TWI789679B

TWI789679B - Anti-human programmed death ligand-1 (pd-l1) antibodies and uses thereof

Info

Publication number: TWI789679B
Application number: TW110101177A
Authority: TW
Inventors: 趙新燕; 鄧婧; 盧士強; 李鑫鑫; 任晉生
Original assignee: 大陸商江蘇先聲藥業有限公司
Priority date: 2021-01-12
Filing date: 2021-01-12
Publication date: 2023-01-11
Also published as: TW202227488A

Abstract

Disclosed are antibodies capable of specifically binding to human programmed death ligand-1 (PD-L1) or antigen-binding fragments thereof. The antibodies or antigen-binding fragments can enhance the function of T cells, up-regulate the immune response mediated by T cells, and is used for treating diseases related to abnormal PD-L1 expression and/or abnormal T cell function, such as tumors.

Description

Antibody against human programmed death ligand-1 (PD-L1) and use thereof

本發明涉及抗人程序性死亡配體-1(PD-L1)的抗體或其抗原結合片段，其編碼核酸、表達載體和表達細胞、製備方法、藥物組合物、以及它們用於增強T細胞的功能，上調T細胞介導的免疫應答和用於治療PD-L1表達異常和T細胞功能異常相關的疾病，如腫瘤的用途。 The present invention relates to an anti-human programmed death ligand-1 (PD-L1) antibody or its antigen-binding fragment, its encoding nucleic acid, expression vector and expression cell, preparation method, pharmaceutical composition, and their use for enhancing T cells Function, up-regulation of T cell-mediated immune response and use in the treatment of diseases related to abnormal expression of PD-L1 and abnormal function of T cells, such as tumors.

免疫治療已成為腫瘤治療中發展最為迅速、最具前景的研究領域之一，而運用免疫檢查點抑制劑，如PD-1/PD-L1單抗，CTLA-4單抗則是腫瘤免疫療法革命性的治療方法，大大地提高了惡行腫瘤患者地生存期。 Immunotherapy has become one of the fastest-growing and most promising research fields in tumor treatment, and the use of immune checkpoint inhibitors, such as PD-1/PD-L1 monoclonal antibody and CTLA-4 monoclonal antibody, is a revolution in tumor immunotherapy Sexual treatment has greatly improved the survival period of patients with malignant tumors.

T細胞介導的免疫反應受到共刺激和共抑制機制的嚴格調控，在抗原免疫反應和維持自我耐受之間保持最佳平衡。該平衡由多種啟動性和抑制性蛋白參與。抑制性蛋白，也被稱為免疫檢查點類蛋白，調節殺傷性T細胞(cytotoxic T lymphocyte,CTL)的啟動和效應功能，以維持自我耐受性。免疫檢查點類抑制性蛋白在腫瘤的調節通路中起著關鍵作用。其中一個重要的免疫檢查點蛋白PD-1，與其配體PD-L1結合後，會傳導免疫抑制信號，降低T細胞的活性。同時腫瘤細胞也可以通過在細胞表面表達PD-L1來抑制T細胞的啟動和增殖，從而逃避CTL的攻擊和殺傷。利用PD-1或PD-L1單抗，阻止PD-1/PD-L1的結合和相互作用，可以部分恢復T細胞的功能，從而增強殺傷腫瘤細胞的能力。2011年，第一個免疫檢測點抑制劑伊普利單抗作為一種抗CTLA-4單克隆抗體，成為成功用於治療黑色素瘤的腫瘤免疫療法，至今治療的很多患者已獲得相比與傳統治療方法更好的5年生存期。之後，FDA又先後批准了3個PD-1單抗和3個PD-L1單抗，成功地用於免疫治療除黑色素瘤之外的十幾種腫瘤，並成為多種癌症的一線治療手段，如非小細胞肺癌(NSCLC)、腎細胞癌(RCC)和膀胱或尿路上皮癌等。中國至今已批准了2個進口PD-1抗體和3個國產PD-1抗體上市，但是還沒有PD-L1抗體獲批，並且鑒於PD-L1抗體與PD-1抗體治療機理上及目前臨床試驗聯用藥物和適用適應症的不同，研發新的PD-L1單抗和基於PD-L1的雙抗仍然有重大的社會和經濟意義。 T cell-mediated immune responses are tightly regulated by co-stimulatory and co-inhibitory mechanisms, maintaining an optimal balance between antigen immune responses and maintaining self-tolerance. This balance is involved in a variety of promoter and repressor proteins. Inhibitory proteins, also known as immune checkpoint proteins, regulate the initiation and effector functions of killer T cells (cytotoxic T lymphocytes, CTLs) to maintain self-tolerance. Immune checkpoint inhibitory proteins play key roles in tumor regulatory pathways. One of the important immune checkpoint proteins, PD-1, when combined with its ligand PD-L1, will transmit immunosuppressive signals and reduce the activity of T cells. At the same time, tumor cells can also inhibit the initiation and proliferation of T cells by expressing PD-L1 on the cell surface, thereby evading the attack and killing of CTL. Using PD-1 or PD-L1 monoclonal antibody to prevent the binding and interaction of PD-1/PD-L1 can partially restore the function of T cells, thereby enhancing the ability to kill tumor cells. In 2011, the first immune checkpoint inhibitor, ipilimumab, as an anti-CTLA-4 monoclonal antibody, became a successful tumor immunotherapy for the treatment of melanoma. Many patients have achieved better 5-year survival compared with traditional treatment methods. Afterwards, the FDA has successively approved 3 PD-1 monoclonal antibodies and 3 PD-L1 monoclonal antibodies, which have been successfully used in the immunotherapy of more than a dozen tumors other than melanoma, and have become the first-line treatment for various cancers, such as Non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC) and bladder or urothelial carcinoma, etc. China has so far approved 2 imported PD-1 antibodies and 3 domestically produced PD-1 antibodies for marketing, but no PD-L1 antibody has been approved yet, and in view of the therapeutic mechanism of PD-L1 antibodies and PD-1 antibodies and current clinical trials Depending on the combination of drugs and applicable indications, the development of new PD-L1 monoclonal antibodies and PD-L1-based double antibodies still has great social and economic significance.

本發明提供特異性結合人程序性死亡配體-1(PD-L1)的抗體及抗原結合片段，編碼這些抗體及抗原結合片段的核酸，包含所述抗體及抗原結合片段和藥物組合物和試劑盒，以及它們用於增強T細胞的功能，上調T細胞介導的免疫應答和用於治療PD-L1表達異常和T細胞功能異常相關的病症，例如腫瘤免疫的用途。該抗體不僅可以與人和食蟹猴PD-L1蛋白結合，還可以阻斷人PD-L1和人PD-1之間的相互作用。 The present invention provides antibodies and antigen-binding fragments specifically binding to human programmed death ligand-1 (PD-L1), nucleic acids encoding these antibodies and antigen-binding fragments, including the antibodies and antigen-binding fragments, pharmaceutical compositions and reagents cassettes, and their use for enhancing the function of T cells, upregulating T cell-mediated immune responses, and treating diseases associated with abnormal expression of PD-L1 and abnormal T cell function, such as tumor immunity. The antibody can not only bind to human and cynomolgus PD-L1 proteins, but also block the interaction between human PD-L1 and human PD-1.

在一些實施方案中，特異性結合人程序性死亡配體-1(PD-L1)的分離的抗體或抗原結合片段，包含重鏈CDRs組合和輕鏈CDRs組合： In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds human programmed death ligand-1 (PD-L1) comprises a combination of heavy chain CDRs and a combination of light chain CDRs:

(1)所述重鏈CDRs組合包含：CDR1-VH、CDR2-VH和CDR3-VH；所述CDR1-VH、CDR2-VH和CDR3-VH具有選自以下的任意序列組合或者與所述序列組合相比具有1、2、3或更多個氨基酸插入、缺失和/或替換的序列組合： (1) The heavy chain CDRs combination includes: CDR1-VH, CDR2-VH and CDR3-VH; the CDR1-VH, CDR2-VH and CDR3-VH have any sequence combination selected from the following or are combined with the sequence Compared to sequence combinations with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions:

和， and,

(2)所述輕鏈CDRs組合包含：CDR1-VL、CDR2-VL和CDR3-VL，所述CDR1-VL、CDR2-VL和CDR3-VL具有選自以下任意序列組合或者與所述序列組合相比具有1、2、3或更多個氨基酸插入、缺失和/或替換的序列組合： (2) The light chain CDRs combination includes: CDR1-VL, CDR2-VL and CDR3-VL, and the CDR1-VL, CDR2-VL and CDR3-VL have any sequence combination selected from the following or match the sequence combination Combinations of sequences with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions than:

各個CDR1-VH、CDR2-VH、CDR3-VH、CDR1-VL、CDR2-VL和CDR3-VL為根據KABAT、Chothia或IMGT的通行分析方法編碼。 Each CDR1-VH, CDR2-VH, CDR3-VH, CDR1-VL, CDR2-VL and CDR3-VL is coded according to the prevailing analysis method of KABAT, Chothia or IMGT.

在一些實施方案中，特異性結合人程序性死亡配體-1(PD-L1)的分離的人源化抗體或抗原結合片段，包含重鏈CDRs組合和輕鏈CDRs組合： In some embodiments, the isolated humanized antibody or antigen-binding fragment that specifically binds human programmed death ligand-1 (PD-L1) comprises a combination of heavy chain CDRs and a combination of light chain CDRs:

(1)所述重鏈CDRs組合包含：CDR1-VH、CDR2-VH和CDR3-VH；所述CDR1-VH、CDR2-VH和CDR3-VH具有選自以下的任意序列組合或者與所述序列組合相比具有1、2、3或更多個氨基酸插入、缺失和/或替換的序列組合： (1) The heavy chain CDRs combination comprises: CDR1-VH, CDR2-VH and CDR3-VH; the CDR1-VH, CDR2-VH and CDR3-VH have the following Any sequence combination or sequence combination with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to said sequence combination:

和， and,

特別地，例如本發明的抗體或其抗原結合片段包含選自以下的重鏈CDRs和輕鏈CDRs組合：VH1+VL1、VH2+VL2、VH3+VL3、VH4+VL4、VH5+VL5、VH6+VL6、VH7+VL7、VH8+VL8、VH9+VL9、VH10+VL10、VH11+VL11、VH12+VL12、VH13+VL13、VH14+VL14、VH15+VL15、VH16+VL16、VH17+VL17、VH18+VL18、VH19+VL19、VH20+VL20、VH21+VL21、VH22+VL22、VH23+VL23、VH24+VL24、VH25+VL25、或VH26+VL26，以及與所述重鏈和輕鏈CDRs組合之序列相比具有1、2、3或更多個氨基酸插入、缺失和/或替換的CDRs組合。 In particular, for example, an antibody of the invention or an antigen-binding fragment thereof comprises a combination of heavy chain CDRs and light chain CDRs selected from the group consisting of: VH1+VL1, VH2+VL2, VH3+VL3, VH4+VL4, VH5+VL5, VH6+VL6 , VH7+VL7, VH8+VL8, VH9+VL9, VH10+VL10, VH11+VL11, VH12+VL12, VH13+VL13, VH14+VL14, VH15+VL15, VH16+VL16, VH17+VL17, VH18+VL18, VH19 +VL19, VH20+VL20, VH21+VL21, VH22+VL22, VH23+VL23, VH24+VL24, VH25+VL25, or VH26+VL26, and have 1, Combinations of CDRs with 2, 3 or more amino acid insertions, deletions and/or substitutions.

