)鹼性的：His，Lys，Arg； (4) Basic: His, Lys, Arg;

(5)影響鏈取向的殘基：Gly，Pro； (5) Residues that affect chain orientation: Gly, Pro;

(6)芳香族的：Trp，Tyr，Phe。 (6) Aromatic: Trp, Tyr, Phe.

非保守取代會需要用這些類別之一的成員替換另一個類別的成員。 Non-conservative substitutions would require the substitution of a member of one of these classes for a member of another class.

一類取代變體涉及取代親本抗體(例如人源化或人抗體)的一個或多個CDR殘基。一般地，經選擇用於進一步研究的所得變體相對於親本抗體會具有某些生物學特性(例如升高的親和力，降低的免疫原性)的改變(例如改善)，和/或會基本上保留親本抗體的某些生物學特性。一種示例性的取代變體是親和力成熟的抗體，可以例如使用基於噬菌體展示的親和力成熟技術(如本文所述的那些技術)，便利地產生該抗體。簡言之，將一個或多個CDR殘基突變，並將變體抗體在噬菌體上展示，並對其篩選特定的生物學活性(例如結合親和力)。可以對CDR做出改變(例如取代)，例如以改善抗體親和力。可以對CDR“熱點”，即在體細胞成熟過程期間高頻率經歷突變的密碼子所編碼的殘基，和/或對接觸抗原的殘基做出此類改變，同時對所得的變體VH或VL測試結合親和力。在親和力成熟的一些實施方案中，藉由多種方法(例如易錯PCR、鏈改組、或寡核苷酸指導的誘變)的任一種，將多樣性引入所選擇用於成熟的可變基因中。然後，創建次級文庫。然後，篩選文庫以鑑定具有期望的親和力的任何抗體變體。另一種引入多樣性的方法涉及CDR定向的方法，其中將幾個CDR殘基(例如4-6個殘基)隨機化。可以例如使用丙胺酸掃描誘變或建模來特異性鑑定涉及抗原結合的CDR殘基。特別地，經常靶向HCDR3和LCDR3。 One type of substitution variant involves the substitution of one or more CDR residues of a parent antibody (eg, a humanized or human antibody). Typically, the resulting variants selected for further study are There will be changes (eg, improvements) in certain biological properties (eg, increased affinity, reduced immunogenicity), and/or certain biological properties of the parent antibody will be substantially retained. One exemplary substitution variant is an affinity-matured antibody, which may 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 (e.g., binding affinity). Changes (eg substitutions) can be made to the CDRs, for example to improve antibody affinity. Such changes can be made to CDR "hotspots," residues encoded by codons that undergo mutations with high frequency during the somatic maturation process, and/or to residues that contact the antigen, while simultaneously modifying the resulting variant VH or VL tests binding affinity. In some embodiments of 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. . Then, create secondary libraries. The library is then screened to identify any antibody variants with the desired affinity. Another way to introduce diversity involves CDR-directed methods, 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. In particular, HCDR3 and LCDR3 are often targeted.

在某些實施方案中，取代、***或缺失可以在一個或多個CDR內進行，只要此類變化不實質性降低抗體結合抗原的能力。例如，可以對CDR做出保守變化(例如保守取代，如表3中提供的)，其不實質性降低結合親和力。此類變化可以例如在CDR中的抗原接觸殘基外部。在上文提供的變體VH和VL序列的某些實施方案中，每個CDR是未改變的，或者含有不超過1、2或3處胺基酸取代。 In certain embodiments, substitutions, insertions, or deletions may be made within one or more CDRs as long as such changes do not substantially reduce the ability of the antibody to bind the antigen. For example, conservative changes (eg, conservative substitutions, as provided in Table 3) can be made to the CDRs that do not substantially reduce binding affinity. Such changes may, for example, be external to the antigen-contacting residues in the CDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or contains no more than 1, 2, or 3 amino acid substitutions.

一種可用於鑑定抗體中可以作為誘變靶位元的殘基或區域的方法稱作“丙胺酸掃描誘變”。在這種方法中，鑑定一個殘基或殘基組(例如帶電荷的殘基，諸如Arg、Asp、His、Lys和Glu)，並且用中性或帶負電荷的胺基酸(例如，Ala或聚丙胺酸)替換以確定該抗體與抗原的相互作用是否受影響。可以在對初始取代顯示功能敏感性的胺基酸位置引入進一步的取代。此外，可藉由研究抗原-抗體複合物的晶體結構來鑑定抗體與抗原間的接觸點。這些接觸殘基及鄰近殘基可以作為取代候選物被打靶或消除。可以篩選變體以確定它們是否含有期望的特性。 One method that can be used to identify residues or regions in an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis." In this method, a residue or group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced with neutral or negatively charged amino acids (e.g., Ala or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. In addition, the contact points between the antibody and the antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine whether they contain the desired properties.

胺基酸序列***包括在多肽的胺基和/或羧基端融合長度範圍為1個殘基至100或更多個殘基，和單個或多個胺基酸殘基的序列內***。末端***的例子包括具有N端甲硫胺醯基殘基的抗體。抗體分子的其它***變體包括抗體的N或C端與酶(或延長抗體的半衰期的多肽)的融合物。 Amino acid sequence insertions include fusion lengths ranging from 1 residue to 100 or more residues at the amino and/or carboxyl termini of the polypeptide, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusions of the N- or C-terminus of the antibody with enzymes (or polypeptides that extend the half-life of the antibody).

Fc區的修飾Modification of Fc region

在一個方面，本揭露的抗ICOSL抗體或抗ICOSL抗體融合蛋白的Fc區包含一個或多個胺基酸取代，該一個或多個胺基酸取代減少其與Fc受體的結合，例如其與Fcγ受體的結合，並且降低或消除效應子功能。天然IgG Fc區，具體地是IgG₁ Fc區或IgG₄ Fc區，可能導致本揭露的融合蛋白靶向表達Fc受體的細胞，而不是表達抗原的細胞。在一些實施方案中，本揭露改造的Fc區表現出降低的對Fc受體的結合親和力和/或降低的效應子功能。在一些實施方案中，改造的Fc區與天然Fc區相比，對Fc受體的結合親和力下降50%、80%、90%或95%以上。在一些實施方案中，該Fc受體是Fcγ受體。在一些實施方案中，該Fc受體是人Fcγ受體，例如FcγRI、FcγRIIa、FcγRIIB、FcγRIIIa。在一些實施方案中，改造的Fc區與天然Fc區相比，對補體，如C1q的結合親和力也降低。在一些實施方案中，改造的Fc區與天然Fc區相比，對新生兒Fc受體 (FcRn)的結合親和力不降低。在一些實施例中，改造的Fc區具有降低的效應子功能，該降低的效應子功能可以包括但不限於以下中的一個或多個：降低的補體依賴性細胞毒性(CDC)、降低的抗體依賴性細胞介導的細胞毒性(ADCC)、降低的抗體依賴性細胞吞噬(ADCP)、減少的細胞因子分泌、減少的免疫複合物介導的抗原呈遞細胞的抗原攝取、減少的與NK細胞的結合、減少的與巨噬細胞的結合、減少的與單核細胞的結合、減少的與多形核細胞的結合、減少的直接信號傳導誘導性細胞凋亡、降低的樹突細胞成熟或減少的T細胞引發。對於IgG₁ Fc區，在238、265、269、270、297、327和329等位置的胺基酸殘基取代可降低的效應子功能。在一些實施方案中，該Fc區是人IgG₁ Fc區，並且在234和235位置的胺基酸殘基為A，編號依據為EU索引。對於IgG₄ Fc區，在228等位置的胺基酸殘基取代可降低的效應子功能。 In one aspect, the Fc region of an anti-ICOSL antibody or anti-ICOSL antibody fusion protein of the present disclosure contains one or more amino acid substitutions that reduce its binding to an Fc receptor, such as its binding to an Fc receptor. Binding of Fcγ receptors and reduction or elimination of effector functions. The native IgG Fc region, specifically the _IgG1 Fc region or the _IgG4 Fc region, may cause the fusion proteins of the present disclosure to target cells expressing Fc receptors rather than cells expressing the antigen. In some embodiments, the engineered Fc region of the present disclosure exhibits reduced binding affinity for Fc receptors and/or reduced effector function. In some embodiments, the engineered Fc region has a binding affinity for the Fc receptor that is reduced by more than 50%, 80%, 90%, or 95% compared to the native Fc region. In some embodiments, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fcγ receptor, such as FcγRI, FcγRIIa, FcγRIIB, FcγRIIIa. In some embodiments, the engineered Fc region also has reduced binding affinity for complement, such as Clq, compared to the native Fc region. In some embodiments, the engineered Fc region has no reduced binding affinity for the neonatal Fc receptor (FcRn) compared to the native Fc region. In some embodiments, the engineered Fc region has reduced effector functions, which may include, but are not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced interaction with NK cells Binding, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced dendritic cell maturation, or reduced T cell priming. For the _IgG1 Fc region, substitution of amino acid residues at positions 238, 265, 269, 270, 297, 327, and 329 may reduce effector function. In some embodiments, the Fc region is a human _IgG1 Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index. For the IgG ₄ Fc region, substitution of amino acid residues at positions such as 228 may reduce effector function.

抗ICOSL抗體或抗ICOSL抗體融合蛋白可包含與Fc區的兩個亞基融合的不同結合域，因此可能導致不期望的同源二聚化。為了提高產率和純度，可以在本揭露的融合蛋白的Fc區中引入促進異源二聚化的修飾將是有利的。在一些實施方案中，本揭露的Fc區包含根據杵臼(knob-into-hole，KIH)技術的改造，該方法涉及在第一亞基的介面處引入凸起結構(knob)以及在第二亞基的介面處引入孔結構(hole)。使得該凸起結構可以定位在孔結構中，促進異源二聚體的形成並抑制同源二聚體的產生。凸起結構是藉由用較大側鏈(例如酪胺酸或色胺酸)取代來自第一亞基的介面的小胺基酸側鏈而構建的。而孔結構是藉由用較小的胺基酸側鏈(例如丙胺酸或蘇胺酸)取代大胺基酸側鏈而在第二亞基的介面中創建的。凸起結構和孔結構藉由改變編碼多肽的核酸來製備，可選的胺基酸取代如下表4所示： Anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins may contain different binding domains fused to the two subunits of the Fc region, thus potentially leading to undesirable homodimerization. To increase yield and purity, it would be advantageous to introduce modifications that promote heterodimerization into the Fc region of the fusion proteins of the present disclosure. In some embodiments, the Fc region of the present disclosure includes modifications according to the knob-into-hole (KIH) technique, which involves introducing a knob at the interface of the first subunit and a knob at the interface of the second subunit. A hole structure (hole) is introduced at the interface of the base. This allows the protruding structure to be positioned in the pore structure, promoting the formation of heterodimers and inhibiting the generation of homodimers. The raised structure is built by replacing small amino acid side chains from the interface of the first subunit with larger side chains, such as tyrosine or tryptophan. The pore structure is created in the interface of the second subunit by replacing large amino acid side chains with smaller amino acid side chains, such as alanine or threonine. The bulge structure and pore structure are prepared by changing the nucleic acid encoding the polypeptide. Optional amino acid substitutions are shown in Table 4 below:

表4. KIH突變組合

Table 4. KIH mutation combinations

除了杵臼技術外，用於修飾抗體的重鏈的CH3結構域以實現異源二聚化的其他技術也是本領域中已知的，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。 In addition to the pestle and mortar technology, other techniques for modifying the CH3 domain of the heavy chain of an antibody to achieve heterodimerization are also known in the art, such as WO96/27011, WO98/050431, EP1870459, WO2007/110205, WO 007/147901, WO2009/089004, WO2010/129304, WO2011/90754, WO2011/143545, WO2012/058768, WO2013/157954 and WO 013/096291.

抗原結合分子還可包含二硫鍵改造，例如Fc區的第一亞基包含354C突變和第二亞基包含349C突變，使Fc區的第一亞基和第二亞基之間產生工程化二硫鍵，促進Fc區第一亞基和第二亞基的異源二聚化。 Antigen-binding molecules can also include disulfide bond modifications, for example, the first subunit of the Fc region contains the 354C mutation and the second subunit contains the 349C mutation, resulting in an engineered disulfide bond between the first subunit and the second subunit of the Fc region. Sulfur bonds promote heterodimerization of the first and second subunits of the Fc region.

抗原結合分子的Fc區還可進一步引入其它的胺基酸改造，例如降低免疫原性的同種異型胺基酸殘基突變。在一些實施方案中，IgG1的Fc引入356E和358M突變。 The Fc region of the antigen-binding molecule can further introduce other amino acid modifications, such as allotype amino acid residue mutations that reduce immunogenicity. In some embodiments, the Fc of IgG1 introduces the 356E and 358M mutations.

Fc區的C末端可以是以胺基酸殘基PGK結束的完整C末端；也可以是縮短的C末端，例如在該縮短的C末端中已經去除了一個或兩個C末端胺基酸殘基。在一個較佳的方面中，重鏈的C末端是以PG結束的縮短的C末端。因此，在一些實施方案中，完整抗體可以包括去除了所有K447殘基和/或G446+K447殘基的抗體混合物。在一些實施方案中，完整抗體可以包括沒有去除K447殘基和/或G446+K447殘基的抗體混合物。在一些實施方案中，完整抗體具有帶有和不帶有K447殘基和/或G446+K447殘基的抗體混合物。 The C-terminus of the Fc region can be a complete C-terminus ending with the amino acid residue PGK; it can also be a shortened C-terminus, for example, one or two C-terminal amino acid residues have been removed from the shortened C-terminus. . In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending in PG. Thus, in some embodiments, intact antibodies may include antibody mixtures with all K447 residues and/or G446+K447 residues removed. In some embodiments, intact antibodies may include antibody mixtures without removal of the K447 residue and/or G446+K447 residues. In some embodiments, intact antibodies have mixtures of antibodies with and without K447 residues and/or G446+K447 residues.

重組方法Recombination method

抗ICOSL抗體或抗ICOSL抗體融合蛋白可以使用重組方法來產生。對於這些方法，提供編碼抗體或融合蛋白的一個或更多個分離的核酸。 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.

在一個實施方案中，本揭露提供了編碼如前所述的抗ICOSL抗體或抗ICOSL抗體融合蛋白的分離的核酸。此類核酸可以給自獨立的編碼前述的任一多肽鏈。在另一方面中，本揭露提供了包含此類核酸的一種或多種載體(例如表達載體)。在另一方面中，本揭露提供了包含此類核酸的宿主細胞。在一個實施方案中，提供製備抗體或融合蛋白的方法，其中該方法包括，在適合表達的條件下，培養包含編碼該抗體或融合蛋白的核酸的宿主細胞，如上文所提供的，和視需要地從宿主細胞(或宿主細胞培養基)回收該抗ICOSL抗體或抗ICOSL抗體融合蛋白。 In one embodiment, the present disclosure provides an isolated nucleic acid encoding an anti-ICOSL antibody or an anti-ICOSL antibody fusion protein as described above. Such nucleic acids may independently encode any of the aforementioned polypeptide chains. In another aspect, the present disclosure provides one or more vectors (eg, expression vectors) comprising such nucleic acids. In another aspect, the present disclosure provides host cells comprising such nucleic acids. In one embodiment, a method of preparing an antibody or fusion protein is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody or fusion protein under conditions suitable for expression, as provided above, and optionally The anti-ICOSL antibody or anti-ICOSL antibody fusion protein is recovered from the host cell (or host cell culture medium).