在另一個具體實施方案中，本發明提供這樣的抗體或其抗原結合片段，其中： In another specific embodiment, the invention provides an antibody or antigen-binding fragment thereof wherein:

1)重鏈可變區和輕鏈可變區分別具有SEQ ID NO：1和SEQ ID NO：2所示序列，或者與所示序列具有70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或更高一致性的序列； 1) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or higher identity sequences;

2)重鏈可變區和輕鏈可變區分別具有SEQ ID NO：3和SEQ ID NO：4所示序列，或者與所示序列具有70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或更高一致性的序列； 2) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or higher identity sequences;

3)重鏈可變區和輕鏈可變區分別具有SEQ ID NO：5和SEQ ID NO：6所示序列，或者與所示序列具有70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或更高一致性的序列； 3) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or higher identity sequences;

4)重鏈可變區和輕鏈可變區分別具有SEQ ID NO：7和SEQ ID NO：8所示序列，或者與所示序列具有70%、75%、80%、85%、90%、95%、 96%、97%、98%、99%或更高一致性的序列； 4) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, Sequences with 96%, 97%, 98%, 99% or higher identity;

5)重鏈可變區和輕鏈可變區分別具有SEQ ID NO：9和SEQ ID NO：10所示序列，或者與所示序列具有70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或更高一致性的序列； 5) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10 respectively, or have 70%, 75%, 80%, 85%, 90% of the sequence shown , 95%, 96%, 97%, 98%, 99% or higher identity sequences;

6)重鏈可變區和輕鏈可變區分別具有SEQ ID NO：11和SEQ ID NO：12所示序列，或者與所示序列具有70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或更高一致性的序列； 6) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12 respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or higher identity sequences;

7)重鏈可變區和輕鏈可變區分別具有SEQ ID NO：13和SEQ ID NO：14所示序列，或者與所示序列具有70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或更高一致性的序列； 7) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively, or have 70%, 75%, 80%, 85%, or 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or higher identity sequences;

8)重鏈可變區和輕鏈可變區分別具有SEQ ID NO：15和SEQ ID NO：16所示序列，或者與所示序列具有70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或更高一致性的序列； 8) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16 respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or higher identity sequences;

9)重鏈可變區和輕鏈可變區分別具有SEQ ID NO：17和SEQ ID NO：18所示序列，或者與所示序列具有70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或更高一致性的序列； 9) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18 respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or higher identity sequences;

10)重鏈可變區和輕鏈可變區分別具有SEQ ID NO：127和SEQ ID NO：128所示序列，或者與所示序列具有70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或更高一致性的序列； 10) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 127 and SEQ ID NO: 128 respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or higher identity sequences;

11)重鏈可變區和輕鏈可變區分別具有SEQ ID NO：129和SEQ ID NO：130所示序列，或者與所示序列具有70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或更高一致性的序列； 11) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 129 and SEQ ID NO: 130, respectively, or have 70%, 75%, 80%, 85%, or 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or higher identity sequences;

12)重鏈可變區和輕鏈可變區分別具有SEQ ID NO：131和SEQ ID NO：132所示序列，或者與所示序列具有70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或更高一致性的序列；或， 12) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 131 and SEQ ID NO: 132, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences; or,

13)重鏈可變區和輕鏈可變區分別具有SEQ ID NO：133和SEQ ID NO：134所示序列，或者與所示序列具有70%、75%、80%、85%、90%、 95%、96%、97%、98%、99%或更高一致性的序列。 13) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 133 and SEQ ID NO: 134, respectively, or have 70%, 75%, 80%, 85%, or 90% of the sequences shown , Sequences with 95%, 96%, 97%, 98%, 99% or higher identity.

在一個優選實施方案中，本發明的抗體或其抗原結合片段是嵌合的或人源化的或全人源的。 In a preferred embodiment, the antibodies or antigen-binding fragments thereof of the invention are chimeric or humanized or fully human.

在一個優選實施方案中，本發明的抗體或其抗原結合片段，其與人程序性死亡配體-1(PD-L1)結合的解離常數(KD)不大於10nM，與食蟹猴程序性死亡配體-1(PD-L1)結合的解離常數(KD)不大於100nM。 In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention, which binds to human programmed death ligand-1 (PD-L1) with a dissociation constant (KD) not greater than 10 nM, is compatible with programmed death in cynomolgus monkeys. The dissociation constant (KD) of ligand-1 (PD-L1) binding is not greater than 100nM.

在一個優選實施方案中，本發明的抗體或其抗原結合片段，包含人或鼠抗體IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD任何其中之一恒定區的序列；優選包含人或鼠抗體IgG1、IgG2、IgG3或IgG4的恒定區的序列；或攜帶突變的人或鼠抗體IgG1、IgG2、IgG3或IgG4的恒定區的序列。 In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises the sequence of any one of the constant regions of human or murine antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; The sequence of the constant region of a murine antibody IgG1, IgG2, IgG3 or IgG4; or the sequence of the constant region of a human or murine antibody IgG1, IgG2, IgG3 or IgG4 carrying a mutation.

在一個優選實施方案中，本發明所述抗原結合片段選自F(ab)2、Fab’、Fab、Fv、scFv、雙特異抗體、納米抗體和抗體最小識別單位中的一種或多種。 In a preferred embodiment, the antigen-binding fragment of the present invention is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibody, nanobody and antibody minimum recognition unit.

在一個優選實施方案中，本發明的抗體或其抗原結合片段可與選自編號34、50、90、130、156、370、373、413、或794的抗體競爭性的結合PD-L1，並且具備以下特性： In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention can competitively bind to PD-L1 with an antibody selected from number 34, 50, 90, 130, 156, 370, 373, 413, or 794, and Has the following characteristics:

1)特異性結合PD-L1重組蛋白及表達PD-L1的細胞； 1) Specifically binding to PD-L1 recombinant protein and cells expressing PD-L1;

2)阻斷PD-L1與PD-1蛋白的結合； 2) Block the combination of PD-L1 and PD-1 protein;

3)抑制PD-1與細胞表面表達的PD-L1的結合； 3) Inhibit the binding of PD-1 to PD-L1 expressed on the cell surface;

4)增強T細胞活性； 4) Enhance T cell activity;

5)介導抗體依賴的細胞殺傷(ADCC)活性；或/和 5) mediate antibody-dependent cell killing (ADCC) activity; or/and

6)抑制腫瘤生長。 6) Inhibit tumor growth.

在一些實施方案中，本發明提供一種分離的核酸分子，所述核酸分子編碼本發明上述所述的抗體、抗原結合片段、或其任意組合。 In some embodiments, the present invention provides an isolated nucleic acid molecule encoding the above-described antibody, antigen-binding fragment, or any combination thereof of the present invention.

在一些實施方案中，本發明提供一種表達載體，其包含本發明上述所述分離的核酸分子。 In some embodiments, the present invention provides an expression vector comprising the above-mentioned Said isolated nucleic acid molecule.

在一些實施方案中，本發明提供一種宿主細胞，其包含本發明上述所述分離的核酸分子或表達載體。 In some embodiments, the present invention provides a host cell comprising the above-mentioned isolated nucleic acid molecule or expression vector of the present invention.

在一個優選實施方案中，所述宿主細胞是真核細胞或原核細胞；更優選，所述宿主細胞來源於哺乳動物細胞、酵母細胞、昆蟲細胞、大腸桿菌和/或枯草桿菌；更優選，所述宿主細胞選自中國倉鼠卵巢細胞(CHO)。 In a preferred embodiment, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli and/or Bacillus subtilis; more preferably, the The host cells are selected from Chinese hamster ovary cells (CHO).

在一些實施方案中，本發明提供一種抗體或抗原結合片段的製備方法，在適當的條件下培養本發明上述所述的宿主細胞，並分離抗體或抗原結合片段。 In some embodiments, the present invention provides a method for preparing an antibody or antigen-binding fragment, culturing the above-mentioned host cells of the present invention under appropriate conditions, and isolating the antibody or antigen-binding fragment.

在一些實施方案中，本發明提供一種藥物組合物，組合物包含本發明上述所述的抗體或抗原結合片段、本發明上述所述分離的核酸分子、本發明上述所述表達載體、本發明上述所述細胞，或本發明上述所述方法製備的產品(例如抗體和抗原結合片段)，以及藥學上可接受的載體。 In some embodiments, the present invention provides a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment of the present invention, the above-mentioned isolated nucleic acid molecule of the present invention, the above-mentioned expression vector of the present invention, the above-mentioned The cells, or the products (such as antibodies and antigen-binding fragments) prepared by the above-mentioned method of the present invention, and a pharmaceutically acceptable carrier.

在一個優選實施方案中，所述藥物組合物還包含額外的抗腫瘤劑。 In a preferred embodiment, the pharmaceutical composition further comprises an additional antineoplastic agent.

在一些實施方案中，本發明提供一種預防和/或治療PD-L1表達異常和/或T細胞功能異常相關的疾病的方法，包含向有此需要的患者施用本發明上述所述的抗體或抗原結合片段、本發明上述所述的分離的核酸分子、本發明上述所述的表達載體、本發明上述所述的細胞、本發明上述所述的方法製備的產品(例如抗體和抗原結合片段)、或本發明上述所述藥物組合物；所述疾病優選腫瘤；所述腫瘤優選結直腸癌。 In some embodiments, the present invention provides a method for preventing and/or treating diseases related to abnormal expression of PD-L1 and/or abnormal T cell function, comprising administering the above-mentioned antibodies or antigens of the present invention to patients in need thereof Binding fragments, isolated nucleic acid molecules as described above in the present invention, expression vectors as described above in the present invention, cells as described above in the present invention, products produced by methods as described above in the present invention (such as antibodies and antigen-binding fragments), Or the above-mentioned pharmaceutical composition of the present invention; the disease is preferably a tumor; the tumor is preferably colorectal cancer.

在一些實施方案中，本發明提供上述所述的抗體或抗原結合片段、本發明上述所述的分離的核酸分子、本發明上述所述的表達載體、本發明上述所述的細胞、本發明上述所述的方法製備的產品(例如抗體和抗原結合片段)、或本發明上述所述藥物組合物在製備預防和/或治療PD-L1表達異常相關的疾病的藥物中的用途，所述疾病優選腫瘤；所述腫瘤優選結直腸癌。 In some embodiments, the present invention provides the above-mentioned antibody or antigen-binding fragment, the above-mentioned isolated nucleic acid molecule of the present invention, the above-mentioned expression vector of the present invention, the above-mentioned cell of the present invention, the above-mentioned The products (such as antibodies and antigen-binding fragments) prepared by the method, or the use of the above-mentioned pharmaceutical composition of the present invention in the preparation of drugs for the prevention and/or treatment of diseases related to abnormal expression of PD-L1, the diseases are preferably A tumor; said tumor is preferably colorectal cancer.

在一些實施方案中，本發明提供一種試劑盒，其包含本發明上述所述的抗體或抗原結合片段、本發明上述所述的分離的核酸分子、本發明上述所述的表達載體、本發明上述所述的細胞、或本發明上述所述的方法製備的產品(例如抗體和抗原結合片段)，以及使用說明。 In some embodiments, the present invention provides a kit comprising the above-mentioned antibody or antigen-binding fragment of the present invention, the above-mentioned isolated nucleic acid molecule of the present invention, the above-mentioned expression vector of the present invention, the above-mentioned The cells, or the products (such as antibodies and antigen-binding fragments) prepared by the above-mentioned methods of the present invention, and instructions for use.

術語和定義： Terms and Definitions:

除非另外說明，本文所用術語具有所屬技術領域普通技術人員通常理解的含義。對於本文中明確定義的術語，則該術語的含義以所述定義為准。 Unless otherwise specified, the terms used herein have the meanings commonly understood by those of ordinary skill in the art. For a term explicitly defined herein, the meaning of that term shall prevail.

如本文所用，術語“抗體”(Ab)是指與目標抗原特異性結合或具有免疫反應性的免疫球蛋白分子，包括抗體的多克隆、單克隆、基因工程化和其他修飾形式(包括但不限於嵌合抗體，人源化抗體，全人源抗體，異源偶聯抗體(例如雙特異性、三特異性和四特異性抗體，雙抗體，三抗體和四抗體)，抗體綴合物)以及抗體的抗原結合片段(包括例如Fab’、F(ab’)2、Fab、Fv、rIgG和scFv片段)。此外，除非另有說明，否則術語“單克隆抗體”(mAb)意指包括能夠特異性結合靶蛋白的完整抗體分子以及不完整的抗體片段(例如Fab和F(ab’)2片段，它們缺少完整抗體的Fc片段(從動物迴圈中更快地清除)，因此缺乏Fc介導的效應功能(effector function)(參見Wahl等人，J.Nucl.Med.24：316,1983；其內容援引加入本文))。 As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with an antigen of interest, including polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies (including but not Limited to chimeric antibodies, humanized antibodies, fully human antibodies, heteroconjugate antibodies (e.g. bispecific, trispecific and tetraspecific antibodies, diabodies, triabodies and tetrabodies), antibody conjugates) As well as antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG and scFv fragments). Furthermore, unless otherwise indicated, the term "monoclonal antibody" (mAb) is intended to include intact antibody molecules capable of specifically binding a target protein as well as incomplete antibody fragments (such as Fab and F(ab')2 fragments, which lack The Fc fragment of an intact antibody (cleared more quickly from the animal cycle) and thus lacks Fc-mediated effector function (see Wahl et al., J.Nucl.Med. 24:316, 1983; the contents of which are cited in Join this article)).

如本文所用，術語“抗原結合片段”是指保留特異性結合靶抗原的能力的一個或更多個抗體片段。抗體的抗原結合功能可以由全長抗體的片段執行。抗體片段可以是Fab、F(ab’)2、scFv、SMIP、雙抗體、三抗體、親和體(affibody)、納米抗體、適體或結構域抗體。涵蓋術語抗體的“抗原結合片段”的結合片段的實例包括但不限於：(i)Fab片段，一種由VL、VH、CL和CH1結構域組成的單價片段；(ii)F(ab)2片段，一種包含由二硫鍵在鉸鏈區連接的兩個Fab片段的雙價片段；(iii)由VH和CH1結構域組成的Fd片段；(iv)由抗體單臂的VL和VH結構域組成的Fv片段；(V)包含VH和VL結構域的dAb；(vi)由VH結構域組成的dAb片段(Ward等人，Nature 341：544-546,1989)；(vii)由VH或VL結構域組成的dAb；(viii)分離的互補決定區(CDR)；以及(ix)兩個或更多個分離的CDR的組合，所述CDR可以任選地由合成接頭連接。此外，雖然Fv片段的兩個結構域VL和VH是通過獨立的基因編碼的，但是這兩個結構域可以使用重組方法通過接頭接合，該接頭能夠使其製成其中VL和VH區配對以形成單價分子的單蛋白質鏈(稱為單鏈Fv(scFv)；參見例如，Bird等人，Science 242：423-426,1988以及Huston等人，Proc.Natl.Acad.Sci.USA 85：5879-5883,1988)。這些抗體片段可以使用本領域技術人員已知的常規技術獲得，並且這些片段被篩選用於與完整抗體相同的方式使用。可以通過重組DNA技術、完整免疫球蛋白的酶促或化學裂解、或在一些實施方式中通過本領域已知的化學肽合成程序來產生抗原結合片段。 As used herein, the term "antigen-binding fragment" refers to one or more antibody fragments that retain the ability to specifically bind a target antigen. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The antibody fragment may be a Fab, F(ab')2, scFv, SMIP, diabody, triabody, affibody, Nanobody, aptamer or domain antibody. Examples of binding fragments encompassing the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragments, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab)2 fragments , a bivalent fragment comprising two Fab fragments connected by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) a set of VL and VH domains consisting of an antibody single arm (V) dAb fragments comprising VH and VL domains; (vi) dAb fragments composed of VH domains (Ward et al., Nature 341:544-546, 1989); (vii) dAb fragments composed of VH or VL (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs, which CDRs may optionally be joined by a synthetic linker. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, these two domains can be joined using recombinant methods through a linker that enables them to be made in which the VL and VH regions pair to form A single protein chain of a monovalent molecule (termed a single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 ,1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as whole antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.

如本文所用，術語“PD-L1”是指程序性死亡配體-1，也稱為CD279(分化簇279)，是一種重要的免疫抑制分子。所述PD-L1優選地是人PD-L1。 As used herein, the term "PD-L1" refers to programmed death ligand-1, also known as CD279 (cluster of differentiation 279), an important immunosuppressive molecule. The PD-L1 is preferably human PD-L1.

如本文所用，術語“抗-程序性死亡配體-1抗體”、“程序性死亡配體-1抗體”、“抗PD-L1抗體”、“PD-L1抗體”、“抗-PD-L1抗體部分”和/或“抗-PD-L1抗體片段”等是指任何包含能夠特異性結合PD-L1的免疫球蛋白分子的至少一部分(例如但不限於重鏈或輕鏈的至少一個互補決定區(CDR)或其配體結合部分、重鏈或輕鏈可變區、重鏈或輕鏈恒定區、框架區或其任何部分)的含蛋白質或肽的分子。PD-L1抗體還包括抗體樣蛋白支架(如第十纖連蛋白III型結構域(10Fn3))，其含有與抗體CDR在結構和溶劑可及性上相似的BC、DE和FG結構環。10Fn3結構域的三級結構類似於IgG重鏈可變區的三級結構，並且通過將10Fn3的BC、DE和FG環的殘基用來自PD-L1單克隆抗體的CDR-H1、 CDR-H2或CDR-H3區的殘基替換，本領域技術人員可以將例如PD-L1單克隆抗體的CDR接枝到纖連蛋白支架上。 As used herein, the terms "anti-programmed death-ligand-1 antibody", "programmed death-ligand-1 antibody", "anti-PD-L1 antibody", "PD-L1 antibody", "anti-PD-L1 Antibody portion" and/or "anti-PD-L1 antibody fragment" etc. refer to any immunoglobulin molecule comprising at least a portion (such as but not limited to at least one complementarity determining protein of a heavy chain or a light chain) capable of specifically binding to PD-L1 region (CDR) or its ligand-binding portion, heavy or light chain variable region, heavy or light chain constant region, framework region or any portion thereof). PD-L1 antibodies also include antibody-like protein scaffolds such as the tenth fibronectin type III domain (10Fn3), which contain BC, DE, and FG structural loops that are similar in structure and solvent accessibility to antibody CDRs. The tertiary structure of the 10Fn3 domain is similar to the tertiary structure of the IgG heavy chain variable region, and by using the residues of the BC, DE and FG loops of 10Fn3 with the CDR-H1, For residue replacement in the CDR-H2 or CDR-H3 region, those skilled in the art can graft, for example, the CDR of the PD-L1 monoclonal antibody onto the fibronectin scaffold.

如本文所用，術語“雙特異性抗體”是指對至少兩種不同的抗原具有單克隆結合特異性的抗體，其通常是人或人源化的抗體。在本發明中，結合特異性之一可以針對PD-L1的抗原表位而被檢測，另一個可以針對PD-L1的另一個抗原表位或任何其他抗原，例如針對細胞表面蛋白、受體、受體亞基、組織特異性抗原、病毒來源蛋白、病毒編碼的包膜蛋白、細菌來源蛋白或細菌表面蛋白等而被檢測。 As used herein, the term "bispecific antibody" refers to an antibody, typically a human or humanized antibody, that has monoclonal binding specificities for at least two different antigens. In the present invention, one of the binding specificities can be detected against an epitope of PD-L1, and the other can be detected against another epitope of PD-L1 or any other antigen, such as for cell surface proteins, receptors, Receptor subunits, tissue-specific antigens, viral-derived proteins, viral-encoded envelope proteins, bacterial-derived proteins, or bacterial surface proteins are detected.

如本文所用，術語“嵌合”抗體是指以下抗體，其具有源自一種來源生物(如大鼠或小鼠)的免疫球蛋白的可變序列以及源自不同生物體(例如人)的免疫球蛋白的恒定區。用於生產嵌合抗體的方法是本領域已知的。參見例如，Morrison,1985,Science 229(4719)：1202-7；Oi等人，1986,Bio Techniques 4：214-221；Gillies等人，1985 J Immunol Methods 125：191-202；以上通過援引加入併入本文。 As used herein, the term "chimeric" antibody refers to an antibody that has variable sequences derived from immunoglobulins of one source organism (such as a rat or mouse) and immunoglobulins derived from a different organism (such as a human). The constant region of a globulin. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, Bio Techniques 4:214-221; Gillies et al., 1985 J Immunol Methods 125:191-202; incorporated by reference above and into this article.

如本文所用，術語“互補決定區”(CDR)指在輕鏈和重鏈可變結構域中均發現的高變區。可變結構域中更高保守性的部分稱為框架區(FR)。如本領域所理解的，表示抗體的高變區的氨基酸位置可以根據上下文和本領域已知的各種定義而變化。可變結構域內的一些位置可以被視為雜合高變位置，因為這些位置可以被認為是在一組標準(如IMGT或KABAT)下的高變區之內，而被認為在不同組的標準(如KABAT或IMGT)下的高變區之外。這些位置中的一個或更多個也可以在延伸的高變區中找到。本發明包括在這些雜合高變的位置中包含修飾的抗體。天然重鏈和輕鏈的可變結構域各自包含主要採用片層構型的四個框架區，其通過三個CDR(CDR1、CDR2和CDR3)連接，這三個CDR形成連接片層結構的環，並且在一些情況下形成片層結構的一部分。每條鏈中的CDR通過FR區按順序FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4緊密保持在一起，並且與來自其他抗體鏈的CDR促成了抗體的抗原結合位點的形成(參見Kabat等人，Sequences of Protein sofImmunological Interest,National Institute of Health,Bethesda,Md.1987；其通過援引加入併入本文)。例如在本文中，CDR1-VH、CDR2-VH和CDR3-VH分別是指重鏈可變區(VH)的第一個CDR、第二個CDR和第三個CDR，這三個CDR構成了重鏈(或其可變區)的CDR組合(VHCDR組合)；CDR1-VL、CDR2-VL和CDR3-VL分別是指輕鏈可變區(VL)的第一個CDR、第二個CDR和第三個CDR，這三個CDR構成了輕鏈(或其可變區)的CDR組合(VLCDR組合)。 As used herein, the term "complementarity determining regions" (CDRs) refers to hypervariable regions found in both light and heavy chain variable domains. The more conserved portions of variable domains are called the framework regions (FR). As understood in the art, the amino acid positions representing the hypervariable regions of an antibody may vary according to the context and various definitions known in the art. Some positions within variable domains can be considered heterozygous hypervariable positions because these positions can be considered to be within hypervariable regions under one set of criteria (such as IMGT or KABAT) but not within a different set. Outside of hypervariable regions under criteria (such as KABAT or IMGT). One or more of these positions may also be found in extended hypervariable regions. The invention includes antibodies comprising modifications in these hybrid hypervariable positions. The variable domains of the native heavy and light chains each comprise four framework regions predominantly in a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3) that form loops connecting the sheets , and in some cases form part of the lamellar structure. The CDRs in each chain are held tightly together by the FR regions in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and are The CDRs of other antibody chains contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Protein of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; incorporated herein by reference). For example, in this paper, CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, the second CDR and the third CDR of the heavy chain variable region (VH), respectively, and these three CDRs constitute the CDR combination (VHCDR combination) of the chain (or its variable region); CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, second CDR and second CDR of the light chain variable region (VL), respectively Three CDRs, which make up the CDR combination (VLCDR combination) of the light chain (or its variable region).

如本文所用，術語“抗體綴合物”是指抗體分子直接或者通過連接接頭與另一個分子化學鍵合而形成的偶聯體/綴合物。例如抗體-藥物綴合物(ADC)，其中藥物分子就是所述的另一個分子。 As used herein, the term "antibody conjugate" refers to a couple/conjugate formed by chemically bonding an antibody molecule to another molecule either directly or through a linker. An example is an antibody-drug conjugate (ADC), wherein the drug molecule is said other molecule.

如本文所用，術語“單克隆抗體”是指來源於單個克隆(包括任何真核、原核、或噬菌體克隆)的抗體，而不限於該抗體的產生方法。 As used herein, the term "monoclonal antibody" refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), without limitation by the method by which the antibody was produced.

如本文所用，術語“VH”是指抗體的免疫球蛋白重鏈(包括Fv、scFv或Fab的重鏈)的可變區。術語“VL”是指免疫球蛋白輕鏈(包括Fv、scFv、dsFv或Fab的輕鏈)的可變區。 As used herein, the term "VH" refers to the variable region of an immunoglobulin heavy chain of an antibody, including that of an Fv, scFv or Fab. The term "VL" refers to the variable region of an immunoglobulin light chain (including the light chain of an Fv, scFv, dsFv or Fab).