為了重組產生抗ICOSL抗體或抗ICOSL抗體融合蛋白，將編碼蛋白的核酸分離並***一個或更多個載體中，用於在宿主細胞中進一步選殖和/或表達。此類核酸可以使用常規程式容易地分離和測序，或者藉由重組方法產生或藉由化學合成獲得。 To recombinantly produce anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins, the nucleic acid encoding the protein is isolated and inserted into one or more vectors for further selection and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures, or produced by recombinant methods or obtained by chemical synthesis.

用於選殖或表達編碼抗ICOSL抗體或抗ICOSL抗體融合蛋白的載體的適當宿主細胞包括本文描述的原核或真核細胞。例如，可在細菌中產生，特別是當不需要糖基化和Fc效應子功能時。在表達後，可以在可溶級分中從細菌細胞糊狀物分離，並且可進一步純化。 Suitable host cells for selection or expression of 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, the bacterial cell paste can be isolated in a soluble fraction and further purified.

除了原核生物以外，真核微生物諸如絲狀真菌或酵母也是用於編碼抗體或融合蛋白的載體的合適的株或表達宿主，包括真菌和酵母菌株。適於表達抗體或融合蛋白的合適的宿主細胞也可源自多細胞生物體(無脊椎動物和脊椎動物)；無脊椎動物細胞的例子包括植物和昆蟲細胞。已經鑑定了許多杆狀病毒株，其可與昆蟲細胞聯合使用，特別是用於草地貪夜蛾(Spodoptera frugiperda)細胞的轉染；還可利用植物細胞培養物作為宿主，例如US5959177、US6040498、US6420548、US7125978和US6417429；也可將脊椎動物細胞用作宿主，例如適應於在懸浮液中生長的哺乳動物細胞系。適宜的哺乳動物宿主細胞系的其它例子是經SV40轉化的猴腎CV1系(COS-7)；人胚腎系(293或293T細胞)；幼倉鼠腎細胞(BHK)；小鼠塞托利(sertoli)細胞(TM4細胞)；猴腎細胞(CV1)；非洲綠猴腎細胞(VERO-76)；人宮頸癌細胞(HELA)；犬腎細胞(MDCK)；水牛鼠(buffalo rat)肝細胞(BRL3A)；人肺細胞(W138)；人肝細胞(Hep G2)；小鼠***腫瘤(MMT 060562)；TRI細胞；MRC 5細胞；和FS4細胞。其它適宜的哺乳動物宿主細胞系包括中國倉鼠卵巢(CHO)細胞，包括DHFR-CHO細胞；以及骨髓瘤細胞系，如Y0、NS0和Sp2/0。關於適合產生抗體或融合蛋白的某些哺乳動物宿主細胞系的綜述參見例如Yazaki，P.和Wu，A.M.，Methods in Molecular Biology，Vol.248，Lo，B.K.C.(編)，Humana Press，Totowa，NJ(2004)，第255-268頁。 In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable strains 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. Many baculovirus strains have been identified that can be used in combination with insect cells, especially for transfection of Spodoptera frugiperda cells; plant cell cultures can also be used as hosts, such as US5959177, US6040498, US6420548 , US7125978 and US6417429; vertebrate cells can also be used as hosts, such as mammalian cell lines adapted to growth in suspension. Other examples of 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); mouse Sertoli ( 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 liver cells (Hep G2); mouse breast tumors (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines, such as Y0, NSO, and Sp2/0. For a review of certain mammalian host cell lines suitable for the production of antibodies or fusion proteins see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology , Vol. 248, Lo, BKC (Eds.), Humana Press, Totowa, NJ (2004), pp. 255-268.

測定Determination

本文提供的抗ICOSL抗體或抗ICOSL抗體融合蛋白可以藉由本領域已知的多種測定法對其物理/化學特徵和/或生物學活性進行鑑定、篩選或表徵。 The 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 a variety of assays known in the art.

在一個方面中，例如藉由已知方法如ELISA、蛋白印跡法等，測試本揭露的抗ICOSL抗體或抗ICOSL抗體融合蛋白活性。 In one aspect, the activity of the anti-ICOSL antibody or anti-ICOSL antibody fusion protein of the present disclosure is tested, for example, by known methods such as ELISA, Western blotting, etc.

治療方法與施用途徑Treatment methods and routes of administration

本文提供的任何抗ICOSL抗體或抗ICOSL抗體融合蛋白可用於治療方法。 Any anti-ICOSL antibody or anti-ICOSL antibody fusion protein provided herein may be used in the therapeutic methods.

在又一個方面，本揭露提供抗ICOSL抗體或抗ICOSL抗體融合蛋白在藥物的製造或製備中的用途。在一些實施方案中，該藥物用於治療自體免疫性疾病、炎性疾病、B細胞障礙或T細胞障礙，該疾病是與ICOSL過表達相關的疾病。 In yet another aspect, the present disclosure provides use of an anti-ICOSL antibody or anti-ICOSL antibody fusion protein in the manufacture or preparation of a medicament. In some embodiments, the medicament is used to treat an autoimmune disease, an inflammatory disease, a B cell disorder, or a T cell disorder that is associated with overexpression of ICOSL.

在一些實施方案中，該自體免疫性疾病或炎性疾病選自：系統性紅斑狼瘡、類風濕性關節炎、移植物抗宿主病、哮喘、免疫性血小板減少性紫癜、多發性硬化、糖尿病引起的炎性疾病、銀屑病、炎性腸病、克羅恩病、潰瘍性結腸炎、格雷夫斯病和橋本氏甲狀腺炎等。在一些實施方案中，該B細胞障礙或T細胞障礙為腫瘤疾病。在一些實施方案中，該腫瘤選自：頭頸癌、非小細胞肺癌、尿路上皮癌、白血病、肉瘤、黑色素瘤、腺癌、結直腸癌、***腫瘤、乳腺癌和小細胞肺癌等。在一些實施方案中，該自身免疫性疾病為系統性紅斑狼瘡。在一個此類實施方案中，該用途進一步包括向受試者施用有效量的至少一種另外的治療劑(例如一種、兩種、三種、四種、五種或六種另外的治療劑)。根據任意以上實施方案的“受試者”可以是人。 In some embodiments, the autoimmune or inflammatory disease is selected from: systemic lupus erythematosus, rheumatoid arthritis, graft versus host disease, asthma, immune thrombocytopenic purpura, multiple sclerosis, diabetes Caused by inflammatory diseases, psoriasis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Graves' disease, and Hashimoto's thyroiditis, among others. In some embodiments, the B cell disorder or T cell disorder is a neoplastic disease. In some embodiments, 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 tumors, breast cancer, small cell lung cancer, and the like. In some embodiments, the autoimmune disease is systemic lupus erythematosus. In one such embodiment, the use further includes administering to the subject an effective amount of at least one additional therapeutic agent (eg, one, two, three, four, five, or six additional therapeutic agents). A "subject" according to any of the above embodiments may be a human.

在又一個的方面，提供包含該抗ICOSL抗體或抗ICOSL抗體融合蛋白的醫藥組成物，例如，其用於以上任何製藥用途或治療方法。在一個實施方案中，醫藥組成物包含本文提供的任何抗體或融合蛋白和藥學上可接受的載體。在另一個實施方案中，醫藥組成物還包含至少一種另外的治療劑。 In yet another aspect, pharmaceutical compositions comprising the anti-ICOSL antibody or anti-ICOSL antibody fusion protein are provided, for example, for use in any of the above pharmaceutical uses or treatment methods. In one embodiment, a pharmaceutical composition includes any antibody or fusion protein provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further includes at least one additional therapeutic agent.

本揭露的抗ICOSL抗體或抗ICOSL抗體融合蛋白可單獨使用或與其他試劑聯合用於治療。例如，本揭露的抗ICOSL抗體或抗ICOSL抗體融合蛋白可與至少一種另外的治療劑聯合施用。 The anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins of the present disclosure can be used alone or in combination with other agents for treatment. For example, 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.

“聯合施用”是指為了達到治療目的，而將兩種或兩種以上活性化合物同時或先後提供給受試者的方法。當涉及“聯合施用”時，每次施用之間的時間間隔，足以實現施用的各活性化合物之間的協同作用。兩種或兩種以上活性化合物在相同的不同容器中。 "Combined administration" refers to a method of providing two or more active compounds to a subject simultaneously or successively for the purpose of treatment. When "co-administration" is concerned, the time between each application is sufficient to achieve a synergistic effect between the active compounds administered. Two or more active compounds in the same and different containers.

本揭露的抗ICOSL抗體或抗ICOSL抗體融合蛋白(和任何另外的治療劑)可藉由任何合適的手段施用，包括腸胃外、肺內和鼻內，並且如果需要局部治療，則病灶內施用。腸胃外輸注包括肌肉內、靜脈內、動脈內、腹膜內或皮下施用。給藥可以藉由任何適當的途徑，例如，藉由注射，諸如靜脈內或皮下注射，這部分取決於施用是短期的還是長期的。本文考慮多種給藥時間方案，包括但不限於，單次或在多個時間點多次施用，推注施用和脈衝輸注。 The anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins of the present disclosure (and any additional therapeutic agents) may be administered by any suitable means, including parenterally, intrapulmonary, and intranasal, and, if local treatment is desired, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulse infusion.

本揭露的抗ICOSL抗體或抗ICOSL抗體融合蛋白將以符合良好醫療實踐(Good Manufacturing Practice)的方式配製、給藥和施用。在此背景下考慮的因素包括所治療的具體病症、所治療的具體哺乳動物、個體患者的臨床狀況、病症的起因、試劑的遞送部位、施用方法、施用時間安排以及醫學從業者已知的其他因素。多肽或融合蛋白可以與或不與目前用於預防或治療該病症的一種或更多種試劑一起配製。此類其它試劑的有效量取決於醫藥組成物中存在的量、病症或治療的類型以及其它因素。這些通常以與本文所述相同的劑量和施用路徑使用，或以本文所述劑量的約1至99%使用，或以其它劑量使用，並藉由經驗/臨床確定為合適的任何途徑使用。 The anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins of the present disclosure will be formulated, administered and administered in a manner consistent with Good Manufacturing Practice. Factors considered in this context include the specific condition being treated, the specific 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. The 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 condition 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 by any route empirically/clinically determined to be appropriate.

具體實施方案中的有效量，可以從來源於動物模型測試系統的劑量-應答曲線而得到，並允許根據醫生的判斷和每位元患者的情況來決定。受試者一次施用所需的藥物的量可以方便地藉由計算受試者體重和一次用藥所需單位體重劑量得到。可以藉由實驗動物與人的單位體重劑量之間的等效劑量換算關係來確定用藥量(Freireich等人1966，Cancer Chemother Rep 50：219)。例如，可以根據FDA、SFDA等藥品管理機構提出的指導意見。在一些實施方式中，可以使用按照人和鼠的體表面積折算係數，來換算人和鼠的劑量(如參照測試例9-11中的劑量)。 Effective amounts in specific embodiments can be derived from dose-response curves derived from animal model test systems and are allowed to be determined based on the physician's judgment and the circumstances of each patient. The amount of drug required by a subject for one administration can be conveniently obtained by calculating the subject's weight and the dose per unit body weight required for one administration. The dosage can be determined by the equivalent dose conversion relationship between the dose per unit body weight in experimental animals and humans (Freireich et al. 1966, Cancer Chemother Rep 50: 219). For example, it can be based on the guidance issued by drug regulatory agencies such as FDA and SFDA. In some embodiments, a conversion factor based on the body surface area of humans and mice can be used to convert the doses for humans and mice (such as the doses in Test Examples 9-11).

為了預防或治療疾病，本揭露的抗ICOSL抗體或抗ICOSL抗體融合蛋白(當單獨使用或與一種或更多種其他另外的治療劑組合使用時)的適當的劑量將取決於待治療的疾病的類型、治療分子的類型、疾病的嚴重性和病程、是為預防還是治療目的施用、之前的治療、患者的臨床病史和對治療分子的響應和主治醫師的判斷。治療分子恰當地以一次或經過一系列治療施用於患者。 To prevent or treat a disease, appropriate dosages of the anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins 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, type of therapeutic molecule, severity and course of the disease, whether administration is for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the therapeutic molecule and the judgment of the attending physician. The therapeutic molecules are appropriately administered to the patient in one session or over a series of treatments.

製品Products

在本揭露的另一方面中，提供一種製品(如藥盒或試劑盒)，該製品包含可用於治療、預防和/或診斷上述病症的材料。該製品包含容器和在容器上或與容器聯合的標籤或包裝插頁(package insert)。合適的容器包括，例如，瓶子、管形瓶、注射器、IV溶液袋等。容器可以自各種材料諸如玻璃或塑膠形成。容器裝有單獨的抗體或其融合蛋白、或與另一種成分組合，並且可具有無菌的存取口(例如，容器可以是具有塞子的靜脈內溶液袋或管形瓶)。容器中的至少一種活性試劑是本揭露的抗ICOSL抗體或抗ICOSL抗體融合蛋白。標籤或包裝插頁指示使用該抗體或其融合蛋白是來治療選擇的病況。 In another aspect of the present disclosure, an article of manufacture (eg, a kit or kit) is provided that contains materials useful in treating, preventing, and/or diagnosing the conditions described above. The article includes 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 a variety of materials such as glass or plastic. The container holds the antibody or fusion protein thereof alone, or in combination with another component, and may have a sterile access port (eg, the container may be an intravenous solution bag or vial with a stopper). At least one active agent in the container is an anti-ICOSL antibody or anti-ICOSL antibody fusion protein of the present disclosure. The label or package insert indicates the use of the antibody or fusion protein thereof to treat the selected condition.

此外，製品可以包含：(a)其中裝有組成物的第一容器，其中該組成物包含本揭露的抗ICOSL抗體或抗ICOSL抗體融合蛋白；和(b)其中裝有組成物的第二容器，其中該組成物包含另外的細胞毒性劑或其他方面的治療劑。 Additionally, 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 , wherein the composition includes an additional cytotoxic agent or other therapeutic agent.

備選地，製品可進一步包含第二(或第三)容器，該第二(或第三)容器包含藥學上可接受的緩衝液。從商業和用戶立場，它可進一步包括所需的其他材料，包括其他緩衝劑、稀釋劑、濾器、針頭和注射器。 Alternatively, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer. It may further include other materials required from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

應當理解，對於數值範圍的表述，例如“10至100”或“至少90%”是一種簡潔的書寫方式。儘管沒有給出該範圍內的每一個點值(包括整數和分數)，但視為已經在文中明確得以披露。 It should be understood that the expression of a numerical range, such as "10 to 100" or "at least 90%" is a concise way of writing. Although every point value within this range (including whole numbers and fractions) is not given, it is deemed to have been expressly disclosed in the text.