如本文所用，術語“百分比(%)序列一致性”是指在為達到最大百分比序列一致性而比對序列和引入空位(如果需要)(例如，為了最佳比對，可以在候選和參比序列中的一個或兩個中引入空位，並且出於比較的目的，可以忽略非同源序列)之後，候選序列的氨基酸(或核苷酸)殘基與參比序列的氨基酸(或核苷酸)殘基相同的百分比。出於確定百分比序列一致性的目的，可以用本領域技術人員熟知的多種方式來實現比對，例如使用公眾可得的電腦軟體，如BLAST、ALIGN或Megalign(DNASTAIi)軟體。本領域技術人員可以確定用於測量比對的適當參數，包括需要在被比較序列的全長範圍實現最大比對的任何演算法。例如，用於與候選序列進行比較而比對的參比序列可以顯示候選序列在候選序列的全長或候選序列的連續氨基酸(或核苷酸)殘基的選定部分上表現出從50%至100%的序列同一性。出於比較目的而比對的候選序列的長度可以是例如參比序列的長度的至少30%(例如30%、40%、50%、60%、70%、80%、90%或100%)。當候選序列中的位置被與在參比序列中的相應位置相同的氨基酸(或核苷酸)殘基佔據時，則這些分子在那個位置是相同的。 As used herein, the term "percent (%) sequence identity" refers to the sequence identity after aligning sequences and introducing gaps (if necessary) for maximum percent sequence identity (e.g., for optimal alignment, the After introducing a gap in one or both of the sequences, and for comparison purposes, non-homologous sequences can be ignored), the amino acid (or nucleotide) residues of the candidate sequence are different from the amino acid (or nucleotide) residues of the reference sequence ) residues are the same percentage. For purposes of determining percent sequence identity, alignment can be achieved in a variety of ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAIi) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, a reference sequence aligned for comparison with a candidate sequence can show that the candidate sequence The list exhibits from 50% to 100% sequence identity over the full length of the candidate sequence or a selected portion of contiguous amino acid (or nucleotide) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of the reference sequence . When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

如本文所用，術語“特異性結合”是指一種結合反應，其決定抗原在蛋白質和其他生物分子的一個異質性群體中的存在狀況，所述蛋白質和其他生物分子例如被抗體或其抗原結合片段特異性識別。與抗原特異性結合的抗體或其抗原結合片段將以小於100nM的KD與抗原結合。例如，與抗原特異性結合的抗體或其抗原結合片段將以高達100nM((例如，1pM至100nM之間)的KD與抗原結合。不顯示與特定抗原或其表位特異性結合的抗體或其抗原結合片段將顯示對該特定抗原或其表位的大於100nM(例如，大於500nM、1μM、100μM、500μM或1mM)的KD。可以使用多種免疫測定方式來選擇與特定蛋白或碳水化合物進行特異性免疫反應的抗體。例如，常規地使用固相ELISA免疫測定法來選擇與蛋白質或碳水化合物進行特異性免疫反應的抗體。參見，Harlow & Lane，Antibodies,ALaboratory Manual,Cold Spring Harbor Press,NewYork(1988)以及Harlow & Lane,Using Antibodies，A Laboratory Manual,Cold Spring Harbor Press,NewYork(1999)，其描述了可以用於確定特異免疫反應性的免疫測定方式和條件。 As used herein, the term "specific binding" refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules, such as those bound by antibodies or antigen-binding fragments thereof. specific recognition. An antibody or antigen-binding fragment thereof that specifically binds an antigen will bind the antigen with a KD of less than 100 nM. For example, an antibody or antigen-binding fragment thereof that specifically binds an antigen will bind the antigen with a KD as high as 100 nM (eg, between 1 pM and 100 nM). Antibodies or antigen-binding fragments thereof that do not exhibit specific binding to a particular antigen or epitope thereof Antigen-binding fragments will exhibit a KD of greater than 100 nM (e.g., greater than 500 nM, 1 μM, 100 μM, 500 μM, or 1 mM) for that particular antigen or epitope thereof. Various immunoassay formats can be used to select for specificity to a particular protein or carbohydrate Immunoreactive antibodies. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that are specifically immunoreactive with proteins or carbohydrates. See, Harlow & Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor Press, New York (1988 ) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), which describe immunoassay formats and conditions that can be used to determine specific immunoreactivity.

如本文所用，術語“載體”包括核酸載體，例如DNA載體(如質粒)，RNA載體，病毒或其他適合的複製子(例如病毒載體)。已經開發了多種載體用於將編碼外源蛋白質的多核苷酸遞送到原核或真核細胞中。本發明的表達載體含有多核苷酸序列以及例如用於表達蛋白質和/或將這些多核苷酸序列整合到哺乳動物細胞基因組中的附加序列元件。可以用於表達本發明的抗體和抗體片段的某些載體包括含有指導基因轉錄的調控序列(如啟動子和增強子區域)的質粒。用於表達抗體和抗體片段的其他有用的載體含有多核苷酸序列，其增強這些基因的翻譯速率或改善由基因轉錄產生的mRNA的穩定性或核輸出。這些序列元件包括例如5’和3’非翻譯區、內部核糖體進入位點(IRES)和聚腺苷酸化信號位元點，以便指導表達載體上攜帶的基因的有效轉錄。本發明的表達載體還可以含有以下多核苷酸，該多核苷酸編碼用於選擇含有這種載體的細胞的標記。適合的標記的實例包括編碼抗生素(如氨苄青黴素、氯黴素、卡那黴素或諾爾絲菌素)抗性的基因。 As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (eg, plasmids), RNA vectors, viruses or other suitable replicons (eg, viral vectors). A variety of vectors have been developed for the delivery of polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells. Expression vectors of the invention contain polynucleotide sequences together with additional sequence elements, eg, for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the antibodies and antibody fragments of the invention include vectors containing vectors directing gene transcription Plasmids for regulatory sequences such as promoter and enhancer regions. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of mRNA resulting from transcription of the genes. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosomal entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of genes carried on the expression vector. The expression vector of the present invention may also contain a polynucleotide encoding a marker for selection of cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourthricin.

如本文所用，術語“受試者”、“對象”和“患者”是指接受對如本文所述的特定疾病或病症(如癌症或傳染性疾病)的治療的生物體。物件和患者的實例包括接受疾病或病症(例如細胞增殖性病症，如癌症或傳染性疾病)的治療的哺乳動物，如人、靈長類動物、豬、山羊、兔、倉鼠、貓、狗、豚鼠、牛科家族成員(如家牛、野牛、水牛、麋鹿和犛牛等)、牛、綿羊、馬和野牛等。 As used herein, the terms "subject", "subject" and "patient" refer to an organism receiving treatment for a particular disease or condition, such as cancer or an infectious disease, as described herein. Examples of articles and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, Guinea pigs, members of the bovid family (such as domestic cattle, bison, buffalo, elk, and yaks), cattle, sheep, horses, and bison.

如本文所用，術語“治療”是指外科手術或藥物處理(surgical or therapeutic treatment)，其目的是預防、減緩(減少)治療物件中不希望的生理變化或病變，如細胞增殖性病症(如癌症或傳染性疾病)的進展。有益的或所希望的臨床結果包括但不限於症狀的減輕、疾病程度減弱、疾病狀態穩定(即，未惡化)、疾病進展的延遲或減慢、疾病狀態的改善或緩和、以及緩解(無論是部分緩解或完全緩解)，無論是可檢測的或不可檢測的。需要治療的物件包括已患有病症或疾病的物件以及易於患上病症或疾病的物件或打算預防病症或疾病的對象。當提到減緩、減輕、減弱、緩和、緩解等術語時，其含義也包括消除、消失、不發生等情況。 As used herein, the term "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) an undesired physiological change or pathology in the subject of treatment, such as a cell proliferative disorder (such as cancer or infectious disease). Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminished extent of disease, stable disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of disease state, and remission (whether partial response or complete response), whether detectable or undetectable. Those in need of treatment include those already with the condition or disease as well as those prone to have the condition or disease or those in which the condition or disease is to be prevented. When referring to the terms slow down, lessen, weaken, moderate, alleviate, etc., the meaning of eliminate, disappear, not occur, etc. is also included.

下面通過對本發明的詳細描述以及附圖來清楚地說明本發明前面敘述的方面以及其他方面。本文中附圖是為了舉例說明本發明的一些優選的實施方案，然而，可以理解，本發明並不限於所公開的特定實施方案。 Below by the detailed description of the present invention and accompanying drawing clearly illustrate the front of the present invention Narrative and other aspects. The drawings herein are for the purpose of illustrating some preferred embodiments of the invention, however, it is to be understood that the invention is not limited to the particular embodiments disclosed.

圖1、末次免疫後測定的小鼠血清結合人PD-L1-mFc(A)和PD-L1-His(B)重組蛋白的滴度。 Figure 1. The titers of mouse serum binding to human PD-L1-mFc (A) and PD-L1-His (B) recombinant proteins determined after the last immunization.

圖2、PD-L1特異性B細胞的流式細胞染色分選(FACS)和圈門策略圖。 Figure 2. Diagram of flow cytometry staining and sorting (FACS) and gating strategy of PD-L1-specific B cells.

圖3、競爭性ELISA方法測定抗PD-L1抗體阻斷PD-L1蛋白與PD-1蛋白結合。 Figure 3. The competitive ELISA method was used to determine the blocking of the binding of PD-L1 protein to PD-1 protein by anti-PD-L1 antibody.

圖4、FACS測定抗PD-L1抗體結合細胞表面PD-L1蛋白的EC50。 Figure 4. FACS determination of the EC50 of anti-PD-L1 antibody binding to cell surface PD-L1 protein.

圖5、抗PD-L1抗體增加Jurkat-PD-1-CHO-PD-L1-NFAT體系中報告基因的表達和活性。 Figure 5. Anti-PD-L1 antibody increases the expression and activity of reporter gene in Jurkat-PD-1-CHO-PD-L1-NFAT system.

圖6、抗PD-L1抗體促進混合淋巴細胞反應中IFN-γ的分泌。 Figure 6. Anti-PD-L1 antibody promotes IFN-γ secretion in mixed lymphocyte reaction.

圖7、抗PD-L1抗體對A431細胞的抗體依賴的細胞殺傷(ADCC)活性。 Figure 7. Antibody-dependent cell killing (ADCC) activity of anti-PD-L1 antibody on A431 cells.

圖8、鼠源抗PD-L1抗體抑制人PD-1/PD-L1轉基因小鼠體內MC38-hPD-L1結腸癌腫瘤生長。 Figure 8. Murine anti-PD-L1 antibody inhibits the growth of MC38-hPD-L1 colon cancer tumors in human PD-1/PD-L1 transgenic mice.

圖9、人源化抗PD-L1抗體抑制人PD-L1轉基因小鼠體內MC38-hPD-L1結腸癌腫瘤生長。 Figure 9. Humanized anti-PD-L1 antibody inhibits the growth of MC38-hPD-L1 colon cancer tumors in human PD-L1 transgenic mice.