實施例與測試例Examples and test cases

以下結合實施例和測試例進一步描述本揭露，但這些實施例和測試例並非限制著本揭露的範圍。本揭露實施例和測試例中未註明具體條件的實驗方法，通常按照常規條件，如冷泉港的抗體技術實驗手冊，分子選殖手冊；或按照原料或商品製造廠商所建議的條件。未註明具體來源的試劑，為市場購買的常規試劑。 The disclosure is further described below in conjunction with embodiments and test examples, but these embodiments and test examples do not limit the scope of the disclosure. Experimental methods without specifying specific conditions in the disclosed embodiments and test examples usually follow conventional conditions, such as Cold Spring Harbor's Antibody Technology Experiment Manual and Molecular Cloning Manual; or follow the conditions recommended by the raw material or product manufacturer. Reagents whose specific sources are not indicated are conventional reagents purchased in the market.

實施例1：小鼠抗人ICOSL單株抗體的製備Example 1: Preparation of mouse anti-human ICOSL monoclonal antibody

使用人ICOSL蛋白(Sino Biological，11559-H08H)免疫SJL小鼠。免疫3次後取血測定血清中抗體的效價，選擇血清中抗體滴度高並且滴度趨於平臺的小鼠進行脾細胞融合，將融合好的融合瘤細胞鋪在96孔細胞培養板中，置於37℃，5% CO₂培養箱中進行培養。取細胞培養上清液藉由酶聯免疫吸附分析法(ELISA)進行檢測。將篩選出的陽性株進行擴增凍存保種和二到三次亞選殖直至獲得單細胞株。選擇的融合瘤株用無血清細胞培養法進一步製備和純化抗體。得到的融合瘤抗體用FACS檢測抗體與細胞表面人ICOSL蛋白的結合和對受體的阻斷情況(方法見本公開測試例1和測試例3)，挑選出結合活性和阻斷活性好的融合瘤細胞株。 SJL mice were immunized with human ICOSL protein (Sino Biological, 11559-H08H). After 3 immunizations, blood was taken to determine the titer of antibodies in the serum. Mice with high antibody titers in the serum and titers tending to a plateau were selected for spleen cell fusion. The fused fusion tumor cells were spread in a 96-well cell culture plate. , placed in a 37°C, 5% CO ₂ incubator for culture. The cell culture supernatant was taken and detected by enzyme-linked immunosorbent assay (ELISA). The selected positive strains will be amplified, cryopreserved and sub-cultured two to three times until a single cell strain is obtained. The selected fusion tumor strains were further used to prepare and purify antibodies using serum-free cell culture method. The obtained fusion tumor antibody was used to detect the binding of the antibody to the human ICOSL protein on the cell surface and the blocking of the receptor by FACS (for methods, see Test Example 1 and Test Example 3 of this disclosure), and select fusion tumors with good binding activity and blocking activity. Cell lines.

獲得單株融合瘤細胞株mAb201和mAb259選殖單株抗體的序列。過程如下：收集對數生長期融合瘤細胞，用Trizol(Invitrogen，Cat# 15596-018)提取RNA，反轉錄為cDNA。用cDNA為範本進行PCR擴增後送測序公司測序，得到的DNA序列對應的抗體胺基酸序列如下表5、表6所示： The sequences of monoclonal antibodies selected from single fusion tumor cell lines mAb201 and mAb259 were obtained. The process is as follows: Collect fusion tumor cells in the logarithmic growth phase, extract RNA with Trizol (Invitrogen, Cat# 15596-018), and reverse-transcribe into cDNA. Use cDNA as a template to perform PCR amplification and send it to a sequencing company for sequencing. The antibody amino acid sequences corresponding to the obtained DNA sequences are shown in Table 5 and Table 6 below:

表5. 鼠源抗ICOSL抗體的可變區序列

Table 5. Variable region sequences of mouse anti-ICOSL antibodies

表6. 抗體CDR序列

Table 6. Antibody CDR sequences

將上述mAb201和mAb259候選分子可變區序列藉由PCR擴增VH/VL序列，再與表達載體pHr(帶信號肽及hIgG4/hkappa恆定區基因(CH1-Fc/CL)片段)進行同源重組。示範性地，人重鏈IgG4恆定區序列如SEQ ID NO：43所示，人輕鏈κ恆定區序列如SEQ ID NO：44所示，構建重組嵌合抗體全長表達質粒VH-CH1-Fc-pHr/VL-CL-pHr，進而獲得其嵌合抗體Ch201和Ch259。 The above mAb201 and mAb259 candidate molecule variable region sequences were amplified by PCR to amplify the VH/VL sequence, and then homologous recombination was performed with the expression vector pHr (with signal peptide and hIgG4/hkappa constant region gene (CH1-Fc/CL) fragment) . Exemplarily, the human heavy chain IgG4 constant region sequence is shown in SEQ ID NO: 43, the human light chain kappa constant region sequence is shown in SEQ ID NO: 44, and the recombinant chimeric antibody full-length expression plasmid VH-CH1-Fc- is constructed. pHr/VL-CL-pHr, and then obtained its chimeric antibodies Ch201 and Ch259.

實施例2：鼠源抗人ICOSL單株抗體的人源化Example 2: Humanization of mouse anti-human ICOSL monoclonal antibody

藉由比對IMGT人類抗體重輕鏈可變區種系基因資料庫，分別挑選同源性高的重輕鏈可變區種系基因作為範本，將鼠源抗體的CDR分別Grafted(嫁接)到相應的人源範本中，形成次序為FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可變區序列，然後將可變區序列與人恆定區序列融合，獲得人源化抗體。以下示範性地描述mAb201和mAb259鼠源抗體的人源化，範例中抗體的CDR胺基酸殘基由Kabat編號系統確定並註釋。 By comparing the IMGT human antibody heavy and light chain variable region germline gene database, the heavy and light chain variable region germline genes with high homology were selected as templates, and the CDRs of the mouse antibody were Grafted (grafted) to the corresponding In the human template, a variable region sequence in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 is formed, and then the variable region sequence is fused with the human constant region sequence to obtain a humanized antibody. The humanization of mAb201 and mAb259 murine antibodies is exemplarily described below. The CDR amino acid residues of the antibodies in the example are determined and annotated by the Kabat numbering system.

1、鼠源抗體mAb201的人源化1. Humanization of mouse antibody mAb201

挑選同源性高的重輕鏈可變區種系基因作為範本。鼠源抗體mAb201的人源化輕鏈範本選自IGKV2-28*01和IGKJ4*01，即選擇人種系輕鏈IGKV2-28*01的FR1、FR2、FR3和IGKJ4*01的JK4區(作為FR4)作為人源化抗體輕鏈框架區；人源化重鏈範本選自IGHV1-46*01和IGHJ6*01，即選擇人種系重鏈IGHV1-46*01的FR1、FR2、FR3和IGHJ6*01的JH6區(作為FR4)作為人源化抗體重鏈框架區。 Select heavy and light chain variable region germline genes with high homology as templates. The humanized light chain template of the mouse 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 are selected (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, FR3 and IGHJ6 of the human germline heavy chain IGHV1-46*01 are selected. The JH6 region of *01 (as FR4) serves as the humanized antibody heavy chain framework region.

首先，將鼠源抗體mAb201的CDR分別移植到相應的人源範本中，替換人源範本的CDR區；然後，對人源化抗體的輕鏈可變區的第1、2和/或4位元(根據Kabat編號系統編號)胺基酸殘基進行突變，重鏈可變區的第1、24、69、71、73和/或78位元(根據Kabat編號系統編號)胺基酸殘基進行突變；此外，還對輕鏈可變區的LCDR2：LVSNRFS(SEQ ID NO：9)中的第1位胺基酸殘基由L突變為K，獲得新的LCDR2：KVSNRFS(SEQ ID NO：17)；對輕鏈可變區的LCDR3：FQSNYLPLT(SEQ ID NO：10)中的第8位胺基酸殘基由L突變為P，獲得新的LCDR3：FQSNYLPPT(SEQ ID NO：18)；對重鏈可變區的HCDR2：MIHPNGGSTNYNEKFKS(SEQ ID NO：6)中的第5位胺基酸殘基由N突變為H，獲得新的HCDR2：MIHPHGGSTNYNEKFKS(SEQ ID NO：19)。鼠源抗體mAb201人源化序列如下所示： First, the CDRs of the mouse antibody mAb201 were transplanted into the corresponding human templates to replace the CDR regions of the human templates; then, positions 1, 2, and/or 4 of the light chain variable region of the humanized antibodies were The amino acid residues at positions 1, 24, 69, 71, 73 and/or 78 of the heavy chain variable region (numbered according to the Kabat numbering system) are mutated. Mutation was carried out; in addition, the first amino acid residue in LCDR2: LVSNRFS (SEQ ID NO: 9) in the light chain variable region was mutated from L to K to obtain a new LCDR2: KVSNRFS (SEQ ID NO: 17); Mute the 8th amino acid residue from L to P in LCDR3: FQSNYLPLT (SEQ ID NO: 10) of the light chain variable region to obtain a new LCDR3: FQSNYLPPT (SEQ ID NO: 18); The amino acid residue at position 5 in HCDR2: MIHPNGGSTNYNEKFKS (SEQ ID NO: 6) in the heavy chain variable region was mutated from N to H to obtain a new HCDR2: MIHPHGGSTNYNEKFKS (SEQ ID NO: 19). The humanized sequence of mouse antibody mAb201 is as follows:

>hAb201VL1(Graft)的胺基酸序列(SEQ ID NO：20) >Amino acid sequence of hAb201VL1 (Graft) (SEQ ID NO: 20)

>hAb201VL2(Graft+D1A，I2V，M4L)的胺基酸序列(SEQ ID NO：21) >Amino acid sequence of hAb201VL2 (Graft+D1A, I2V, M4L) (SEQ ID NO: 21)

>hAb201VL3(Graft+L50K)的胺基酸序列(SEQ ID NO：22) >Amino acid sequence of hAb201VL3 (Graft+L50K) (SEQ ID NO: 22)

>hAb201VL4(Graft+D1A，I2V，M4L+L50K)的胺基酸序列(SEQ ID NO：23) >Amino acid sequence of hAb201VL4 (Graft+D1A, I2V, M4L+L50K) (SEQ ID NO: 23)

>hAb201VL5(Graft+L96P)的胺基酸序列(SEQ ID NO：24) >Amino acid sequence of hAb201VL5 (Graft+L96P) (SEQ ID NO: 24)

>hAb201VL6(Graft+D1A，I2V，M4L+L96P)的胺基酸序列(SEQ ID NO：25) >Amino acid sequence of hAb201VL6 (Graft+D1A, I2V, M4L+L96P) (SEQ ID NO: 25)

>hAb201VL7(Graft+L50K，L96P)的胺基酸序列(SEQ ID NO：26) >Amino acid sequence of hAb201VL7 (Graft+L50K, L96P) (SEQ ID NO: 26)

>hAb201VL8(Graft+D1A，I2V，M4L+L50K，L96P)的胺基酸序列(SEQ ID NO：27) >Amino acid sequence of hAb201VL8 (Graft+D1A, I2V, M4L+L50K, L96P) (SEQ ID NO: 27)

>hAb201VH1(Graft+Q1E，A24T，M69L，R71V，T73K，V78A)的胺基酸序列(SEQ ID NO：28) >Amino acid sequence of hAb201VH1 (Graft+Q1E, A24T, M69L, R71V, T73K, V78A) (SEQ ID NO: 28)

>hAb201VH2(Graft+Q1E，R71V，T73K+N53H)的胺基酸序列(SEQ ID NO：29) >Amino acid sequence of hAb201VH2 (Graft+Q1E, R71V, T73K+N53H) (SEQ ID NO: 29)

>hAb201VH3(Graft+Q1E，A24T，M69L，R71V，T73K，V78A+N53H)的胺基酸序列(SEQ ID NO：30) >Amino acid sequence of hAb201VH3 (Graft+Q1E, A24T, M69L, R71V, T73K, V78A+N53H) (SEQ ID NO: 30)

註：上述序列依次為FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中底線部分為CDR序列(根據Kabat編號系統確認)，其餘為FR序列，斜體加粗部分表示突變位元點。 Note: The above sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The bottom line part of the sequence is the CDR sequence (confirmed according to the Kabat numbering system), and the rest is the FR sequence. The bolded italic part indicates the mutation site point. .

2、鼠源抗體mAb259的人源化2. Humanization of mouse antibody mAb259

挑選同源性高的重輕鏈可變區種系基因作為範本，例如，鼠源抗體mAb259的人源化輕鏈範本選自IGKV4-1*01/IGKV1-39*01和IGKJ2*01，即選擇人種系輕鏈IGKV4-1*01或IGKV1-39*01的FR1、FR2、FR3和IGKJ2*01的JK4區(作為FR4)作為人源化抗體輕鏈框架區；人源化重鏈範本選自IGHV3-74*03和IGHJ1*01，即選擇人種系重鏈IGHV3-74*03的FR1、FR2、FR3和IGHJ1*01的JH1區(作為FR4)作為人源化抗體重鏈框架區。首先，將鼠源抗體mAb259的CDR分別移植到相應的人源範本中，替換人源範本的CDR區；然後，對人源化抗體的輕鏈可變區的第1、2、3、42、43、60和/或85位元(根據Kabat編號系統編號)胺基酸殘基進行突變，重鏈可變區的第37、43和/或49(根據Kabat編號系統編號)胺基酸殘基進行突變。此外，對重鏈可變區的HCDR2：YISSSSGKVYADAVKG(SEQ ID NO：12)中的第3位胺基酸殘基由S突變為D，獲得新的HCDR2：YIDSSSGKVYADAVKG(SEQ ID NO：31)。鼠源抗體mAb259人源化序列如下所示： Select heavy and light chain variable region germline genes with high homology as templates. For example, the humanized light chain template of mouse antibody mAb259 is selected from IGKV4-1*01/IGKV1-39*01 and IGKJ2*01, that is, Select the FR1, FR2, FR3 of the human germline light chain IGKV4-1*01 or IGKV1-39*01 and the JK4 region (as FR4) of IGKJ2*01 as the humanized antibody light chain framework region; humanized heavy chain template Selected from IGHV3-74*03 and IGHJ1*01, that is, FR1, FR2, FR3 of human germline heavy chain IGHV3-74*03 and the JH1 region (as FR4) of IGHJ1*01 were selected as the humanized antibody heavy chain framework region . First, the CDRs of the mouse antibody mAb259 were transplanted into the corresponding human templates to replace the CDR regions of the human templates; then, the 1, 2, 3, 42, 42, and Mutation of amino acid residues at positions 43, 60 and/or 85 (numbered according to Kabat numbering system), amino acid residues 37, 43 and/or 49 (numbered according to Kabat numbering system) of the heavy chain variable region Make mutations. In addition, the amino acid residue at position 3 in HCDR2: YISSSSGKVYADAVKG (SEQ ID NO: 12) in the heavy chain variable region was mutated from S to D to obtain a new HCDR2: YIDSSSGKVYADAVKG (SEQ ID NO: 31). The humanized sequence of mouse antibody mAb259 is as follows:

>hAb259VL1(Graft(IGKV4-1*01-IGKJ2*01))的胺基酸序列(SEQ ID NO：32) >Amino acid sequence of hAb259VL1 (Graft(IGKV4-1*01-IGKJ2*01)) (SEQ ID NO: 32)

>hAb259VL2(Graft(IGKV4-1*01-IGKJ2*01)+D1N，I2T，P43S)的胺基酸序列(SEQ ID NO：33) >Amino acid sequence of hAb259VL2 (Graft(IGKV4-1*01-IGKJ2*01)+D1N, I2T, P43S) (SEQ ID NO: 33)

>hAb259VL3(Graft(IGKV1-39*01-IGKJ2*01)的胺基酸序列(SEQ ID NO：34) >Amino acid sequence of hAb259VL3 (Graft(IGKV1-39*01-IGKJ2*01) (SEQ ID NO: 34)

>hAb259VL4(Graft(IGKV1-39*01-IGKJ2*01+D1N，I2T，A43S，S60D，T85V)的胺基酸序列(SEQ ID NO：35) >Amino acid sequence of hAb259VL4 (Graft(IGKV1-39*01-IGKJ2*01+D1N, I2T, A43S, S60D, T85V) (SEQ ID NO: 35)

>hAb259VL5(Graft(IGKV1-39*01-IGKJ2*01+I2T)的胺基酸序列(SEQ ID NO：36) >Amino acid sequence of hAb259VL5 (Graft(IGKV1-39*01-IGKJ2*01+I2T) (SEQ ID NO: 36)

>hAb259VL6(Graft(IGKV1-39*01-IGKJ2*01+D1N，I2T，Q3V)的胺基酸序列(SEQ ID NO：37) >Amino acid sequence of hAb259VL6 (Graft(IGKV1-39*01-IGKJ2*01+D1N, I2T, Q3V) (SEQ ID NO: 37)

>hAb259VL7(Graft(IGKV1-39*01-IGKJ2*01+D1N，I2T，Q3V，K42Q)的胺基酸序列(SEQ ID NO：38) >Amino acid sequence of hAb259VL7 (Graft(IGKV1-39*01-IGKJ2*01+D1N, I2T, Q3V, K42Q) (SEQ ID NO: 38)

>hAb259VH1(Graft)的胺基酸序列(SEQ ID NO：39) >Amino acid sequence of hAb259VH1 (Graft) (SEQ ID NO: 39)

>hAb259VH2(Graft+V37I，S49A)的胺基酸序列(SEQ ID NO：40) >Amino acid sequence of hAb259VH2 (Graft+V37I, S49A) (SEQ ID NO: 40)

>hAb259VH3(Graft+V37I，K43E，S49A)的胺基酸序列(SEQ ID NO：41) >Amino acid sequence of hAb259VH3 (Graft+V37I, K43E, S49A) (SEQ ID NO: 41)

>hAb259VH4(Graft+V37I，K43E，S49A+S52D)的胺基酸序列(SEQ ID NO：42) >Amino acid sequence of hAb259VH4 (Graft+V37I, K43E, S49A+S52D) (SEQ ID NO: 42)

註：序列順序為FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，其中底線部分為CDR序列(根據Kabat編號系統確認)，其餘為FR序列，斜體加粗部分表示突變位元點。 Note: The sequence sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where the bottom line is the CDR sequence (confirmed according to the Kabat numbering system), and the rest is the FR sequence. The bolded italic part indicates the mutation site point.

3、人源化抗體的製備3. Preparation of humanized antibodies

分別構建抗體輕鏈和重鏈的表達載體，將人源化的抗體輕/重鏈分別交叉配對組合，轉染293E細胞後收集培養上清純化即得到人源化的全長抗體。人源化抗體重鏈恆定區可選自IgG1、IgG2、IgG3、IgG4及其變體的恆定區，示例性的，使用人重鏈IgG4恆定區(如SEQ ID NO：43所示)與前述人源化重鏈可變區融合形成抗體全長重鏈；人源化抗體輕鏈恆定區可選自人源κ、λ鏈或其變體的恆定區，示例性的，使用人輕鏈恆定區κ鏈(如SEQ ID NO：44所示)與前述人源化輕鏈可變區融合形成抗體全長輕鏈。 Expression vectors for the antibody light chain and heavy chain were constructed respectively, and the humanized antibody light and heavy chains were cross-matched and combined respectively. After transfection into 293E cells, the culture supernatant was collected and purified to obtain the humanized full-length antibody. The humanized antibody heavy chain constant region can be selected from the constant regions of IgG1, IgG2, IgG3, IgG4 and variants thereof. Exemplarily, the human heavy chain IgG4 constant region (shown in SEQ ID NO: 43) and the aforementioned human The humanized heavy chain variable region is fused to form an antibody full-length heavy chain; the humanized antibody light chain constant region can be selected from the constant regions of human kappa, lambda chains or their variants. For example, the human light chain constant region k is used. The chain (shown as SEQ ID NO: 44) is fused with the aforementioned humanized light chain variable region to form an antibody full-length light chain.

示例性的抗體的恆定區序列如下： Exemplary antibody constant region sequences are as follows:

人IgG4的重鏈恆定區的胺基酸序列(SEQ ID NO：43)： Amino acid sequence of heavy chain constant region of human IgG4 (SEQ ID NO: 43):

人κ鏈的輕鏈恆定區的胺基酸序列(SEQ ID NO：44)： Amino acid sequence of the light chain constant region of human kappa chain (SEQ ID NO: 44):

示例性的，將前述mAb201的人源化抗體重鏈可變區羧基端與人重鏈IgG4恆定區(如SEQ ID NO：43)胺基端連接形成抗體全長重鏈，同時將人源化抗體輕鏈可變區羧基端與人輕鏈κ恆定區(如SEQ ID NO：44)胺基端連接形成抗體全長輕鏈，得到如下表7-1所示的mAb201的人源化抗體： Exemplarily, the carboxyl terminus of the humanized antibody heavy chain variable region of the aforementioned mAb201 is connected to the amino terminus of the human heavy chain IgG4 constant region (such as SEQ ID NO: 43) to form an antibody full-length heavy chain, and the humanized antibody is simultaneously The carboxyl terminus of the light chain variable region is connected to the amino terminus of the human light chain kappa constant region (such as SEQ ID NO: 44) to form the full-length light chain of the antibody, and the humanized antibody of mAb201 is obtained as shown in Table 7-1 below:

表7-1. mAb201人源化抗體

Table 7-1. mAb201 humanized antibody

另外，將前述來自mAb259的人源化抗體重鏈可變區羧基端與人重鏈IgG4恆定區胺基端連接形成抗體全長重鏈，同時將人源化抗體輕鏈可變區羧基端與人輕鏈κ恆定區胺基端連接形成抗體全長輕鏈，得到如下表7-2所示的mAb259人源化抗體： In addition, the carboxy terminus of the humanized antibody heavy chain variable region from mAb259 was connected to the amino terminus of the human heavy chain IgG4 constant region to form the full-length heavy chain of the antibody. At the same time, the carboxy terminus of the humanized antibody light chain variable region was connected to the human The amino terminus of the light chain kappa constant region is connected to form the full-length light chain of the antibody, and the mAb259 humanized antibody is obtained as shown in Table 7-2 below:

表7-2. mAb259人源化抗體

Table 7-2. mAb259 humanized antibody

示例性的，人源化抗體201-H3L3和259-H4L7的輕鏈/重鏈序列如下表8所示： Exemplarily, the light chain/heavy chain sequences of humanized antibodies 201-H3L3 and 259-H4L7 are shown in Table 8 below:

表8. 人源化抗體的輕/重鏈序列

Table 8. Light/heavy chain sequences of humanized antibodies

藉由本揭露測試例1方法檢測抗ICOSL抗體對hICOSL-CHOK1的結合，藉由本揭露測試例3方法檢測抗ICOSL抗體對ICOSL與ICOS結合的阻斷活性的結合實驗結果見下表9、表10： The binding test results of the anti-ICOSL antibody to hICOSL-CHOK1 were detected by the method of Test Example 1 of the disclosure, and the blocking activity of the anti-ICOSL antibody to the binding of ICOSL to ICOS was detected by the method of Test Example 3 of the disclosure. The results are shown in Table 9 and Table 10 below:

表9. 抗ICOSL抗體與hICOSL-CHOK1結合實驗結果

Table 9. Experimental results of anti-ICOSL antibody binding to hICOSL-CHOK1

表10. 抗ICOSL抗體阻斷ICOSL與ICOS結合實驗結果

Table 10. Experimental results of anti-ICOSL antibody blocking the binding of ICOSL and ICOS

實施例3：抗ICOSL抗體融合蛋白的構建Example 3: Construction of anti-ICOSL antibody fusion protein

將前述抗ICOSL抗體與TACI多肽融合，構建抗ICOSL抗體融合蛋白。示例性地，抗ICOSL抗體為人源化抗體259-H4L7或201-H3L3。TACI多肽可以是任意適宜的TACI多肽，示例性地，TACI多肽為來源於專利申請PCT/CN2022/084256及其優先權202110348497.6(發明名稱：一種新型TACI多肽，其融合蛋白及用途；申請日：2021/3/31)(藉由援引完整收入本文)中所述的TACI多肽，包括例如下表11中所示的TACI多肽： The aforementioned anti-ICOSL antibody and TACI polypeptide are fused to construct an anti-ICOSL antibody fusion protein. Exemplarily, the anti-ICOSL antibody is humanized antibody 259-H4L7 or 201-H3L3. The TACI polypeptide can be any suitable TACI polypeptide. For example, the TACI polypeptide is derived from patent application PCT/CN2022/084256 and its priority 202110348497.6 (Invention title: A novel TACI polypeptide, its fusion protein and use; Application date: 2021 /3/31) (incorporated herein by reference in its entirety), include, for example, the TACI polypeptides shown in Table 11 below:

表11. TACI多肽的胺基酸序列

Table 11. Amino acid sequence of TACI polypeptides

示例性的，將TACI多肽(例如TACI-9-15c)融合到抗ICOSL抗體(例如201-H3L3或259-H4L7)重鏈的C端、輕鏈的N端或C端，並在融合抗體重鏈或輕鏈的C端的TACI多肽的C末端添加QK作為保護胺基酸，以防止TACI末端的胺基酸被羧肽酶切除。構建獲得多種抗ICOSL抗體融合蛋白，其中259H4L7-T11和201H3L3-T11的結構示意圖見圖1、201H3L3-T7和259H4L7-T7的結構示意圖見圖2、259H4L7-T9的結構示意圖見圖3，該抗ICOSL抗體融合蛋白包括2條相同的第一鏈和2條相同的第二鏈，具體如下所示： Exemplarily, a TACI polypeptide (eg, TACI-9-15c) is fused to the C-terminus of the heavy chain, the N-terminus or the C-terminus of the light chain of an anti-ICOSL antibody (eg, 201-H3L3 or 259-H4L7), and the TACI polypeptide is fused to the heavy chain of the fused antibody. QK is added to the C-terminus of the TACI polypeptide as a protective amino acid to prevent The amino acid at the end of TACI is cleaved by carboxypeptidase. A variety of 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 diagram of 259H4L7-T9 is shown in Figure 3. ICOSL antibody fusion protein includes 2 identical first chains and 2 identical second chains, as shown below:

>201H3L3-T7第一鏈的胺基酸序列(SEQ ID NO：84) >Amino acid sequence of the first chain of 201H3L3-T7 (SEQ ID NO: 84)

>201H3L3-T7第二鏈的胺基酸序列同201-H3L3的輕鏈(SEQ ID NO：46)。 >The amino acid sequence of the second chain of 201H3L3-T7 is the same as the light chain of 201-H3L3 (SEQ ID NO: 46).

>201H3L3-T11第一鏈的胺基酸序列同201H3L3-T7第一鏈的胺基酸序列(SEQ ID NO：84)； >The 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);

>201H3L3-T11第二鏈的胺基酸序列(SEQ ID NO：85) >Amino acid sequence of the second chain of 201H3L3-T11 (SEQ ID NO: 85)

>259H4L7-T7第一鏈的胺基酸序列(SEQ ID NO：86) > Amino acid sequence of the first chain of 259H4L7-T7 (SEQ ID NO: 86)

>259H4L7-T7第二鏈的胺基酸序列同259-H4L7的輕鏈(SEQ ID NO：48)。 >The amino acid sequence of the second chain of 259H4L7-T7 is the same as the light chain of 259-H4L7 (SEQ ID NO: 48).

>259H4L7-T9第一鏈的胺基酸序列同259H4L7-T7第一鏈的胺基酸序列(SEQ ID NO：86)； >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);

>259H4L7-T9第二鏈的胺基酸序列(SEQ ID NO：87) > Amino acid sequence of the second chain of 259H4L7-T9 (SEQ ID NO: 87)

>259H4L7-T11第一鏈的胺基酸序列同259H4L7-T7第一鏈的胺基酸序列(SEQ ID NO：86)； >The 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);

>259H4L7-T11第二鏈的胺基酸序列(SEQ ID NO：88) >259H4L7-T11 second chain amino acid sequence (SEQ ID NO: 88)

註：上述序列中，單底線為TACI序列，雙底線為抗體可變區序列，斜體為抗體恆定區序列，粗體為連接子序列。 Note: In the above sequence, the single underline is the TACI sequence, the double underline is the antibody variable region sequence, italics are the antibody constant region sequence, and bold is the linker sequence.

本揭露測試例中所用對照分子為AMG-570(抗ICOSL抗體與結合BAFF多肽融合而成抗體融合蛋白)的序列如下： The control molecule used in this disclosed test example is AMG-570 (anti-ICOSL antibody fused with a BAFF-binding polypeptide to form an antibody fusion protein). The sequence is as follows:

>AMG-570的重鏈的胺基酸序列(SEQ ID NO：49) >Amino acid sequence of heavy chain of AMG-570 (SEQ ID NO: 49)

>AMG-570的輕鏈的胺基酸序列(SEQ ID NO：50) >Amino acid sequence of the light chain of AMG-570 (SEQ ID NO: 50)

備註：序列中單底線為可變區，雙底線為BAFF結合多肽，粗體為連接胺基酸，其餘為恆定區。 Note: The single bottom line in the sequence is the variable region, the double bottom line is the BAFF-binding polypeptide, bold fonts are the connecting amino acids, and the rest are constant regions.