下面結合實施例和附圖對本發明進行詳細描述，本文中附圖是為了舉例說明本發明的一些優選的實施方案，然而，可以理解，本發明並不限於所公開的特定實施方案或看作對本發明範圍的限制。實施例中未注明具體條件者，按照常規條件或製造商建議的條件進行。所用試劑或儀器未注明生產廠商者，均為可以通過市售購買獲得的常規產品。 The present invention will be described in detail below in conjunction with embodiment and accompanying drawing, and accompanying drawing herein is in order to illustrate some preferred embodiments of the present invention, but, it can be understood that the present invention is not limited to disclosed specific embodiment or is regarded as the embodiment of the present invention. Limitation on Scope of Invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

實施例1 小鼠免疫產生PD-L1抗體Example 1 Mice were immunized to produce PD-L1 antibody

對6-8周齡的雌性SJL小鼠(購自北京維通利華實驗動物技術有限公司)或者Balb/c小鼠(購自上海斯萊克實驗動物有限公司)，使用融合了小鼠Fc的人PD-L1蛋白(PD-L1-mFc,Novoprotein,Cat.CM06，或Sino Biological,Cat.10084-H05H)或人PD-L1-His(Novoprotein,Cat.C315或Sino Biological，Cat：.10084-H08H)與弗氏完全佐劑(complete freund's adjuvant,CFA,Sigma，Cat.F5881)進行第一次免疫；使用上述PD-L1-mFc或人PD-L1-His與弗氏不完全佐劑(incomplete freund's adjuvant，IFA,Sigma，Cat.F5506)和加未甲基化的胞嘧啶鳥嘌呤二核苷酸(CpGODN1826，合成自上海生工生物)進行後三次免疫，免疫時注射50μg/只/次通過乳化操作形成的均一穩定的乳劑。特別地，第一次和第二次免疫注射後足墊和背部，第三次和第四次免疫注射尾部皮下及背部，以獲得高滴度高親和力高特異性的抗血清及特異性的免疫細胞。在末次免疫(第四次免疫)後的第5-7天，安樂死小鼠並無菌取出脾臟，無菌分離提取小鼠脾臟淋巴細胞，分裝至凍存管中，凍存於液氮中。分別在二次免疫，三次免疫後10天及安樂死小鼠當天進行小鼠的采血操作，分離血清，使用酶聯免疫吸附(ELISA)方法測定血清中抗PD-L1特異性抗體的滴度。 For 6-8 week-old female SJL mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) or Balb/c mice (purchased from Shanghai Slack Experimental Animal Co., Ltd.), human mice fused with mouse Fc were used. PD-L1 protein (PD-L1-mFc, Novoprotein, Cat.CM06, or Sino Biological, Cat.10084-H05H) or human PD-L1-His (Novoprotein, Cat.C315 or Sino Biological, Cat: .10084-H08H ) with Freund's complete adjuvant (complete freund's adjuvant, CFA, Sigma, Cat.F5881) for the first immunization; use the above PD-L1-mFc or human PD-L1-His with Freund's incomplete adjuvant (incomplete freund's adjuvant, IFA, Sigma, Cat.F5506) and unmethylated cytosine guanine dinucleotide (CpGODN1826, synthesized from Shanghai Sangon Biology) for the last three immunizations, injecting 50 μg/piece/time through emulsification during immunization Uniform and stable emulsion formed by operation. In particular, the first and second immunizations were injected into the foot pads and back, and the third and fourth immunizations were injected under the skin of the tail and back to obtain high-titer, high-affinity, high-specificity antisera and specific immunity. cell. On the 5th to 7th day after the last immunization (the fourth immunization), the mice were euthanized and the spleen was aseptically removed, and the spleen lymphocytes of the mice were aseptically separated and extracted, aliquoted into cryopreservation tubes, and frozen in liquid nitrogen. After the second immunization, 10 days after the third immunization, and the day of euthanasia, the mice were blood-collected, the serum was separated, and the titer of the anti-PD-L1 specific antibody in the serum was determined by enzyme-linked immunosorbent assay (ELISA).

實驗結果如圖1，顯示經過四次免疫後，所免疫小鼠的血清與人PD-L1-mFc和PD-L1-His結合的滴度都很高。說明瞭使用此方法進行小鼠的免疫，可以使小鼠產生高滴度的抗PD-L1抗體。 The experimental results are shown in Figure 1, which shows that after four immunizations, the serum of the immunized mice has a high binding titer to human PD-L1-mFc and PD-L1-His. It shows that using this method to immunize mice can make mice produce high titer anti-PD-L1 antibody.

實施例2 PD-L1特異性單個B細胞的流式細胞螢光分選(Fluorescence-activated cell sorting,FACS)Example 2 Fluorescence-activated cell sorting (FACS) of PD-L1-specific single B cells

PD-L1蛋白免疫的小鼠脾臟細胞，經抗原PD-L1-His蛋白(Novoprotein,Cat.C315或Sino Biological，Cat.10084-H08H)及間接標記抗體anti-His-APC(R&D Systems,Cat.IC050A)和針對小鼠B細胞表面特有標誌的抗體(anti-mouse B220-Pacfic Blue，R&D Systems, Cat.553089；anti-mouse IgD-PE，R&D Systems,Cat.558597；anti-mouse IgM-PE Cy7,R&D Systems,Cat.552867)染色，並在分選前加入區分死細胞和活細胞的染料7-AAD(R&D Systems,Cat.51-68981E)，用AriaIII(BD公司)流式細胞分選儀分選PD-L1特異性的單個B細胞(7AAD^-B220⁺IgD^-IgM^-PDL1-His⁺)至含有細胞裂解液，RNA酶抑制劑的PCR孔中，每個孔收集一個細胞。結果顯示(圖2)空白對照未免疫的小鼠脾臟(A)，或使用帶His的不相關蛋白CREG-His染色的PD-L1免疫的小鼠脾臟(B)均未檢測到明顯的PD-L1⁺抗原特異性B細胞亞群，而使用PD-L1-His染色的PD-L1免疫的小鼠脾臟(C)則檢測到一群PD-L1⁺B細胞，每10⁶個脾臟細胞中約有114個PD-L1⁺B細胞。 Mouse spleen cells immunized with PD-L1 protein, antigen PD-L1-His protein (Novoprotein, Cat.C315 or Sino Biological, Cat.10084-H08H) and indirectly labeled antibody anti-His-APC (R&D Systems, Cat. IC050A) and antibodies against specific markers on the surface of mouse B cells (anti-mouse B220-Pacfic Blue, R&D Systems, Cat.553089; anti-mouse IgD-PE, R&D Systems, Cat.558597; anti-mouse IgM-PE Cy7 , R&D Systems, Cat.552867) staining, and before sorting, add the dye 7-AAD (R&D Systems, Cat.51-68981E) to distinguish between dead cells and living cells, and use AriaIII (BD company) flow cytometer Single PD-L1-specific B cells (7AAD ^- B220 ⁺ IgD ^- IgM ^- PDL1-His ⁺ ) were sorted into PCR wells containing cell lysate and RNase inhibitor, and one cell was collected from each well. The results showed (Figure 2) that no obvious PD- L1 ⁺ antigen-specific B cell subsets, while the spleen of PD-L1-immunized mice stained with PD-L1-His (C) detected a population of PD-L1 ⁺ B cells, about ¹⁰⁶ spleen cells 114 PD-L1 ⁺ B cells.

實施例3 單克隆抗體的擴增和高通量表達Example 3 Amplification and high-throughput expression of monoclonal antibodies

採用專利“一種用於巢式擴增的組合引物及其應用”專利申請號：201811618134.4中實施例1的方法，將單細胞的mRNA反轉錄成cDNA。然後以cDNA為範本進行巢式PCR，分別進行抗體重鏈和輕鏈擴增。擴增得到抗體重鏈可變區和輕鏈可變區，分別通過同源重組方法克隆到重鏈表達載體和輕鏈表達載體。重鏈表達載體和輕鏈表達載體的恒定區都來自於人IgG1。完整重鏈表達序列是信號肽-VH-CH1-鉸鏈區-CH2-CH3，完整輕鏈表達序列是信號肽-Vκ-Cκ。以上所述單B細胞抗體克隆和表達皆在96孔板內以高通量方式達到抗體的快速鑒定和發現。經過一系列理化和功能篩選324對克隆表達的抗體重鏈和輕鏈後，共獲得9個與已上市的PD-L1抗體Avelumab或Atezolizumab理化和功能活性相當或更好的候選鼠源抗體分子，其序列的CDRs分別用IMGT和KABAT軟體分析，對應的序列資訊如下表1至2所示，其中表1示出鼠源抗體分子的VH和VL序列，表2示出鼠源抗體分子的IMGT和KABAT分析結果。 Single-cell mRNA was reverse-transcribed into cDNA using the method described in Example 1 of the patent "A Combination Primer for Nested Amplification and Its Application" patent application number: 201811618134.4. Then nested PCR was performed using cDNA as a template to amplify the antibody heavy chain and light chain respectively. The heavy chain variable region and the light chain variable region of the antibody are amplified, and cloned into the heavy chain expression vector and the light chain expression vector by homologous recombination method respectively. The constant regions of both the heavy chain expression vector and the light chain expression vector were derived from human IgG1. The complete heavy chain expression sequence is signal peptide-VH-CH1-hinge region-CH2-CH3, and the complete light chain expression sequence is signal peptide-Vκ-CK. The cloning and expression of the single B cell antibody described above are all performed in a 96-well plate in a high-throughput manner to achieve rapid identification and discovery of antibodies. After a series of physical, chemical and functional screening of 324 pairs of antibody heavy chains and light chains expressed by clones, a total of 9 candidate mouse antibody molecules with physical, chemical and functional activities comparable to or better than those of the marketed PD-L1 antibodies Avelumab or Atezolizumab were obtained. The CDRs of its sequence were analyzed by IMGT and KABAT software, and the corresponding sequence information is shown in Tables 1 to 2 below, where Table 1 shows the VH and VL sequences of the mouse antibody molecule, and Table 2 shows the IMGT and VL sequences of the mouse antibody molecule. KABAT analysis results.

表1.鼠源抗PD-L1抗體重鏈可變區和輕鏈可變區的具體序列資訊Table 1. Specific sequence information of the heavy chain variable region and light chain variable region of the murine anti-PD-L1 antibody

分別使用KABAT和IMGT軟體分析各抗體的CDRs，具體的序列資訊如下表2： The CDRs of each antibody were analyzed using KABAT and IMGT software respectively. The specific sequence information is shown in Table 2:

實施例4 抗體人源化Example 4 Antibody humanization

首先採用經典“CDRs移植”方法進行抗體人源化，即通過序列挑選同源性最高的人源性抗體提供抗體骨架區(FRs)，把目標抗體的基於Kabat命名方法的抗原結合片段互補決定區(CDRs)，移植到前者形成人源化抗體。其次，為更好保持抗體活性和親和力，基於抗體結構建模分析(MOE軟體)：1).選擇抗體骨架區位於VH-VL介面、靠近或與CDRs有直接相互作用等氨基酸殘基進行回復突變，這類氨基酸殘基對保持CDRs區構象多較重要；2).考慮到免疫原性，儘量選擇包埋在蛋白內部的氨基酸進行回復突變；3).考慮到抗體穩定性和表達水準，優先考慮分子能量降低突變；4).在人源化過程中通過CDRs區的氨基酸定點突變嘗試進一步提升人源化抗體親和力。通過測試含有不同突變的人源化抗體與人PD-L1的親和力以及和表面表達PD-L1的細胞的結合，篩選與鼠源PD-L1抗體親和力、抗體表徵和活性功能相當或更好的人源化抗體。 First, the classic "CDRs transplantation" method is used for antibody humanization, that is, the human antibody with the highest homology is selected through sequence to provide the antibody framework region (FRs), and the antigen-binding fragment complementarity-determining region of the target antibody based on the Kabat naming method (CDRs), transplanted to the former to form humanized antibodies. Secondly, in order to better maintain antibody activity and affinity, based on antibody structure modeling analysis (MOE software): 1). Select amino acid residues in the antibody framework region located at the VH-VL interface, close to or directly interacting with CDRs for back mutation , such amino acid residues are more important to maintain the conformation of the CDRs region; 2). Considering immunogenicity, try to select the amino acids embedded in the protein for back mutation; 3). Considering the stability and expression level of the antibody, it is preferred Consider molecular energy reduction mutations; 4). During the humanization process, try to further improve the affinity of the humanized antibody by site-directed mutation of amino acids in the CDRs region. by testing different mutations The affinity of humanized antibodies to human PD-L1 and the binding to cells expressing PD-L1 on the surface are screened for humanized antibodies with comparable or better affinity, antibody characterization and activity functions to murine PD-L1 antibodies.

其中，PDL1-156抗體經過人源化後的優選候選抗體分子的序列的CDRs如下，分別用IMGT和KABAT軟體分析，對應的序列資訊如下表3和表4所示，其中表3示出人源化抗體分子的VH和VL序列，表4示出人源化抗體分子的IMGT和KABAT分析結果)。 Among them, the CDRs of the sequence of the preferred candidate antibody molecule after humanization of the PDL1-156 antibody are as follows, which are analyzed by IMGT and KABAT software respectively. The corresponding sequence information is shown in Table 3 and Table 4 below, and Table 3 shows the human source VH and VL sequences of humanized antibody molecules, Table 4 shows the IMGT and KABAT analysis results of humanized antibody molecules).