測試例test case

測試例1：FACS結合實驗Test example 1: FACS binding experiment

為了檢測待測分子對ICOSL的結合情況，藉由FACS(流式細胞術)的方法檢測其與過表達人ICOSL的CHOK1細胞(ATCC，CCL-61)hICOSL-CHOK1的結合活性。用0.25%胰酶消化hICOSL-CHOK1細胞，300g離心5分鐘，用PBS(源培生物，B320)+2% FBS(Biosun，BS-0002-500)重新懸浮細胞。取50μL細胞懸液加入到96孔U型板(Corning，3795)中，使每孔細胞數為10⁵個。每孔加入50μL梯度稀釋的待測樣品溶液，於4℃避光孵育1小時。用PBS+2% FBS洗板2次，每孔加入100μL Alexa羊抗人IgG(H+L)FITC螢光二抗(Thermo，A-11013，1：500稀釋)，4℃避光孵育1小時。洗板2次後，每孔加入100μL PBS重新懸浮細胞，用流式細胞儀(BD Biosciences，FACS Canto II，338962)讀取FITC通道的螢光值，用軟體擬合出待測分子與細胞表面ICOSL的結合曲線，計算出EC₅₀值。實驗結果見下表12所示。 In order to detect the binding of the test molecule to ICOSL, the FACS (flow cytometry) method was used to detect its binding activity to CHOK1 cells (ATCC, CCL-61) overexpressing human ICOSL hICOSL-CHOK1. Digest hICOSL-CHOK1 cells with 0.25% trypsin, centrifuge at 300g for 5 minutes, and resuspend the cells in PBS (Biosun, B320) + 2% FBS (Biosun, BS-0002-500). Take 50 μL of cell suspension and add it to a 96-well U-shaped plate (Corning, 3795), so that the number of cells in each well is 10 ⁵ . Add 50 μL of gradient diluted sample solution to be tested into each well, and incubate for 1 hour at 4°C in the dark. Wash the plate twice with PBS+2% FBS, add 100 μL Alexa goat anti-human IgG (H+L) FITC fluorescent secondary antibody (Thermo, A-11013, 1:500 dilution) to each well, and incubate for 1 hour at 4°C in the dark. After washing the plate twice, add 100 μL PBS to each well to resuspend the cells. Use a flow cytometer (BD Biosciences, FACS Canto II, 338962) to read the fluorescence value of the FITC channel, and use software to fit the molecules to be measured and the cell surface. Binding curve of ICOSL and calculated EC ₅₀ value. The experimental results are shown in Table 12 below.

表12. 抗ICOSL抗體及其融合蛋白對hICOSL-CHOK1的結合實驗結果

Table 12. Binding experimental results of anti-ICOSL antibodies and their fusion proteins to hICOSL-CHOK1

實驗結果表明，本揭露構建的抗ICOSL抗體及其融合蛋白對hICOSL-CHOK1具有很強的結合活性。 Experimental results show that the anti-ICOSL antibody and its fusion protein constructed in this disclosure have strong binding activity to hICOSL-CHOK1.

測試例2：ELISA結合實驗Test example 2: ELISA binding experiment

藉由ELISA的方法(包被待測分子)檢測待測分子對BAFF和APRIL蛋白的結合活性。具體方法如下： The binding activity of the test molecules to BAFF and APRIL proteins is detected by ELISA method (coating the test molecules). The specific methods are as follows:

用pH 7.4的PBS緩衝液將待測分子稀釋至2μg/mL，以100μL/孔的體積加入96孔酶標板(Corning，3590)中，4℃過夜孵育。棄去液體後，每孔加入300μL用PBS稀釋的1% Casein(37528，Thermo)進行封閉，37℃孵育2小時。封閉結束後，棄去封閉液，並用PBST緩衝液(pH 7.4 PBS含0.1% tween-20)洗板3次後，每孔加入100μL梯度稀釋的人BAFF(ACROBiosystems，BAF-H52D4)或人APRIL(R&D Systems，5860-AP-010/CF)溶液，於37℃孵育1小時，孵育結束後用PBST洗板3次。檢測與BAFF的結合時，每孔加入100μL抗-his-HRP 二抗(Sino biological，105327-MM02T-H，1：2000稀釋)，檢測與APRIL的結合時，每孔加入100μL抗-HA-HRP(Abcam，ab1190，1：4000稀釋)，37℃孵育1小時。用PBST洗板3次後，每孔加入100μL TMB顯色受質(KPL，5120-0077)，室溫孵育10至15分鐘，每孔加入50μl 1M H₂SO₄終止反應，用酶標儀讀取在450nm處的吸收值，用軟體擬合出待測分子與抗原的結合曲線，計算出EC₅₀值。實驗結果見下表13、表14。 The molecules to be tested were diluted to 2 μg/mL with PBS buffer at pH 7.4, added to a 96-well enzyme plate (Corning, 3590) at a volume of 100 μL/well, and incubated at 4°C overnight. After discarding the liquid, 300 μL of 1% Casein (37528, Thermo) diluted in PBS was added to each well for blocking, and incubated at 37°C for 2 hours. After blocking, discard the blocking solution, wash the plate three times with PBST buffer (pH 7.4 PBS containing 0.1% tween-20), and add 100 μL of gradient diluted human BAFF (ACROBiosystems, BAF-H52D4) or human APRIL ( R&D Systems, 5860-AP-010/CF) solution, incubate at 37°C for 1 hour, and wash the plate three times with PBST after the incubation. When detecting binding to BAFF, add 100 μL anti-his-HRP secondary antibody (Sino biological, 105327-MM02T-H, 1:2000 dilution) to each well. When detecting binding to APRIL, add 100 μL anti-HA-HRP to each well. (Abcam, ab1190, 1:4000 dilution), incubate at 37°C for 1 hour. After washing the plate three times with PBST, add 100 μL TMB chromogenic substrate (KPL, 5120-0077) to each well, incubate at room temperature for 10 to 15 minutes, add 50 μL 1M H ₂ SO ₄ to each well to terminate the reaction, and read with a microplate reader. Take the absorption value at 450nm, use software to fit the binding curve between the molecule to be tested and the antigen, and calculate the EC ₅₀ value. The experimental results are shown in Table 13 and Table 14 below.

表13. 抗ICOSL抗體融合蛋白對APRIL蛋白的結合實驗結果

Table 13. Binding experimental results of anti-ICOSL antibody fusion protein to APRIL protein

表14. 抗ICOSL抗體融合蛋白對BAFF蛋白的結合實驗結果

Table 14. Binding experimental results of anti-ICOSL antibody fusion protein to BAFF protein

實驗結果表明，本揭露構建的抗ICOSL抗體融合蛋白對BAFF和APRIL蛋白均具有較強的結合活性，而對照分子AMG-570對APRIL蛋白沒有結合活性。 Experimental results show that the anti-ICOSL antibody fusion protein constructed in this disclosure has strong binding activity to both BAFF and APRIL proteins, while the control molecule AMG-570 has no binding activity to APRIL protein.

測試例3：阻斷ICOSL與ICOS的結合實驗Test Example 3: Experiment on blocking the combination of ICOSL and ICOS

藉由FACS的方法檢測待測分子對ICOSL與ICOS結合的阻斷活性。用0.25%胰酶消化hICOSL-CHOK1細胞(同測試例1)，300g離心5分鐘，用PBS(源培生物，B320)+2% FBS(Biosun，BS-0002-500)重新懸浮細胞。取50μL細胞懸液加入到96孔板 The blocking activity of the test molecule on the binding between ICOSL and ICOS was detected by FACS method. Digest hICOSL-CHOK1 cells with 0.25% trypsin (same as test example 1), and centrifuge at 300g for 5 minutes. Resuspend the cells with PBS (Biosun, B320) + 2% FBS (Biosun, BS-0002-500). Add 50 μL of cell suspension to a 96-well plate

U型板中，使每孔細胞數為10⁵個。將梯度稀釋的待測分子加入96孔板中，輕輕混勻，4℃避光孵育40分鐘。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小時。洗板2次，每孔加入100μL羊抗兔IgG Fc FITC螢光二抗(Invitrogen，A16125，1：500稀釋)，4℃避光孵育40分鐘。洗板2次後每孔加100μL PBS重新懸浮細胞，用流式細胞儀讀取螢光數值。用軟體擬合出抑制配體和受體結合的曲線，計算出IC₅₀值。實驗結果見下表15。 In the U-shaped plate, the number of cells per well was 10 ⁵ . Add the gradient diluted molecules to be tested into the 96-well plate, mix gently, and incubate at 4°C in the dark for 40 minutes. Wash the plate twice with PBS+2% FBS, add 100 μL of 20 nM hICOS-rabbit Fc protein (Sino Biological, 10344-H31H) to each well, and incubate for 1 hour at 4°C in the dark. Wash the plate twice, add 100 μL goat anti-rabbit IgG Fc FITC fluorescent secondary antibody (Invitrogen, A16125, 1:500 dilution) to each well, and incubate in the dark at 4°C for 40 minutes. After washing the plate twice, add 100 μL PBS to each well to resuspend the cells, and read the fluorescence value with a flow cytometer. Use software to fit the curve that inhibits the binding of ligand and receptor, and calculate the IC ₅₀ value. The experimental results are shown in Table 15 below.

表15. 阻斷ICOSL與ICOS結合的實驗結果

Table 15. Experimental results of blocking the binding of ICOSL and ICOS

實驗結果表明，本揭露構建的抗ICOSL抗體及其融合蛋白對ICOSL與ICOS的結合，相較於對照，具有更強的阻斷活性。 Experimental results show that the anti-ICOSL antibodies and their fusion proteins constructed in this disclosure have stronger blocking activity on the binding of ICOSL and ICOS than the control.

測試例4：阻斷BAFF、APRIL和受體的結合實驗Test Example 4: Experiment to block the binding of BAFF, APRIL and receptors

藉由ELISA方法檢測抗ICOSL抗體融合蛋白對BAFF或APRIL與其受體(BAFF/BAFF-R、BAFF/BCMA、BAFF/TACI、APRIL/BCMA、APRIL/TACI)結合的阻斷活性。具體方法如下： The blocking activity of the anti-ICOSL antibody fusion protein on the binding of BAFF or APRIL to its receptors (BAFF/BAFF-R, BAFF/BCMA, BAFF/TACI, APRIL/BCMA, APRIL/TACI) was detected by ELISA method. The specific method is as follows:

用pH 7.4的PBS緩衝液將受體蛋白稀釋至2μg/mL，以100μL/孔的體積加入96孔酶標板中，4℃過夜孵育。棄去液體後，每孔加入200μL 1% Casein封閉液(Thermo，37528)進行封閉，37℃孵育2小時。封閉結束後，棄去封閉液，並用PBST緩衝液(pH 7.4 PBS含0.1% tween-20)洗板3次後備用。將固定濃度的生物素(Biotin)標記的配體蛋白與梯度稀釋的融合蛋白混合後37℃預孵育30分鐘後加入封閉好的酶標板中，37℃孵育1.5小時。孵育結束後用PBST洗板3次，每孔加入100μL鏈黴親和素-HRP(Invitrogen，434323，1：4000稀釋)，37℃孵育1小時。去上清，用PBST洗板3次後每孔加入100μL TMB顯色受質(KPL，5120-0077)，室溫孵育10至15分鐘，每孔加入50μL 1M H₂SO₄終止反應，用酶標儀讀取在450nm處的吸收值，用軟體擬合出抑制配體和受體結合的曲線，計算出IC₅₀值。本測試例中所用配體蛋白的來源資訊如下：BAFF(Sino biological，10056-HNCH)，APRIL(R&D Systems，5860-AP-010/CF)。所用受體蛋白的來源資訊如下：BAFF-R(Sino biological，16079-H02H)，BCMA(Sino biological，10620-H02H)，TACI(ACROBiosystems，TAI-H5256)。實驗結果見下表16、表17、表18： Dilute the receptor protein to 2 μg/mL with PBS buffer at pH 7.4, add it to a 96-well enzyme plate at a volume of 100 μL/well, and incubate at 4°C overnight. After discarding the liquid, add 200 μL of 1% Casein blocking solution (Thermo, 37528) to each well for blocking, and incubate at 37°C for 2 hours. After blocking, discard the blocking solution and wash the plate three times with PBST buffer (pH 7.4 PBS containing 0.1% tween-20) before use. Mix the fixed concentration of biotin-labeled ligand protein and the gradient diluted fusion protein and pre-incubate at 37°C for 30 minutes, then add them to the blocked microplate and incubate at 37°C for 1.5 hours. After the incubation, the plate was washed three times with PBST, 100 μL of streptavidin-HRP (Invitrogen, 434323, 1:4000 dilution) was added to each well, and incubated at 37°C for 1 hour. Remove the supernatant, wash the plate three times with PBST, add 100 μL TMB chromogenic substrate (KPL, 5120-0077) to each well, incubate at room temperature for 10 to 15 minutes, add 50 μL 1M H ₂ SO ₄ to each well to terminate the reaction, and use enzyme The standard instrument reads the absorption value at 450nm, and the software is used to fit the curve of the binding of the inhibitory ligand and the receptor, and the IC ₅₀ value is calculated. The source information of the ligand proteins used in this test example is as follows: BAFF (Sino biological, 10056-HNCH), APRIL (R&D Systems, 5860-AP-010/CF). The source information of the receptor proteins used is as follows: BAFF-R (Sino biological, 16079-H02H), BCMA (Sino biological, 10620-H02H), TACI (ACROBiosystems, TAI-H5256). The experimental results are shown in Table 16, Table 17 and Table 18 below:

表16. 抗ICOSL抗體融合蛋白阻斷APRIL與其受體結合實驗結果

Table 16. Experimental results of anti-ICOSL antibody fusion protein blocking the binding of APRIL to its receptor

表17. 抗ICOSL抗體融合蛋白阻斷BAFF與BCMA結合實驗結果

Table 17. Experimental results of anti-ICOSL antibody fusion protein blocking the binding of BAFF and BCMA

表18. 抗ICOSL抗體融合蛋白阻斷BAFF與BAFF-R或TACI結合實驗結果

Table 18. Experimental results of anti-ICOSL antibody fusion protein blocking the binding of BAFF to BAFF-R or TACI

實驗結果表明，本揭露構建的抗ICOSL抗體融合蛋白能有效阻斷BAFF或APRIL與其受體的結合，而陽性對照AMG-570不能有效阻斷APRIL與其受體結合。 Experimental results show that the anti-ICOSL antibody fusion protein constructed in this disclosure can effectively block the binding of BAFF or APRIL to its receptor, while the positive control AMG-570 cannot effectively block the binding of APRIL to its receptor.