分別使用KABAT和IMGT軟體分析各人源化抗體的CDRs，具體的序列資訊如下： The CDRs of each humanized antibody were analyzed using KABAT and IMGT software respectively. The specific sequence information is as follows:

實施例5 分子排阻色譜法測定抗體純度Example 5 Size Exclusion Chromatography Determination of Antibody Purity

採用TSKgel G3000SWXL色譜柱(TOSOH，0008541)，預柱Tskgel guard column SWXL(TOSOH，Cat.0008543)進行分子排阻色譜法測定抗體純度。流動相為磷酸鹽緩衝液(NaH₂PO₄-Na₂HPO₄)，配製：稱取8.88g的NaH₂PO₄‧2H₂O，33.33g的Na₂HPO₄‧12H₂O。流動相平衡色譜柱，流速為1mL/min。待基線走平後進樣，其中進樣體積10μL，紫外檢測波長280nm，頻寬16nm，參比波長關閉。測定結果如表5所示。 TSKgel G3000SWXL chromatographic column (TOSOH, 0008541) and pre-column Tskgel guard column SWXL (TOSOH, Cat. 0008543) were used to determine the purity of the antibody by size exclusion chromatography. The mobile phase is phosphate buffer (NaH ₂ PO ₄ -Na ₂ HPO ₄ ). Preparation: Weigh 8.88g of NaH ₂ PO ₄ ‧2H ₂ O and 33.33g of Na ₂ HPO ₄ ‧12H ₂ O. The mobile phase equilibrates the chromatographic column with a flow rate of 1 mL/min. After the baseline leveled off, the sample was injected, the sample volume was 10 μL, the ultraviolet detection wavelength was 280nm, the bandwidth was 16nm, and the reference wavelength was turned off. The measurement results are shown in Table 5.

實施例6 抗體與人以及食蟹猴PD-L1重組蛋白結合的KD測定Example 6 KD Determination of Antibody Binding to Human and Cynomolgus Monkey PD-L1 Recombinant Proteins

使用Biacore T200(GE Healthcare)測定PD-L1抗體對於人和食蟹猴PD-L1-His蛋白的結合親和力。25℃下在CM5晶片(GE Healthcare，Cat.BR-1005-30)上固定anti-human IgG Fc(Genway，Cat.GWB-20A705)。將anti-human Fc(Genway,Cat.GWB-20A705)用Acetate pH5.0(GE Healthcare，BR-1003-51)稀釋至20μg/mL。使用Immobilization method中Amine方法進行固定。或者使用商品化Protein A(GE Healthcare，Cat.29127556)晶片進行檢測。25℃下採用多迴圈動力學法測定抗體與抗原的親和力，在每一個迴圈中，首先將待測抗體捕獲到固定好的CM5晶片，然後注入重組人PD-L1-His(Novoprotein,Cat.315)和食蟹猴PD-L1-His蛋白(Sino Biological，Cat.90251-C08H)，最後用Glycine pH1.5再生。流動相為HBS-EP+Buffer(GE Healthcare， Cat.BR-1006-69)，流速30μL/min，結合時間為300秒。再生流速30μL/min，時間為30秒。應用Biacore T200 Evaluation Software(version 3.0)，以1：1結合模型，分析試驗資料，擬合抗體抗原的平衡解離常數KD，確定結合速率常數ka和解離速率常數kd。 The binding affinities of PD-L1 antibodies to human and cynomolgus monkey PD-L1-His proteins were determined using Biacore T200 (GE Healthcare). Anti-human IgG Fc (Genway, Cat. GWB-20A705) was immobilized on CM5 wafers (GE Healthcare, Cat. BR-1005-30) at 25°C. Anti-human Fc (Genway, Cat. GWB-20A705) was diluted to 20 μg/mL with Acetate pH5.0 (GE Healthcare, BR-1003-51). Immobilization was performed using the Amine method in the Immobilization method. Alternatively, a commercial Protein A (GE Healthcare, Cat. 29127556) wafer is used for detection. At 25°C, the affinity of the antibody to the antigen was determined by a multi-cycle kinetic method. In each cycle, the antibody to be tested was first captured onto the immobilized CM5 chip, and then injected into recombinant human PD-L1-His (Novoprotein, Cat .315) and cynomolgus monkey PD-L1-His protein (Sino Biological, Cat.90251-C08H), and finally regenerated with Glycine pH1.5. Mobile phase is HBS-EP+Buffer (GE Healthcare, Cat.BR-1006-69), the flow rate is 30 μL/min, and the binding time is 300 seconds. The regeneration flow rate is 30 μL/min, and the time is 30 seconds. Biacore T200 Evaluation Software (version 3.0) was used to analyze the experimental data with a 1:1 binding model, and the equilibrium dissociation constant KD of the antibody antigen was fitted to determine the association rate constant ka and dissociation rate constant kd.

從結果可知所測試的PD-L1抗體對人PD-L1重組蛋白的結合，都表現出nM或更高的親和力，且都與食蟹猴的PD-L1重組蛋白的親和力在62.5nM到0.375nM之間，詳見下表6。 From the results, it can be seen that the tested PD-L1 antibodies have an affinity of nM or higher for the binding of the human PD-L1 recombinant protein, and all of them have an affinity of 62.5nM to 0.375nM for the PD-L1 recombinant protein of cynomolgus monkeys. For details, see Table 6 below.

表7則顯示鼠源抗體PDL1-156來源的人源化抗體156-1H、156-7H、156-10H和156-11H對人PD-L1蛋白的結合，表現出與PDL1-156相當的親和力。 Table 7 shows that the humanized antibodies 156-1H, 156-7H, 156-10H and 156-11H derived from the murine antibody PDL1-156 bind to human PD-L1 protein, showing an affinity comparable to that of PDL1-156.

實施例7 抗體阻斷PD-L1和PD-1相互作用的IC50測定Example 7 IC50 Determination of Antibody Blocking Interaction Between PD-L1 and PD-1

通過競爭性ELISA方法確定抗PD-L1抗體阻斷PD-L1蛋白與PD-1蛋白結合的IC50。 The IC50 of blocking the binding of PD-L1 protein to PD-1 protein by anti-PD-L1 antibody was determined by competitive ELISA method.

使用碳酸鹽緩衝液稀釋人PD-L1重組蛋白(Sino Biological,Cat.10084-H05H)，加入96孔酶標板，終濃度為1μg/ml。用含3% BSA的PBS溶液封閉，加入梯度稀釋的抗PD-L1抗體(6000ng/ml~2ng/ml)以及人PD-1-His重組蛋白(Sino Biological,Cat.10377-H08H)進行共孵育後，加入HRP標記的抗His標籤抗體(MBL,Cat.D291-7)，TMB顯色，1M硫酸終止後讀取OD值(雙波長450nm-630nm)。將抗體濃度與OD值對應即可繪製出測試抗體的競爭結合曲線，計算出IC50值。圖3顯示了抗PD-L1抗體與人PD-L1重組蛋白的競爭結合曲線。結果表明，圖中所示與沒有任何阻斷作用的抗體同型陰性對照anti-Hel(百英生物製備)相比，被測試的9個鼠源抗PD-L1抗體(A)和4個人源化抗體(B)均可以有效的阻斷人PD-L1蛋白與人PD-1蛋白的相互作用，且人源化抗體與人源化之前的鼠源PDL1-156的抑制活性相當(B)，IC50分別為197.0ng/mL(156-1H)、230.5ng/mL(156-7H)、250.1ng/mL(156-10H)、207.2ng/mL(156-11H)。鼠源PD-L1-156的IC50則為221.3ng/ml，陽性對照Atezolizumab(百英生物製備)為446.4ng/ml、Avelumab(Pfizer,lot AU020322)為190.3ng/ml。 Human PD-L1 recombinant protein (Sino Biological, Cat.10084-H05H) was diluted with carbonate buffer and added to a 96-well microtiter plate with a final concentration of 1 μg/ml. Block with PBS solution containing 3% BSA, add serially diluted anti-PD-L1 antibody (6000ng/ml~2ng/ml) and human PD-1-His recombinant protein (Sino Biological, Cat.10377-H08H) for co-incubation Afterwards, add HRP-labeled anti-His tag antibody (MBL, Cat.D291-7), TMB color development, and read OD value (dual wavelength 450nm-630nm) after termination of 1M sulfuric acid. Comparing the antibody concentration with the OD value, the competition binding curve of the test antibody can be drawn, and the IC50 value can be calculated. Figure 3 shows the competition binding curve of anti-PD-L1 antibody to human PD-L1 recombinant protein. The results showed that the tested 9 murine anti-PD-L1 antibodies (A) and 4 humanized Antibodies (B) can effectively block the interaction between human PD-L1 protein and human PD-1 protein, and the inhibitory activity of humanized antibody is comparable to that of mouse PDL1-156 before humanization (B), IC50 They are 197.0ng/mL (156-1H), 230.5ng/mL (156-7H), 250.1ng/mL (156-10H), and 207.2ng/mL (156-11H), respectively. The IC50 of murine PD-L1-156 was 221.3ng/ml, the positive control Atezolizumab (Baiying Bio-manufacturing) was 446.4ng/ml, and Avelumab (Pfizer, lot AU020322) was 190.3ng/ml.

實施例8 FACS測定PD-L1抗體對細胞表面PD-L1結合的ECExample 8 FACS determination of the EC of PD-L1 antibody binding to cell surface PD-L1 ₅₀₅₀

將梯度濃度的待檢測抗體(抗體終濃度：10000ng/ml-0.1ng/ml，10倍系列稀釋)與細胞表面高表達PD-L1的CHO-PD-L1細胞(南京勇山生物科技有限公司，10⁵個/孔)，4℃共同孵育30min。孵育結束後，加入1：250稀釋的anti-human IgG PE螢光抗體(eBioscience，Cat.12-4998-8)，4℃下共同孵育30min，螢光抗體與待檢測抗體的Fc段產生特異性結合，通過FACS檢測PE螢光強度的高低而對待檢測抗體的結合細胞表面高表達的PD-L1蛋白的能力進行分析。圖4結果顯示，待測的鼠源PD-L1抗體EC50均與本次實驗的陽性對照Avelumab(EC50為58.2ng/ml)和Atezolizumab(~99.73ng/ml)相近，其中PDL1-156和PDL1-370的EC50最低，被檢測的人源化抗體156-1H，156-7H，156-10H和156-11H的EC50分別為49.42ng/ml，78.37ng/ml，63.5ng/ml和49.97ng/ml，與本次實驗的陽性對照Avelumab(EC50為56.88ng/ml)相近。該檢測定量地證實了待測各PD-L1抗體對細胞表面上PD-L1靶點劑量依賴性結合的能力。MFI fold=實驗組MFI值/未加藥物的對照組MFI值。 The antibody to be detected at gradient concentrations (antibody final concentration: 10000ng/ml-0.1ng/ml, 10-fold serial dilution) was mixed with CHO-PD-L1 cells with high expression of PD-L1 on the cell surface (Nanjing Yongshan Biotechnology Co., Ltd., 10 ⁵ cells/well), and incubated together at 4°C for 30 min. After the incubation, add anti-human IgG PE fluorescent antibody (eBioscience, Cat.12-4998-8) diluted 1:250, and incubate together at 4°C for 30 minutes, and the fluorescent antibody will be specific to the Fc segment of the antibody to be detected For binding, the ability of the antibody to be detected to bind to the highly expressed PD-L1 protein on the cell surface was analyzed by detecting the fluorescence intensity of PE through FACS. The results in Figure 4 show that the EC50 of the murine PD-L1 antibody to be tested is similar to the positive controls Avelumab (EC50 of 58.2 ng/ml) and Atezolizumab (~99.73 ng/ml) in this experiment, among which PDL1-156 and PDL1- 370 has the lowest EC50, and the EC50 of tested humanized antibodies 156-1H, 156-7H, 156-10H and 156-11H are 49.42ng/ml, 78.37ng/ml, 63.5ng/ml and 49.97ng/ml respectively , which is similar to the positive control Avelumab (EC50 of 56.88ng/ml) in this experiment. This assay quantitatively confirms the ability of each PD-L1 antibody to be tested to bind to the PD-L1 target on the cell surface in a dose-dependent manner. MFI fold=MFI value of the experimental group/MFI value of the control group without drugs.