測試例5：Jurkat-ICOS-NFAT報告基因實驗Test example 5: Jurkat-ICOS-NFAT reporter gene experiment

藉由Jurkat-ICOS-NFAT報告基因實驗檢測待測分子對ICOSL-ICOS信號通路的抑制活性。實驗方法如下： The Jurkat-ICOS-NFAT reporter gene assay was used to detect the inhibitory activity of the test molecule on the ICOSL-ICOS signaling pathway. The experimental method is as follows:

將穩定表達人ICOSL的CHOK1細胞hICOSL-CHOK1重新懸浮，按每孔40μL 12500個細胞鋪在96孔細胞板中。將ICOS的全長序列與CD3 zeta的胞內區融合，在Jurkat細胞(ATCC，TIB-152)表面穩定表達，獲得Jurkat-NFAT-ICOS-CD3 zeta細胞。將Jurkat-NFAT-ICOS-CD3 zeta細胞重新懸浮後按每孔40μL 2×10⁵個細胞鋪在含hICOSL-CHOK1的96孔細胞板中。將待測樣品梯度稀釋，每孔20μL加入細胞板中，37℃培養箱中孵育48小時。取出細胞培養板，每孔加入50μL One-glo螢光素酶檢測液(Promega，E6120)，用酶標儀(PerkinElmer，EnVision 2105)檢測生物發光信號。將螢光值用軟體擬合出抑制曲線，計算出IC₅₀值。實驗結果見下表19。 CHOK1 cells stably expressing human ICOSL hICOSL-CHOK1 were resuspended and 12,500 cells were plated in a 96-well cell plate at 40 μL per well. The full-length sequence of ICOS was fused to the intracellular region of CD3 zeta and stably expressed on the surface of Jurkat cells (ATCC, TIB-152) to obtain Jurkat-NFAT-ICOS-CD3 zeta cells. Jurkat-NFAT-ICOS-CD3 zeta cells were resuspended and 40 μL of 2 × 10 ⁵ cells per well were plated in a 96-well cell plate containing hICOSL-CHOK1. Gradient dilute the sample to be tested, add 20 μL per well to the cell plate, and incubate in a 37°C incubator for 48 hours. Take out the cell culture plate, add 50 μL of One-glo luciferase detection solution (Promega, E6120) to each well, and detect the bioluminescence signal with a microplate reader (PerkinElmer, EnVision 2105). Use software to fit the fluorescence values to the inhibition curve and calculate the IC ₅₀ value. The experimental results are shown in Table 19 below.

表19. 抑制Jurkat-ICOS-NFAT報告基因啟動的實驗結果

Table 19. Experimental results of inhibiting the initiation of Jurkat-ICOS-NFAT reporter gene

實驗結果表明，本揭露構建的抗ICOSL抗體及其融合蛋白抑制Jurkat-ICOS-NFAT報告基因啟動的活性比對照分子AMG-570強。 Experimental results show that the anti-ICOSL antibody and its fusion protein constructed in this disclosure have stronger activity in inhibiting the initiation of the Jurkat-ICOS-NFAT reporter gene than the control molecule AMG-570.

測試例6：T細胞增殖實驗Test Example 6: T cell proliferation experiment

藉由T細胞增殖實驗檢測抗ICOSL抗體融合蛋白抑制ICOSL誘導T細胞增殖的活性。實驗方法如下： 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:

在96孔板(Corning，3599)中每孔加入100μL 0.5μg/mL抗人CD3抗體(Ebioscience，16-0037-85)，4℃過夜孵育，用PBS(源培生物，B320)洗滌3次，每孔加入100μL 5μg/mL ICOSL蛋白(R&D，165-B7-100)溶液，37℃孵育4小時。用人Pan T細胞分離試劑盒(Pan T Cell Isolation Kit，human，Miltenyi Biotec，130-096-535)按試劑盒說明書的方法從人外周血單核細胞(PBMC)中分離T細胞，按每孔2×10⁵個細胞鋪在包被好的96孔板中。將待測樣品梯度稀釋，加入細胞板中，37℃孵育48小時後，每孔加入50μL CTG(Cell titer Glo)檢測液(Promega，G7573)，在酶標儀(PerkinElmer，Victor3)上檢測生物發光信號。用軟體擬合出抑制T細胞增殖的曲線，計算出IC₅₀值。實驗結果見下表20： Add 100 μL of 0.5 μg/mL anti-human CD3 antibody (Ebioscience, 16-0037-85) to each well of a 96-well plate (Corning, 3599), incubate at 4°C overnight, and wash 3 times with PBS (Ebioscience, B320). Add 100 μL of 5 μg/mL ICOSL protein (R&D, 165-B7-100) solution to each well and incubate at 37°C for 4 hours. Use Pan T Cell Isolation Kit (Pan T Cell Isolation Kit, human, Miltenyi Biotec, 130-096-535) to isolate T cells from human peripheral blood mononuclear cells (PBMC) according to the instructions of the kit, and press 2 cells per well. ×10 ⁵ cells were plated in a coated 96-well plate. Gradient dilute the sample to be tested and add it to the cell plate. After incubation at 37°C for 48 hours, add 50 μL CTG (Cell titer Glo) detection solution (Promega, G7573) to each well, and detect bioluminescence on a microplate reader (PerkinElmer, Victor3). signal. Use software to fit the curve that inhibits T cell proliferation and calculate the IC ₅₀ value. The experimental results are shown in Table 20 below:

表20. 抑制T細胞增殖的實驗結果

Table 20. Experimental results of inhibiting T cell proliferation

實驗結果表明，本揭露構建的抗ICOSL抗體融合蛋白能有效抑制T細胞增殖。 Experimental results show that the anti-ICOSL antibody fusion protein constructed in this disclosure can effectively inhibit T cell proliferation.

測試例7：B細胞增殖實驗Test Example 7: B cell proliferation experiment

藉由B細胞增殖實驗檢測抗ICOSL抗體融合蛋白抑制BAFF和APRIL誘導的B細胞增殖的活性。實驗方法如下： B cell proliferation assay was used to detect the activity of anti-ICOSL antibody fusion protein in inhibiting B cell proliferation induced by BAFF and APRIL. The experimental method is as follows:

取小鼠脾臟進行研磨，4℃離心5分鐘收集下層細胞，用洗滌溶液(PBS+2% FBS+2mM EDTA)清洗一次並離心，去上清後加入紅細胞裂解液(RBC Lysis Buffer，Invitrogen，00-4333-57)，室溫靜置5分鐘至紅細胞完全裂解。再次離心並重新懸浮細胞進行計數。細胞懸液用B細胞分離試劑盒(B Cell Isolation Kit，Miltenyi Biotec，130-090-862)進行分選，將分離的B細胞用RPMI 1640培養基(Gibco，11875119)+10% FBS(Gibco，10099-141)+50μM 2-巰基乙醇(Sigma-Aldrich，M6250)重新懸浮並計數，細胞鋪在96孔細胞板中備用。將BAFF(R&D Systems，7537-BF)或APRIL(R&D Systems，5860-AP)蛋白稀釋至固定濃度，並加入梯度稀釋的抗ICOSL抗體融合蛋白混勻，37℃預孵育30分鐘後加入96孔細胞板中，37℃細胞培養箱中培養48小時。取出細胞培養板，每孔加入50μL Celltiter Glo檢測液(Promega，G7573)，室溫孵育10分鐘，用酶標儀檢測生物發光信號，將檢測結果用軟體擬合出抑制曲線，計算出IC₅₀值。實驗結果見下表21、表22： Take the mouse spleen and grind it, centrifuge it for 5 minutes at 4°C to collect the lower cells, wash it once with washing solution (PBS+2% FBS+2mM EDTA) and centrifuge, remove the supernatant and add red blood cell lysis buffer (RBC Lysis Buffer, Invitrogen, 00 -4333-57), let stand at room temperature for 5 minutes until red blood cells are completely lysed. Centrifuge again and resuspend cells for counting. The cell suspension was sorted using B Cell Isolation Kit (Miltenyi Biotec, 130-090-862), and the isolated B cells were treated with RPMI 1640 medium (Gibco, 11875119) + 10% FBS (Gibco, 10099 -141) + 50 μM 2-mercaptoethanol (Sigma-Aldrich, M6250) were resuspended and counted, and the cells were plated in a 96-well cell plate for later use. Dilute BAFF (R&D Systems, 7537-BF) or APRIL (R&D Systems, 5860-AP) protein to a fixed concentration, add serially diluted anti-ICOSL antibody fusion protein and mix well, pre-incubate at 37°C for 30 minutes and then add 96-well cells. Plates were cultured in a 37°C cell culture incubator for 48 hours. Take out the cell culture plate, add 50 μL of Celltiter Glo detection solution (Promega, G7573) to each well, and incubate at room temperature for 10 minutes. Use a microplate reader to detect the bioluminescence signal. Use software to fit the detection results to an inhibition curve and calculate the IC ₅₀ value. . The experimental results are shown in Table 21 and Table 22 below:

表21. 抗ICOSL抗體融合蛋白抑制BAFF誘導的B細胞增殖的實驗結果

Table 21. Experimental results of anti-ICOSL antibody fusion protein inhibiting BAFF-induced B cell proliferation

表22. 抗ICOSL抗體融合蛋白抑制APRIL誘導的B細胞增殖的實驗結果

Table 22. Experimental results of anti-ICOSL antibody fusion protein inhibiting APRIL-induced B cell proliferation

實驗結果表明，本揭露構建的抗ICOSL抗體融合蛋白能抑制BAFF或APRIL誘導的B細胞增殖，而陽性對照分子AMG-570對APRIL沒有抑制活性。 Experimental results show that the anti-ICOSL antibody fusion protein constructed in this disclosure can inhibit B cell proliferation induced by BAFF or APRIL, while the positive control molecule AMG-570 has no inhibitory activity on APRIL.

測試例8：親和力測試Test Example 8: Affinity Test

用生物傳感晶片Protein A(GE，29127556)親和捕獲一定量的待測樣品，然後於晶片表面流經一系列濃度梯度的抗原，利用Biacore(GE，8K)即時檢測反應信號從而獲得結合和解離曲線。在每個迴圈解離完成後，用10mM甘胺酸-鹽酸溶液pH 1.5(GE，BR-1003-54)將生物晶片洗淨再生。實驗資料用BIAevaluation version 4.1軟體以1：1模型進行擬合，從而得出親和力數值。 The biosensor chip Protein A (GE, 29127556) is used to affinity capture a certain amount of the sample to be tested, and then a series of concentration gradient antigens flow through the surface of the chip, and Biacore (GE, 8K) is used to detect the reaction signal in real time to obtain binding and dissociation. curve. After each cycle of dissociation is completed, the biowafer is washed and regenerated with 10mM glycine-hydrochloric acid solution pH 1.5 (GE, BR-1003-54). The experimental data were fitted with a 1:1 model using BIAevaluation version 4.1 software to obtain the affinity value.

本測試中用到的相關抗原蛋白如下：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)。親和力的測試結果見下表23至表30： 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 crab 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 affinity test results are shown in Tables 23 to 30 below:

表23. 抗ICOSL抗體融合蛋白與人APRIL結合的親和力實驗結果

Table 23. Affinity experiment results of anti-ICOSL antibody fusion protein binding to human APRIL

表24. 抗ICOSL抗體融合蛋白與猴APRIL結合的親和力實驗結果

Table 24. Affinity experiment results of anti-ICOSL antibody fusion protein binding to monkey APRIL

表25. 抗ICOSL抗體融合蛋白與鼠APRIL結合的親和力實驗結果

Table 25. Affinity experimental results of anti-ICOSL antibody fusion protein binding to mouse APRIL

表26. 抗ICOSL抗體融合蛋白與人BAFF結合的親和力實驗結果

Table 26. Affinity experiment results of anti-ICOSL antibody fusion protein binding to human BAFF

表27. 抗ICOSL抗體融合蛋白與猴BAFF結合的親和力實驗結果

Table 27. Affinity experiment results of anti-ICOSL antibody fusion protein binding to monkey BAFF

表28. 抗ICOSL抗體融合蛋白與鼠BAFF結合的親和力實驗結果

Table 28. Affinity experiment results of anti-ICOSL antibody fusion protein binding to mouse BAFF

表29. 抗ICOSL抗體及其融合蛋白與人ICOSL結合的親和力實驗結果

Table 29. Affinity experimental results of anti-ICOSL antibodies and their fusion proteins binding to human ICOSL

表30. 抗ICOSL抗體融合蛋白與猴ICOSL/鼠ICOSL結合的親和力實驗結果

Table 30. Affinity experiment results of anti-ICOSL antibody fusion protein binding to monkey ICOSL/mouse ICOSL

實驗結果表明，本揭露構建的抗ICOSL抗體融合蛋白對人、猴和鼠的APRIL均具有有很強的親和力，而陽性對照分子AMG-570與人、猴和鼠的APRIL均不結合。本揭露構建的抗ICOSL抗體融合蛋白與人、猴和鼠的BAFF的親和力優於陽性對照分子AMG-570。本揭露構建的抗ICOSL抗體及其融合蛋白與人ICOSL、和猴ICOSL均具有良好的結合活性，但對鼠ICOSL沒有交叉結合活性。 Experimental results show that the anti-ICOSL antibody fusion protein constructed in this disclosure has strong affinity to human, monkey and mouse APRIL, while the positive control molecule AMG-570 does not bind to human, monkey and mouse APRIL. The anti-ICOSL antibody fusion protein constructed in this disclosure has better affinity to human, monkey and mouse BAFF than the positive control molecule AMG-570. The anti-ICOSL antibody and its fusion protein constructed in this disclosure White has good binding activity with human ICOSL and monkey ICOSL, but has no cross-binding activity with mouse ICOSL.

測試例9：體內抑制細胞因子分泌活性評價Test Example 9: Evaluation of In vivo Cytokine Secretion Inhibitory Activity

用人hICOSL-CHOK1細胞和人BAFF蛋白混合刺激小鼠，誘導小鼠體內產生IFNγ、IL-10和IgA等細胞因子，藉由檢測這些細胞因子的水準來評價抗ICOSL抗體融合蛋白的體內活性。實驗過程如下： Human hICOSL-CHOK1 cells and human BAFF protein were mixed to stimulate mice to induce the production of IFNγ, IL-10, IgA and other cytokines in the mice. 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:

SPF級雌性C57BL/6小鼠(浙江維通利華實驗動物技術有限公司)，8週齡，將小鼠進行隨機分4組，每組8隻，藉由腹腔混合注射hICOSL-CHOK1細胞(10⁶個細胞/小鼠)和1mg/kg人BAFF蛋白(Sino biological，10056-HNCH)，每天注射一次，持續四天。各組(陽性對照(AMG-570 12mpk)組，259H4L7-T7 11.7mpk組，259H4L7-T11 12.4mpk組，陰性對照(PBS)組)分別在第一天和第三天在注射hICOSL-CHOK1細胞和BAFF蛋白前1小時腹腔注射待測樣品。第五天收集各組小鼠血漿樣品，分別檢測IFNγ、IL-10和IgA的水準。其中AMG-570作為陽性對照，PBS作為陰性對照，其中AMG-570 12mpk(mpk也即給藥劑量為mg/kg)，259H4L7-T7 11.7mpk和259H4L7-T11 12.4mpk的藥物莫耳濃度相同。本測試例中所用的檢測試劑盒來源：Mouse IFN-gamma Quantikine ELISA Kit(R&D Systems，MIF00)，Mouse IL-10 Quantikine ELISA Kit(R&D Systems，M1000B)，Mouse IgA ELISA Kit(Abcam，ab157717)。實驗結果如下表31及圖4、圖5、圖6。 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 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) was injected with hICOSL-CHOK1 cells and The sample to be tested was injected intraperitoneally 1 hour before BAFF protein. On the fifth day, plasma samples from mice in each group were collected and the levels of IFNγ, IL-10 and IgA were measured respectively. AMG-570 is used as a positive control, and 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 sources of the detection kits used in this test example are: Mouse IFN-gamma Quantikine ELISA Kit (R&D Systems, MIF00), Mouse IL-10 Quantikine ELISA Kit (R&D Systems, M1000B), Mouse IgA ELISA Kit (Abcam, ab157717). The experimental results are shown in Table 31 and Figures 4, 5, and 6 below.