實施例9 PD-1/PD-L1-NFAT報告基因測試抗PD-L1抗體阻抑PD-1：PD-L1結合和信號傳導Example 9 PD-1/PD-L1-NFAT reporter gene test anti-PD-L1 antibody inhibits PD-1: PD-L1 binding and signal transduction

利用穩定轉染PD-1的Jurkat細胞株(GenScript，Cat.00612)和穩定轉染PD-L1的CHO細胞株(GenScript，Cat.M00613)比較PD-L1抗體對PD-1/PD-L1蛋白相互作用及其信號通路的拮抗作用。當抑制信號通路阻抑，NFAT控制的發光報告基因表達增強，發光信號值增加。通過發光讀值的強弱(relative light units,RLU)反應抗體對PD-L1的阻斷作用強弱。 Using the Jurkat cell line stably transfected with PD-1 (GenScript, Cat.00612) and the CHO cell line stably transfected with PD-L1 (GenScript, Cat.M00613) to compare the effect of PD-L1 antibody on PD-1/PD-L1 protein Antagonism of interactions and their signaling pathways. When the inhibitory signaling pathway is inhibited, the expression of the luminescent reporter gene controlled by NFAT is enhanced, and the value of the luminescent signal increases. The strength of the antibody's blocking effect on PD-L1 is reflected by the intensity of the luminescence reading (relative light units, RLU).

將穩轉PD-L1的CHO細胞株種在96孔白底板上，每孔40000個細胞，100μl/孔，放回培養箱過夜；第二天，取出孔板，吸去培養基，加入穩轉PD-1的細胞株及待測的PD-L1抗體共孵育，PD-1細胞每孔加樣量為16000個/孔，抗體則做梯度稀釋，每個劑量3複孔，孵育體積為100μl/孔，孵育時長為6小時，待孵育完成時，取出孔板，等體積(100μl)加入發光檢測試劑，讀值。根據檢測值用Graphpad進行4參數分析做回歸曲線，得到各抗體的EC50值，結果詳見圖5A，被測的鼠源PD-L1抗體的EC50值均與陽性對照抗體Avelumab和Atezolizumab的EC50值(222.9ng/ml，321.6ng/ml)相近，圖5B和5C則顯示4個人源化PD-L1抗體156-1H，156-7H和156-10H，156-11H的EC50分別為：342ng/ml，313.7ng/ml，357.1ng/ml和282.2ng/ml，和陽性對照Avelumab的EC50相近。該檢測定量地證實了鼠源和人源化的抗PD-L1抗體對細胞表面PD-1：PD-L1相互作用導致的T細胞活性抑制呈現劑量依賴性的阻抑能力，從而劑量依賴地增強Jurkat細胞內報告基因的活性。 Plant the CHO cell strain stably transfected with PD-L1 on a 96-well white bottom plate, with 40,000 cells per well, 100 μl/well, and put it back into the incubator overnight; -1 cell line and the PD-L1 antibody to be tested were co-incubated, and each well of PD-1 cells The sample volume is 16,000/well, and the antibody is serially diluted. Each dose is replicated in 3 wells. The incubation volume is 100 μl/well, and the incubation time is 6 hours. When the incubation is completed, take out the well plate and equal volume (100 μl) Add the luminescent detection reagent and read the value. According to the detection value, Graphpad was used to analyze the regression curve with 4 parameters, and the EC50 value of each antibody was obtained. The results are shown in Figure 5A. 222.9ng/ml, 321.6ng/ml), Figure 5B and 5C show that the EC50 of the four humanized PD-L1 antibodies 156-1H, 156-7H and 156-10H, 156-11H were: 342ng/ml, 313.7ng/ml, 357.1ng/ml and 282.2ng/ml are similar to the EC50 of the positive control Avelumab. This assay quantitatively demonstrates that murine and humanized anti-PD-L1 antibodies have a dose-dependent inhibitory ability to inhibit T cell activity caused by cell surface PD-1:PD-L1 interaction, thereby dose-dependently enhancing Activity of reporter genes in Jurkat cells.

實施例10 ELISA檢測混合淋巴細胞反應中T細胞分泌的IFN-γExample 10 ELISA detection of IFN-γ secreted by T cells in mixed lymphocyte reaction

通過混合淋巴細胞反應(mixed lymphocyte reaction，MLR)來測定PD-L1單抗增強T細胞的活性。從健康人供體1的外周血單核細胞(peripheral blood mononuclear cells,PBMC)中分離CD14⁺單核細胞，應用重組人粒細胞-巨噬細胞集落刺激因數(GM-CSF，Peprotech，Cat.300-03)和重組人白介素4(rhIL-4,Peprotech，Cat.200-04)進行體外誘導分化為樹突狀細胞(dendritic cell,DC)，於培養第6天加入LPS(Sigma，Cat：L4516)刺激成熟DC，第7天將供體1的DC細胞與從健康供體2的PBMC富集的CD4⁺T細胞混合共培養，DC：CD4⁺T細胞數比例為1：10，加入待測抗體或陽性對照抗體Avelumab或Atezolizumab(抗體濃度為1000ng/ml，100ng/ml，10ng/ml，1ng/ml)，共培養4天。4天后收集細胞培養上清，用ELISA方法檢測上清中IFN-γ的含量如圖6顯示，所有測試的鼠源抗體及陽性對照抗體Avelumab、Atezolizumab相比anti-HEL單抗和no treatment(即沒有給藥)陰性對照組，均可明顯增強MLR實驗中CD4⁺T細胞分泌IFN-γ的能力，並且隨著PD-L1抗體藥物濃度降低，增加分泌IFN-γ的活性也降低。該結果表明PD-L1抗體可增強T細胞的功能，且具有劑量依賴性。T-test，*P<0.05，**P<0.01，***P<0.001，****P<0.0001. The activity of PD-L1 monoclonal antibody to enhance T cells was measured by mixed lymphocyte reaction (MLR). CD14 ⁺ monocytes were isolated from peripheral blood mononuclear cells (PBMC) of healthy human donor 1, and recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF, Peprotech, Cat.300 -03) and recombinant human interleukin 4 (rhIL-4, Peprotech, Cat.200-04) were induced to differentiate into dendritic cells (dendritic cells, DC) in vitro, and LPS (Sigma, Cat: L4516) was added on the 6th day of culture ) to stimulate mature DC. On the 7th day, DC cells from donor 1 were mixed with CD4 ⁺ T cells enriched from PBMCs from healthy donor 2. The ratio of DC: CD4 ⁺ T cells was 1:10. Antibody or positive control antibody Avelumab or Atezolizumab (antibody concentration is 1000ng/ml, 100ng/ml, 10ng/ml, 1ng/ml), co-cultured for 4 days. After 4 days, the cell culture supernatant was collected, and the ELISA method was used to detect the content of IFN-γ in the supernatant as shown in Figure 6. Compared with the anti-HEL monoclonal antibody and no treatment (ie, all tested mouse antibodies and positive control antibodies Avelumab and Atezolizumab No drug administration) negative control group can significantly enhance the ability of CD4 ⁺ T cells to secrete IFN-γ in the MLR experiment, and as the concentration of PD-L1 antibody drug decreases, the activity of increasing the secretion of IFN-γ also decreases. This result indicates that PD-L1 antibody can enhance the function of T cells in a dose-dependent manner. T-test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

實施例11 抗PD-L1抗體的抗體依賴的細胞殺傷(Antibody Dependent Cell-mediated Cytotoxicit，ADCC)活性檢測Example 11 Detection of antibody-dependent cell killing (Antibody Dependent Cell-mediated Cytotoxicit, ADCC) activity of anti-PD-L1 antibody

通過分離純化的正常人PBMC中的天然殺傷(natural killer,NK)細胞對高表達人PD-L1的A431細胞的殺傷實驗來測定抗PD-L1單抗介導的抗體依賴的細胞殺傷活性。 The antibody-dependent cell killing activity mediated by anti-PD-L1 monoclonal antibody was determined by the killing experiment of natural killer (natural killer, NK) cells isolated and purified from normal human PBMC on A431 cells highly expressing human PD-L1.

效應細胞的製備：復蘇凍存的人PBMC細胞(Stemexpress公司)後，在補充有100IU/ml重組人白介素2(rhIL-2,Peprotech,Cat.200-02)的RPMI1640培養基(Invitrogen，Cat.11835030)中孵育過夜。其後，收集細胞後進行活細胞計數。然後使用NK細胞磁珠分離試劑盒(Stemcell,Cat.17955)，從PBMC中純化NK細胞，將NK細胞用補充有10%滅活的胎牛血清的DMEM(Invitrogen,Cat.11965084)重懸後進行計數，用作效應細胞。 Preparation of effector cells: After thawing frozen human PBMC cells (Stemexpress), RPMI1640 medium (Invitrogen, Cat.11835030) supplemented with 100 IU/ml recombinant human interleukin 2 (rhIL-2, Peprotech, Cat.200-02) ) overnight. Thereafter, viable cell counts were performed after harvesting the cells. Then use the NK cell magnetic bead separation kit (Stemcell, Cat.17955) to purify NK cells from PBMC, and resuspend the NK cells with DMEM (Invitrogen, Cat.11965084) supplemented with 10% inactivated fetal bovine serum Count and use as effector cells.

靶細胞的製備：靶細胞A431用含10%胎牛血清的DMEM培養基培養，ADCC實驗前一天，加上終濃度為500IU幹擾素IFN-γ(Peprotech,Cat.300-02)進行刺激培養過夜後待用。 Preparation of target cells: target cell A431 was cultured in DMEM medium containing 10% fetal bovine serum, and the day before ADCC experiment, plus a final concentration of 500 IU interferon IFN-γ (Peprotech, Cat.300-02) for stimulation and culture overnight stand-by.

用含有10%胎牛血清的DMEM培養基稀釋抗PD-L1抗體，稀釋好的抗體以25μl/孔分至白壁96孔板(Corning，Cat.3610)中，並且將靶細胞加入孔中(25μl/孔，10000細胞/孔)。使板在37℃，5%CO₂培養箱中孵育30分鐘。隨後，將效應細胞加入孔中(25μl/孔，40000細胞/孔，即效靶比NK：A431為4：1)，總體積為100μl，使板在37℃，5%CO₂培養箱中孵育4個小時。 Dilute the anti-PD-L1 antibody with DMEM medium containing 10% fetal bovine serum, distribute the diluted antibody to a white-walled 96-well plate (Corning, Cat.3610) at 25 μl/well, and add target cells to the well (25 μl/well well, 10000 cells/well). Incubate the plate in a 37°C, 5% _CO2 incubator for 30 minutes. Subsequently, effector cells were added to the wells (25 μl/well, 40,000 cells/well, the immediate target ratio NK:A431 was 4:1), the total volume was 100 μl, and the plate was incubated at 37°C, 5% _CO2 incubator 4 hours.

需同時設置培養基背景對照孔(即孔中加入100μl培養基)，靶細胞自發死亡釋放孔(即孔中只有靶細胞)，效應細胞自發死亡釋放孔(即孔中只有NK細胞)，靶細胞最大死亡釋放孔(即靶細胞加CytoTox-Glo^TM Cytotoxicity試劑盒(Promega，Cat.G9291)中提供的lysis buffer，所有對照孔的體積最後用培養基統一補充至100μl。培養4小時後，將板取出，每孔加入50μl CytoTox-Glo^TM Cytotoxicity Assay Reagent，放於搖床上混勻後，室溫放置15分鐘後進行讀數化學發光(luminescence,RLU)。 At the same time, it is necessary to set the culture medium background control well (that is, add 100 μl medium to the well), target cell spontaneous death release well (that is, only target cells in the well), effector cell spontaneous death release well (that is, only NK cells in the well), and the maximum death of target cells Release wells (i.e. target cells plus the lysis buffer provided in the CytoTox-Glo ^TM Cytotoxicity kit (Promega, Cat.G9291), and the volume of all control wells was finally uniformly supplemented with medium to 100 μl. After culturing for 4 hours, the plate was taken out, and every Add 50 μl of CytoTox-Glo ^TM Cytotoxicity Assay Reagent to the wells, place on a shaker to mix well, and then place at room temperature for 15 minutes to read chemiluminescence (luminescence, RLU).