表31. 抗ICOSL抗體融合蛋白抑制細胞因子分泌實驗結果

Table 31. Experimental results of anti-ICOSL antibody fusion protein inhibiting cytokine secretion

實驗結果表明，本揭露構建的抗ICOSL抗體融合蛋白能顯著抑制IFNγ、IL-10、IgA的分泌，抑制活性強於陽性對照分子AMG-570。 Experimental results show that the anti-ICOSL antibody fusion protein constructed in this disclosure can significantly inhibit the secretion of IFNγ, IL-10, and IgA, and the inhibitory activity is stronger than the positive control molecule AMG-570.

測試例10：小鼠體內免疫抑制實驗Test Example 10: In vivo immunosuppression experiment in mice

藉由免疫抑制實驗評估了融合蛋白在體內抑制免疫反應的活性。實驗過程如下：SPF級雌性human ICOSL/human ICOS C57BL/6轉基因小鼠(百奧賽圖江蘇基因生物技術有限公司)，小鼠體重15-18g，6至8週齡。在第1天用1mg/kg KLH(Keyhole Limpet Hemocyanin，血藍蛋白)(Sigma，H7017)加佐劑(Thermo，77161)乳化混勻免疫小鼠，在第21天用5mg/kg KLH單獨免疫小鼠。KLH是一種具有強免疫原性的蛋白，可以引起小鼠產生很強的免疫反應並產生KLH特異性的抗體。在KLH免疫前一天開始給藥，每5天給藥一次，總共持續35天。給藥劑量為AMG-570 15mpk、259H4L7-T7 14.6mpk、259H4L7-T11 15.6mpk，各組的藥物莫耳濃度相同，AMG-570作為陽性對照，PBS作為陰性對照。每週取各組小鼠的血清樣品，藉由檢測小鼠血清中KLH特異性的IgG和IgM含量來評估融合蛋白抑制免疫反應的強度。本測試例中所用檢測試劑盒如下：Mouse anti-KLH IgG ELISA Kit(Life diagnostics，KLHG-1)，Mouse anti-KLH IgM ELISA Kit(Life Diagnostics，KLHM-1)。實驗結果見圖7、圖8。 The activity of the fusion protein in suppressing immune responses in vivo was evaluated by immunosuppression experiments. The experimental process is as follows: SPF grade female human ICOSL/human ICOS C57BL/6 transgenic mice (Biocytogen Jiangsu Gene Biotechnology Co., Ltd.), mice weighing 15-18g, 6 to 8 weeks old. On day 1, mice were immunized with 1 mg/kg KLH (Keyhole Limpet Hemocyanin, hemocyanin) (Sigma, H7017) plus adjuvant (Thermo, 77161) emulsified and mixed, and on day 21, mice were immunized with 5 mg/kg KLH alone. . KLH is a protein with strong immunogenicity that can cause mice to produce strong immune responses and produce KLH-specific antibodies. Dosing began one day before KLH immunization and was administered every 5 days for a total of 35 days. The dosages were AMG-570 15mpk, 259H4L7-T7 14.6mpk, and 259H4L7-T11 15.6mpk. The drug molar concentrations in each group were the same. AMG-570 was used as a positive control and PBS was used as a negative control. Serum samples from mice in each group were taken every week, and the intensity of the fusion protein in suppressing the immune response was evaluated by detecting the KLH-specific IgG and IgM content in the mouse serum. The detection kit used in this test example 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 Figures 7 and 8.

KLH特異性IgG的檢測結果表明，所有的給藥組在第28天和第35天這兩個時間點都能夠顯著的抑制KLH特異性IgG的產生，其中259H4L7-T7和259H4L7-T11組完全沒有出現免疫反應，KLH特異性IgG的水準與正常小鼠相同，免疫抑制活性要強於AMG-570。KLH特異性IgM的檢測結果表明，AMG-570只在第7-14天具有顯著的抑制活性，而259H4L7-T7和259H4L7-T11在所有的時間點都顯示出了顯著的抑制活性，抑制活性要顯著的強於AMG-570。 The test results of KLH-specific IgG showed that all administration groups were able to significantly inhibit the production of KLH-specific IgG at the two time points of day 28 and day 35, among which the 259H4L7-T7 and 259H4L7-T11 groups did not at all. An immune response occurred. 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. Significantly stronger than the AMG-570.

測試例11.大鼠體內藥物代謝動力學實驗Test Example 11. In vivo pharmacokinetic experiment in rats

用SD大鼠進行體內藥物代謝動力學測試。雄性SD大鼠(浙江維通利華實驗動物技術有限公司)隨機分組，每組4隻，靜脈注射給藥，給藥組於給藥前及給藥後5分鐘、8小時、24小時、48小時、84小時、9天、10天、14天、21天、28天採集全血0.2mL，不加抗凝，取血後在4℃放置30分鐘，1000g離心15分鐘，取上層血清置於EP管中，於-80℃保存。用ELISA法檢測血清中的血藥濃度，用Winnolin軟體計算受試藥物的ㄍ藥物代謝動力學參數和體內半衰期。 In vivo pharmacokinetic testing was performed using SD rats. Male SD rats (Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) were randomly divided into groups, 4 in each group, and administered intravenously. The administration group was administered intravenously before and 5 minutes, 8 hours, 24 hours, and 48 hours after administration. , 84 hours, 9 days, 10 days, 14 days, 21 days, and 28 days, collect 0.2 mL of whole blood without anticoagulation. After blood collection, place it at 4°C for 30 minutes, centrifuge at 1000g for 15 minutes, and take the upper serum and place it in EP tube and store at -80°C. Use ELISA method to detect the blood drug concentration in serum, and use Winnolin software to calculate the pharmacokinetic parameters and in vivo half-life of the test drug.

大鼠體內藥物代謝動力學實驗結果見下表32，實驗結果表明，本揭露獲得的融合蛋白具備較好的穩定性。 The results of the pharmacokinetics experiment in rats are shown in Table 32 below. The experimental results show that the fusion protein obtained in this disclosure has good stability.

表32.大鼠體內藥物代謝動力學實驗結果

Table 32. Results of pharmacokinetic experiments in rats

TW202334225A_111143243_SEQL.xmlTW202334225A_111143243_SEQL.xml

Claims

一種抗ICOSL抗體融合蛋白，其包含抗ICOSL抗體和TACI多肽； An anti-ICOSL antibody fusion protein comprising an anti-ICOSL antibody and a TACI polypeptide;

其中該抗ICOSL抗體包含重鏈可變區和輕鏈可變區，其中， Wherein the anti-ICOSL antibody comprises a heavy chain variable region and a light chain variable region, wherein,

該重鏈可變區包含HCDR1、HCDR2和HCDR3， The heavy chain variable region includes HCDR1, HCDR2 and HCDR3,

該輕鏈可變區包含LCDR1、LCDR2和LCDR3， The light chain variable region includes LCDR1, LCDR2 and LCDR3,

其中， in,

(i)該重鏈可變區的HCDR1、HCDR2和HCDR3分別包含SEQ ID NO：42、3、39、40或41中的HCDR1、HCDR2和HCDR3的胺基酸序列，和 (i) HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 42, 3, 39, 40 or 41, and

該輕鏈可變區的LCDR1、LCDR2和LCDR3分別包含SEQ ID NO：38、4、32、33、34、35、36或37中的LCDR1、LCDR2和LCDR3的胺基酸序列；或 LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 38, 4, 32, 33, 34, 35, 36 or 37; or

(ii)該重鏈可變區的HCDR1、HCDR2和HCDR3分別包含SEQ ID NO：30、1、28或29中的HCDR1、HCDR2和HCDR3的胺基酸序列，和 (ii) HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 30, 1, 28 or 29, and

該輕鏈可變區的LCDR1、LCDR2和LCDR3分別包含SEQ ID NO：22、2、20、21、23、24、25、26或27中的LCDR1、LCDR2和LCDR3的胺基酸序列。 LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 22, 2, 20, 21, 23, 24, 25, 26 or 27.

如請求項1所述的抗ICOSL抗體融合蛋白，其中， The anti-ICOSL antibody fusion protein as described in claim 1, wherein,

(i)該重鏈可變區的HCDR1包含SEQ ID NO：11的胺基酸序列，HCDR2包含SEQ ID NO：31或12的胺基酸序列，和HCDR3包含SEQ ID NO：13的胺基酸序列，和 (i) HCDR1 of the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 11, HCDR2 includes the amino acid sequence of SEQ ID NO: 31 or 12, and HCDR3 includes the amino acid sequence of SEQ ID NO: 13 sequence, and

該輕鏈可變區的LCDR1包含SEQ ID NO：14的胺基酸序列，LCDR2包含SEQ ID NO：15的胺基酸序列，和LCDR3包含SEQ ID NO：16的胺基酸序列；或 LCDR1 of the light chain variable region includes the amino acid sequence of SEQ ID NO: 14, LCDR2 includes the amino acid sequence of SEQ ID NO: 15, and LCDR3 includes the amino acid sequence of SEQ ID NO: 16; or

(ii)該重鏈可變區的HCDR1包含SEQ ID NO：5的胺基酸序列，HCDR2包含SEQ ID NO：19或6的胺基酸序列，和HCDR3包含SEQ ID NO：7的胺基酸序列，和 (ii) HCDR1 of the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 5, HCDR2 includes the amino acid sequence of SEQ ID NO: 19 or 6, and HCDR3 includes the amino acid sequence of SEQ ID NO: 7 sequence, and

該輕鏈可變區的LCDR1包含SEQ ID NO：8的胺基酸序列，LCDR2包含SEQ ID NO：17或9的胺基酸序列，和LCDR3包含SEQ ID NO：10或18的胺基酸序列； LCDR1 of the light chain variable region includes the amino acid sequence of SEQ ID NO: 8, LCDR2 includes the amino acid sequence of SEQ ID NO: 17 or 9, and LCDR3 includes the amino acid sequence of SEQ ID NO: 10 or 18 ;

較佳地， Preferably,

(i)該重鏈可變區的HCDR1包含SEQ ID NO：11的胺基酸序列，HCDR2包含SEQ ID NO：31的胺基酸序列，和HCDR3包含SEQ ID NO：13的胺基酸序列，和 (i) HCDR1 of the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 11, HCDR2 includes the amino acid sequence of SEQ ID NO: 31, and HCDR3 includes the amino acid sequence of SEQ ID NO: 13, and

(ii)該重鏈可變區的HCDR1包含SEQ ID NO：5的胺基酸序列，HCDR2包含SEQ ID NO：19的胺基酸序列，和HCDR3包含SEQ ID NO：7的胺基酸序列，和 (ii) HCDR1 of the heavy chain variable region includes the amino acid sequence of SEQ ID NO:5, HCDR2 includes the amino acid sequence of SEQ ID NO:19, and HCDR3 includes the amino acid sequence of SEQ ID NO:7, and

該輕鏈可變區的LCDR1包含SEQ ID NO：8的胺基酸序列，LCDR2包含SEQ ID NO：17的胺基酸序列，和LCDR3包含SEQ ID NO：10的胺基酸序列。 LCDR1 of the light chain variable region includes the amino acid sequence of SEQ ID NO:8, LCDR2 includes the amino acid sequence of SEQ ID NO:17, and LCDR3 includes the amino acid sequence of SEQ ID NO:10.

如請求項1或2所述的抗ICOSL抗體融合蛋白，其中， The anti-ICOSL antibody fusion protein as described in claim 1 or 2, wherein,

(i)該重鏈可變區包含與SEQ ID NO：42、39、40或41具有至少90%序列同一性的胺基酸序列，和該輕鏈可變區包含與SEQ ID NO：38、32、33、34、35、36或37具有至少90%序列同一性的胺基酸序列；或 (i) The heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 42, 39, 40 or 41, and the light chain variable region comprises an amino acid sequence with SEQ ID NO: 38, 32, 33, 34, 35, 36 or 37 amino acid sequences having at least 90% sequence identity; or

(ii)該重鏈可變區包含與SEQ ID NO：30、28或29具有至少90%序列同一性的胺基酸序列，和該輕鏈可變區包含與SEQ ID NO：22、20、21、23、24、25、26或27具有至少90%序列同一性的胺基酸序列；或 (ii) The heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 30, 28 or 29, and the light chain variable region comprises an amino acid sequence with SEQ ID NO: 22, 20, 21, 23, 24, 25, 26 or 27 have an amino acid sequence with at least 90% sequence identity; or

(iii)該重鏈可變區包含與SEQ ID NO：3具有至少90%序列同一性的胺基酸序列，和該輕鏈可變區包含與SEQ ID NO：4具有至少90%序列同一性的胺基酸序列；或 (iii) the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 3, and the light chain variable region comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 4 The amino acid sequence; or

(iv)該重鏈可變區包含與SEQ ID NO：1具有至少90%序列同一性的胺基酸序列，和該輕鏈可變區包含與SEQ ID NO：2具有至少90%序列同一性的胺基酸序列； (iv) the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, and the light chain variable region comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2 The amino acid sequence;

較佳地，(i)該重鏈可變區包含SEQ ID NO：42的胺基酸序列，和該輕鏈可變區包含SEQ ID NO：38的胺基酸序列；或 Preferably, (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 42, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 38; or

如請求項1至3中任一項所述的抗ICOSL抗體融合蛋白，其中該抗ICOSL抗體還包含抗體重鏈恆定區和輕鏈恆定區； The anti-ICOSL antibody fusion protein as described in any one of claims 1 to 3, wherein the anti-ICOSL antibody further comprises an antibody heavy chain constant region and a light chain constant region;

較佳地，該重鏈恆定區選自人IgG1、IgG2、IgG3和IgG4恆定區，該輕鏈恆定區選自人抗體κ或λ鏈恆定區； Preferably, the heavy chain constant region is selected from human IgG1, IgG2, IgG3 and IgG4 constant regions, and the light chain constant region is selected from the human antibody kappa or lambda chain constant region;

更佳地，該重鏈恆定區包含SEQ ID NO：43的胺基酸序列，該輕鏈恆定區包含SEQ ID NO：44的胺基酸序列。 More preferably, the heavy chain constant region includes the amino acid sequence of SEQ ID NO: 43, and the light chain constant region includes the amino acid sequence of SEQ ID NO: 44.

如請求項1至4中任一項所述的抗ICOSL抗體融合蛋白，其中， The anti-ICOSL antibody fusion protein as described in any one of claims 1 to 4, wherein,

(i)該抗ICOSL抗體的重鏈包含與SEQ ID NO：47具有至少90%序列同一性的胺基酸序列，和該抗ICOSL抗體的輕鏈包含與SEQ ID NO：48具有至少90%序列同一性的胺基酸序列；或 (i) The heavy chain of the anti-ICOSL antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 47, and the light chain of the anti-ICOSL antibody comprises at least 90% sequence identity with SEQ ID NO: 48 Identity of the amino acid sequence; or

(ii)該抗ICOSL抗體的重鏈包含與SEQ ID NO：45具有至少90%序列同一性的胺基酸序列，和該抗ICOSL抗體的輕鏈包含與SEQ ID NO：46具有至少90%序列同一性的胺基酸序列； (ii) The heavy chain of the anti-ICOSL antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 45, and the light chain of the anti-ICOSL antibody comprises at least 90% sequence identity with SEQ ID NO: 46 Identity of amino acid sequence;

較佳地， Preferably,

如請求項1至5中任一項所述的抗ICOSL抗體融合蛋白，其中該TACI多肽為包含SEQ ID NO：58的第48位至第85位胺基酸殘基的多肽或其變體；其中，該變體包含在選自第49、52、53、57、65、82和83位中的一個或更多個位點上具有胺基酸替換，該胺基酸替換的位點為相對於序列SEQ ID NO：58的自然順序編號的胺基酸殘基位點； The anti-ICOSL antibody fusion protein as described in any one of claims 1 to 5, wherein the TACI polypeptide is a polypeptide comprising amino acid residues 48 to 85 of SEQ ID NO: 58 or a variant thereof; Wherein, the variant includes an amino acid substitution at one or more positions selected from positions 49, 52, 53, 57, 65, 82 and 83, and the amino acid substitution positions are relative to Amino acid residue positions numbered in the natural sequence of sequence SEQ ID NO: 58;

較佳地，該變體包含選自由49T或49R、52S、53E或53Q、65T或65A、82A或82R、57E和83Y組成的組中的一個或更多個胺基酸替換，該胺基酸替換的位點為相對於序列SEQ ID NO：58的自然順序編號的胺基酸殘基位點。 Preferably, the variant comprises one or more amino acid substitutions selected from the group consisting of 49T or 49R, 52S, 53E or 53Q, 65T or 65A, 82A or 82R, 57E and 83Y, the amino acid The replaced positions are amino acid residue positions numbered relative to the natural sequence of sequence SEQ ID NO:58.

如請求項1至6中任一項所述的抗ICOSL抗體融合蛋白，其中該TACI多肽的胺基酸序列如SEQ ID NO：51至83中任一所示； The anti-ICOSL antibody fusion protein as described in any one of claims 1 to 6, wherein the amino acid sequence of the TACI polypeptide is as shown in any one of SEQ ID NO: 51 to 83;

較佳地，該TACI多肽的胺基酸序列如SEQ ID NO：83所示。 Preferably, the amino acid sequence of the TACI polypeptide is shown in SEQ ID NO: 83.

如請求項1至7中任一項所述的抗ICOSL抗體融合蛋白，其包括： The anti-ICOSL antibody fusion protein as described in any one of claims 1 to 7, which includes:

第二鏈：[TACI多肽2]-[連接子2]-[抗ICOSL抗體的輕鏈]；或 Second chain: [TACI polypeptide 2]-[linker 2]-[light chain of anti-ICOSL antibody]; or

第二鏈：抗ICOSL抗體的輕鏈；或 Second chain: light chain of anti-ICOSL antibody; or

第二鏈：[抗ICOSL抗體的輕鏈]-[連接子2]-[TACI多肽2]； Second chain: [light chain of anti-ICOSL antibody]-[linker 2]-[TACI polypeptide 2];

其中，該(A)、(B)或(C)中，其中該TACI多肽1和TACI多肽2是相同或不相同的，該連接子1和連接子2是相同或不相同的； Wherein, in (A), (B) or (C), the TACI polypeptide 1 and TACI polypeptide 2 are the same or different, and the linker 1 and the linker 2 are the same or different;

較佳地，該連接子1和連接子2各自獨立地具有(G_xS)_y的結構，其中，x選自1-5的整數，y選自0-6的整數， Preferably, the linker 1 and the linker 2 each independently have the structure of (G _x S) _y , where x is selected from an integer of 1-5, and y is selected from an integer of 0-6,

更佳地，該連接子1和連接子2各自獨立地如SEQ ID NO：89或90所示； More preferably, the linker 1 and the linker 2 are each independently as shown in SEQ ID NO: 89 or 90;

視需要地，在(A)、(B)或(C)中，在第一鏈的C端還包括保護胺基酸； Optionally, in (A), (B) or (C), a protected amino acid is further included at the C-terminus of the first chain;

視需要地，在(C)中，在第二鏈的C端還包括保護胺基酸； Optionally, in (C), a protected amino acid is further included at the C-terminus of the second chain;

較佳地，該保護胺基酸是QK。 Preferably, the protected amino acid is QK.

如請求項1至8中任一項所述的抗ICOSL抗體融合蛋白，其中， The anti-ICOSL antibody fusion protein as described in any one of claims 1 to 8, wherein,

該抗ICOSL抗體融合蛋白具有：包含SEQ ID NO：86的胺基酸序列的第一鏈，和包含SEQ ID NO：88、87或48的胺基酸序列的第二鏈；或 The anti-ICOSL antibody fusion protein has: a first chain comprising the amino acid sequence of SEQ ID NO: 86, and a second chain comprising the amino acid sequence of SEQ ID NO: 88, 87 or 48; or

該抗ICOSL抗體融合蛋白具有：包含SEQ ID NO：84的胺基酸序列的第一鏈，和包含SEQ ID NO：85或46的胺基酸序列的第二鏈； The anti-ICOSL antibody fusion protein has: a first chain including the amino acid sequence of SEQ ID NO: 84, and a second chain including the amino acid sequence of SEQ ID NO: 85 or 46;

較佳地，該抗ICOSL抗體融合蛋白具有2條包含SEQ ID NO：86的胺基酸序列的第一鏈，和2條包含SEQ ID NO：88的胺基酸序列的第二鏈。 Preferably, the anti-ICOSL antibody fusion protein has 2 first chains comprising the amino acid sequence of SEQ ID NO: 86, and 2 second chains comprising the amino acid sequence of SEQ ID NO: 88.

一種抗ICOSL抗體，其包含重鏈可變區和輕鏈可變區，該重鏈可變區包含HCDR1、HCDR2和HCDR3，該輕鏈可變區包含LCDR1、LCDR2和LCDR3，其中， An anti-ICOSL antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein,

如請求項10所述的抗ICOSL抗體，其中， The anti-ICOSL antibody as described in claim 10, wherein,

較佳地， Preferably,

如請求項10或11所述的抗ICOSL抗體，其中， The anti-ICOSL antibody as described in claim 10 or 11, wherein,

如請求項10至12中任一項所述的抗ICOSL抗體，其中該抗ICOSL抗體還包含抗體重鏈恆定區和輕鏈恆定區； The anti-ICOSL antibody as described in any one of claims 10 to 12, wherein the anti-ICOSL antibody further comprises an antibody heavy chain constant region and a light chain constant region;

如請求項10至13中任一項所述的抗ICOSL抗體，其中， The anti-ICOSL antibody according to any one of claims 10 to 13, wherein,

較佳地， Preferably,

如請求項1至9中任一項所述的抗ICOSL抗體融合蛋白或如請求項10至14中任一項所述的抗ICOSL抗體，其具有一種或更多種以下特性： The anti-ICOSL antibody fusion protein as described in any one of claims 1 to 9 or the anti-ICOSL antibody as described in any one of claims 10 to 14, which has one or more of the following properties:

A.與人ICOSL和食蟹猴ICOSL特異性結合，不與鼠ICOSL特異性結合；較佳地，以小於5.00E-09M的KD值與人ICOSL結合，和/或以小於8.00E-09M的KD值與食蟹猴ICOSL結合，該KD值藉由表面電漿共振測定法測量； A. Specifically binds to human ICOSL and cynomolgus ICOSL, but does not specifically bind to mouse ICOSL; preferably, it binds to human ICOSL with a KD value of less than 5.00E-09M, and/or with a KD of less than 8.00E-09M value combined with cynomolgus monkey ICOSL, the KD value was measured by surface plasmon resonance assay;

B.與人APRIL、食蟹猴APRIL和/或鼠APRIL特異性結合；較佳地，以小於2.00E-11M的KD值與人APRIL結合，以小於2.00E-10M的KD值與食蟹猴APRIL結合，和/或以小於2.00E-11M的KD值與鼠APRIL結合，該KD值藉由表面電漿共振測定法所測量； B. Specifically binds to human APRIL, cynomolgus monkey APRIL and/or mouse APRIL; preferably, binds to human APRIL with a KD value of less than 2.00E-11M, and binds to cynomolgus monkey with a KD value of less than 2.00E-10M Binds to APRIL, and/or binds to murine APRIL with a KD value less than 2.00E-11M, as measured by surface plasmon resonance assay;

C.與人BAFF、食蟹猴BAFF和/或鼠BAFF特異性結合；較佳地，以小於7.00E-11M的KD值與人BAFF結合，以小於4.00E-10M的KD值與食蟹猴BAFF 結合，和/或以小於6.00E-11M的KD值與鼠BAFF結合，該KD值藉由表面電漿共振測定法所測量； C. Specifically binds to human BAFF, cynomolgus BAFF and/or mouse BAFF; preferably, binds to human BAFF with a KD value of less than 7.00E-11M, and binds to cynomolgus monkey with a KD value of less than 4.00E-10M BAFF Binds to, and/or binds to mouse BAFF with a KD value of less than 6.00E-11M, as measured by surface plasmon resonance assay;

D.具有阻斷ICOSL與ICOS結合的活性；較佳地，阻斷人ICOSL與ICOS結合的IC₅₀值小於0.16nM，該IC₅₀值藉由FACS方法檢測； D. Has the activity of blocking the binding of ICOSL to ICOS; preferably, the IC ₅₀ value of blocking the binding of human ICOSL to ICOS is less than 0.16nM, and the IC ₅₀ value is detected by FACS method;

E.具有阻斷APRIL與BCMA結合的活性；較佳地，阻斷APRIL與BCMA結合的IC₅₀值小於40.00nM，該IC₅₀值藉由ELISA方法檢測； E. Has the activity of blocking the binding of APRIL to BCMA; preferably, the IC ₅₀ value of blocking the binding of APRIL to BCMA is less than 40.00nM, and the IC ₅₀ value is detected by ELISA method;

F.具有阻斷APRIL與TACI結合的活性；較佳地，阻斷APRIL與TACI結合的IC₅₀值小於40.00nM，該IC₅₀值藉由ELISA方法檢測； F. Has the activity of blocking the binding of APRIL and TACI; preferably, the IC ₅₀ value of blocking the binding of APRIL and TACI is less than 40.00nM, and the IC ₅₀ value is detected by ELISA method;

G.具有阻斷BAFF與BCMA結合的活性；較佳地，阻斷BAFF與BCMA結合的IC₅₀值小於2.00nM，該IC₅₀值藉由ELISA方法檢測； G. Has the activity of blocking the binding of BAFF and BCMA; preferably, the IC ₅₀ value of blocking the binding of BAFF and BCMA is less than 2.00nM, and the IC ₅₀ value is detected by ELISA method;

H.具有阻斷BAFF與BAFF-R結合的活性；較佳地，阻斷BAFF與BAFF-R結合的IC₅₀值小於2.00nM，該IC₅₀值藉由ELISA方法檢測； H. Has the activity of blocking the binding of BAFF and BAFF-R; preferably, the IC ₅₀ value of blocking the binding of BAFF and BAFF-R is less than 2.00nM, and the IC ₅₀ value is detected by ELISA method;

I.具有阻斷BAFF與TACI結合的活性；較佳地，阻斷BAFF與TACI結合的IC₅₀值小於1.00nM，該IC₅₀值藉由ELISA方法檢測； I. Have the activity of blocking the binding of BAFF and TACI; preferably, the IC ₅₀ value of blocking the binding of BAFF and TACI is less than 1.00nM, and the IC ₅₀ value is detected by ELISA method;

J.抑制細胞因子的分泌，較佳地，細胞因子選自以下任一項或其組合：IFNγ、IL-10和/或IgA； J. Inhibit the secretion of cytokines. Preferably, the cytokines are selected from any one of the following or a combination thereof: IFNγ, IL-10 and/or IgA;

K.抑制B細胞增殖和/或T細胞增殖。 K. Inhibit B cell proliferation and/or T cell proliferation.

一種醫藥組成物，其包含： A pharmaceutical composition containing:

如請求項1至9或15中任一項所述的抗ICOSL抗體融合蛋白或如請求項10至15中任一項所述的抗ICOSL抗體，以及 An anti-ICOSL antibody fusion protein as described in any one of claims 1 to 9 or 15 or an anti-ICOSL antibody as described in any one of claims 10 to 15, and

一種或多種藥學上可接受的載體、稀釋劑或賦形劑。 One or more pharmaceutically acceptable carriers, diluents or excipients.

一種核酸分子，其編碼如請求項1至9或15中任一項所述的抗ICOSL抗體融合蛋白或如請求項10至15中任一項所述的抗ICOSL抗體。 A nucleic acid molecule encoding an anti-ICOSL antibody fusion protein as described in any one of claims 1 to 9 or 15 or an anti-ICOSL antibody as described in any one of claims 10 to 15.

一種宿主細胞，其含有如請求項17所述的核酸分子。 A host cell containing the nucleic acid molecule of claim 17.

一種治療自體免疫性疾病、炎性疾病、B細胞障礙或T細胞障礙的方法，該方法包括向有需要的受試者施用治療有效量的如請求項1至9或15中任一項所述的抗ICOSL抗體融合蛋白或如請求項10至15中任一項所述的抗ICOSL抗體或如請求項16所述的醫藥組成物的步驟； 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 one of claims 1 to 9 or 15. The steps of the above-mentioned anti-ICOSL antibody fusion protein or the anti-ICOSL antibody as described in any one of claims 10 to 15 or the pharmaceutical composition as described in claim 16;

較佳地，該自體免疫性疾病或炎性疾病選自：系統性紅斑狼瘡、類風濕性關節炎、移植物抗宿主病、哮喘、免疫性血小板減少性紫癜、多發性硬化、糖尿病引起的炎性疾病、銀屑病、炎性腸病、克羅恩病、潰瘍性結腸炎、格雷夫斯病和橋本氏甲狀腺炎；該B細胞障礙或T細胞障礙為腫瘤； Preferably, the autoimmune disease or inflammatory disease is selected from: systemic lupus erythematosus, rheumatoid arthritis, graft-versus-host disease, asthma, immune thrombocytopenic purpura, multiple sclerosis, diabetes-induced Inflammatory diseases, 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;

更佳地，該腫瘤選自：頭頸癌、非小細胞肺癌、尿路上皮癌、白血病、肉瘤、黑色素瘤、腺癌、結直腸癌、***腫瘤、乳腺癌和小細胞肺癌；該自體免疫性疾病為系統性紅斑狼瘡。 More preferably, the tumor is selected from: head and neck cancer, non-small cell lung cancer, urothelial cancer, leukemia, sarcoma, melanoma, adenocarcinoma, colorectal cancer, prostate tumor, breast cancer and small cell lung cancer; the autoimmune The disease is systemic lupus erythematosus.