根據下面步驟進行計算由ADCC活性引起的細胞裂解率：先將所有孔的讀值減去培養基背景對照孔的平均讀值，然後計算裂解率%=(實驗孔讀值-靶細胞自發死亡釋放孔讀值-效應細胞自發死亡釋放孔)/(靶細胞最大死亡釋放孔讀值-靶細胞自發死亡釋放孔讀值)*100 Calculate the cell lysis rate caused by ADCC activity according to the following steps: first subtract the average reading value of the culture medium background control well from the reading value of all wells, and then calculate the lysis rate %=(reading value of the experimental well-target cell spontaneous death release well Reading value-effector cell spontaneous death release hole)/(target cell maximum death release hole reading value-target cell spontaneous death release hole reading value)*100

結果顯示於圖7中，實驗結果顯示所測試的3個鼠源PD-L1抗體和FDA已批准上市的陽性對照抗PD-L1藥物Avelumab均顯示出對靶細胞A431細胞的抗體濃度依賴性的裂解殺傷活性和相近的EC50數值，同時，陰性同型對照抗體hIgG1,K(Abdserotec,PHP010)即使在最高濃度時也顯示沒有ADCC活性。 The results are shown in Figure 7. The experimental results show that the three tested murine PD-L1 antibodies and the FDA-approved positive control anti-PD-L1 drug Avelumab all showed antibody concentration-dependent lysis of target cells A431 cells Killing activity and similar EC50 values, meanwhile, the negative isotype control antibody hIgG1, K (Abdserotec, PHP010) showed no ADCC activity even at the highest concentration.

實施例12 鼠源PD-L1抗體PDL1-156的小鼠體內藥效檢測Example 12 In vivo drug efficacy test of mouse PD-L1 antibody PDL1-156

利用B-hPD-1/hPD-L1轉基因小鼠(北京百奧賽圖基因生物技術有限公司)建立MC38-hPD-L1結腸癌動物模型並進行PD-L1抗體藥效實驗。小鼠右側皮下接種5×10⁵MC38-hPD-L1結腸癌細胞。當腫瘤體積達到~110mm³時，挑選個體腫瘤體積適中的小鼠入組，將動物按腫瘤體積使用隨機分組軟體分配到3個實驗組：anti-Hel hIgG同型對照組、抗PDL1-156抗體組、陽性藥Avelumab組(Pfizer，lot AU020322)，每組8只，分組當天開始給藥(定義為研究第0天)。各組均按照10mg/kg劑量，每週腹腔注射給藥兩次，共7次。每週兩次測量腫瘤體積和小鼠體重，並記錄測量值，計算腫瘤體積(長徑x短徑2/2)和生長抑制率 (tumor growth inhibition %,TGITV(%)=[1-(Ti-T0)/(Vi-V0)]×100%；Ti：治療組在給藥第i天的腫瘤體積均值，T0：治療組在給藥第0天的腫瘤體積均值；Vi：溶劑對照組在給藥第i天的腫瘤體積均值，V0：溶劑對照組在給藥第0天的腫瘤體積均值)，同時對腫瘤體積進行統計學分析，P<0.05認為有顯著性差異。 B-hPD-1/hPD-L1 transgenic mice (Beijing Biocytogen Biotechnology Co., Ltd.) were used to establish MC38-hPD-L1 colon cancer animal models and conduct PD-L1 antibody efficacy experiments. The right side of the mouse was subcutaneously inoculated with 5×10 ⁵ MC38-hPD-L1 colon cancer cells. When the tumor volume reaches ~110mm ³ , select mice with moderate tumor volume to enter the group, and use the random grouping software to assign the animals to 3 experimental groups according to the tumor volume: anti-Hel hIgG isotype control group, anti-PDL1-156 antibody group 1. Positive drug Avelumab group (Pfizer, lot AU020322), 8 rats in each group, started administration on the day of grouping (defined as the 0th day of the study). Each group was given 10 mg/kg intraperitoneal injection twice a week, 7 times in total. The tumor volume and mouse body weight were measured twice a week, and the measured values were recorded, and the tumor volume (long diameter x short diameter 2/2) and growth inhibition rate (tumor growth inhibition %, TGITV (%)=[1-(Ti -T0)/(Vi-V0)]×100%; Ti: the average tumor volume of the treatment group on day i of administration, T0: the average tumor volume of the treatment group on day 0 of administration; Vi: the solvent control group at The average tumor volume on day i of administration, V0: the average tumor volume of the solvent control group on day 0 of administration), and the tumor volume was statistically analyzed at the same time, and P<0.05 was considered to have a significant difference.

在分組給藥第21天，與對照組相比，陽性藥Avelumab組和PDL1-156鼠源抗PD-L1抗體組在腫瘤體積上有顯著且相近的抑制效果，且具有統計學差異(P<0.05)。見圖8A、B、C，表8。 On the 21st day of group administration, compared with the control group, the positive drug Avelumab group and the PDL1-156 mouse anti-PD-L1 antibody group had significant and similar inhibitory effects on tumor volume, and there were statistical differences (P< 0.05). See Figure 8A, B, C, Table 8.

此外，在實驗過程中，除hIgG對照組有1只小鼠提前異常死亡。其餘實驗動物在給藥期間活動和進食狀態良好，體重均有一定程度的上升，結果說明該抗體安全性較高，見圖8D、表9。 In addition, during the experiment, except for the hIgG control group, one mouse died prematurely and abnormally. The rest of the experimental animals were active and eating well during the administration period, and their body weight all increased to a certain extent. The results showed that the antibody was relatively safe, as shown in Figure 8D and Table 9.

實施例13 人源化抗PD-L1抗體的小鼠體內藥效檢測Example 13 In vivo efficacy test of humanized anti-PD-L1 antibody in mice

MC38-hPD-L1細胞以5×10⁵個/0.1mL濃度接種於雌性6-8周B-hPD-L1轉基因小鼠(百奧賽圖江蘇基因生物技術有限公司)的右側皮下，待腫瘤生長到大約108mm³時按腫瘤體積挑選24只隨機分組，每組8只，共3組，分別為：生理鹽水、Avelumab(5mg/kg)、156-10H(5mg/kg)。所有組給藥途徑均為腹腔注射，每週給藥2次，連續給藥6次，末次給藥5天后結束實驗。給藥和觀察期間每週測量3次小鼠體重和腫瘤體積，並記錄測量值，計算腫瘤體積(長徑x短徑²/2)和生長抑制率(TGI_TV(%)，在分組給藥第21天，與生理鹽水對照組相比，陽性藥Avelumab組和PD-L1抗體156-10H組在腫瘤體積上有顯著且相近的抑制效果，且具有統計學差異(P<0.05)。見圖9A、B、C，表10。 MC38-hPD-L1 cells were inoculated subcutaneously on the right side of female 6-8 week B-hPD-L1 transgenic mice (Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) at a concentration of 5×10 ⁵ cells/0.1 mL. At about 108 mm ³ , 24 rats were selected according to the tumor volume and randomly divided into 3 groups, 8 rats in each group, namely: normal saline, Avelumab (5 mg/kg), and 156-10H (5 mg/kg). The route of administration for all groups was intraperitoneal injection, administered twice a week for 6 consecutive times, and the experiment ended 5 days after the last administration. During administration and observation, the body weight and tumor volume of the mice were measured 3 times a week, and the measured values were recorded, and the tumor volume (long diameter x short diameter ² /2) and growth inhibition rate (TGI _TV (%) were calculated. On the 21st day, compared with the normal saline control group, the positive drug Avelumab group and the PD-L1 antibody 156-10H group had significant and similar inhibitory effects on tumor volume, and there was a statistical difference (P<0.05). See Figure 9A, B, C, Table 10.

實驗動物在給藥期間活動和進食狀態良好，體重均有一定程度的上升，見圖9D、表11。 During the administration period, the experimental animals were in a good state of activity and eating, and their body weight increased to a certain extent, as shown in Figure 9D and Table 11.

以上結果表明人源化的PD-L1抗體156-10H對MC38-hPD-L1腫瘤皮下移植瘤生長具有顯著抑制作用且表現出較高安全性。與陽性對照抗體Avelumab相比，兩者TGI水準相當，且156-10H顯示更均一抗腫瘤效果。 The above results show that the humanized PD-L1 antibody 156-10H has a significant inhibitory effect on the growth of MC38-hPD-L1 tumor subcutaneous xenografts and shows high safety. Compared with the positive control antibody Avelumab, the TGI levels of the two are comparable, and 156-10H shows a more uniform anti-tumor effect.

以上對本發明所提供的抗人程序性死亡配體-1(PD-L1)的抗體及其用途進行了詳細介紹。本文應用了具體個例對本發明的原理及實施方式進行了闡述，以上實施例的說明只是用於幫助理解本發明的方法及其核心思想。應當指出，對於本技術領域技術人員來說，在不脫離本發明原理的前提下，還可以對本發明進行若干改進和修飾，這些改進和修飾也落入本發明請求項的保護範圍內。 The anti-human programmed death ligand-1 (PD-L1) antibody provided by the present invention and its use are described above in detail. This paper has applied specific examples to the principles of the present invention and implementation methods. The formula has been described, and the description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

<110> 大陸商先聲生物醫藥科技有限公司(SIMCERE BIO-PHARMACEUTICAL TECHNOLOGY.,LTD.)；大陸商江蘇先聲藥業有限公司(JIANGSU SIMCERE PHARMACEUTICAL CO.,LTD.) <110> Continental Shangxiansheng Bio-Pharmaceutical Technology Co., Ltd. (SIMCERE BIO-PHARMACEUTICAL TECHNOLOGY.,LTD.);

<120> 抗人程序性死亡配體-1(PD-L1)的抗體及其用途 <120> Antibody against human programmed death ligand-1 (PD-L1) and use thereof

<130> CP0021-TW-0007 <130> CP0021-TW-0007

<160> 182 <160> 182

<170> SIPOSequenceListing 1.0 <170> SIPOSequenceListing 1.0

<210> 1 <210> 1

<211> 118 <211> 118

<212> PRT <212> PRT

<213> 人工序列(Artificial sequence；) <213> Artificial sequence (Artificial sequence;)

<400> 1

<400> 1

<210> 2 <210> 2

<211> 107 <211> 107

<212> PRT <212> PRT

<400> 2

<400> 2

<210> 3 <210> 3

<211> 118 <211> 118

<212> PRT <212> PRT

<400> 3

<400> 3

<210> 4 <210> 4

<211> 107 <211> 107

<212> PRT <212> PRT

<400> 4

<400> 4

<210> 5 <210> 5

<211> 118 <211> 118

<212> PRT <212> PRT

<400> 5

<400> 5

<210> 6 <210> 6

<211> 107 <211> 107

<212> PRT <212> PRT

<400> 6

<400> 6

<210> 7 <210> 7

<211> 118 <211> 118

<212> PRT <212> PRT

<400> 7

<400> 7

<210> 8 <210> 8

<211> 113 <211> 113

<212> PRT <212> PRT

<400> 8

<400> 8

<210> 9 <210> 9

<211> 114 <211> 114

<212> PRT <212> PRT

<400> 9

<400> 9

<210> 10 <210> 10

<211> 106 <211> 106

<212> PRT <212> PRT

<400> 10

<400> 10

<210> 11 <210> 11

<211> 118 <211> 118

<212> PRT <212> PRT

<400> 11

<400> 11

<210> 12 <210> 12

<211> 113 <211> 113

<212> PRT <212> PRT

<400> 12

<400> 12

<210> 13 <210> 13

<211> 118 <211> 118

<212> PRT <212> PRT

<400> 13

<400> 13

<210> 14 <210> 14

<211> 107 <211> 107

<212> PRT <212> PRT

<400> 14

<400> 14

<210> 15 <210> 15

<211> 118 <211> 118

<212> PRT <212> PRT

<400> 15

<400> 15

<210> 16 <210> 16

<211> 113 <211> 113

<212> PRT <212> PRT

<400> 16

<400> 16

<210> 17 <210> 17

<211> 118 <211> 118

<212> PRT <212> PRT

<400> 17

<400> 17

<210> 18 <210> 18

<211> 113 <211> 113

<212> PRT <212> PRT

<400> 18

<400> 18

<210> 19 <210> 19

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 19

<400> 19

<210> 20 <210> 20

<211> 17 <211> 17

<212> PRT <212> PRT

<400> 20

<400> 20

<210> 21 <210> 21

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 21

<400> 21

<210> 22 <210> 22

<211> 11 <211> 11

<212> PRT <212> PRT

<400> 22

<400> 22

<210> 23 <210> 23

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 23

<400> 23

<210> 24 <210> 24

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 24

<400> 24

<210> 25 <210> 25

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 25

<400> 25

<210> 26 <210> 26

<211> 17 <211> 17

<212> PRT <212> PRT

<400> 26

<400> 26

<210> 27 <210> 27

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 27

<400> 27

<210> 28 <210> 28

<211> 11 <211> 11

<212> PRT <212> PRT

<400> 28

<400> 28

<210> 29 <210> 29

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 29

<400> 29

<210> 30 <210> 30

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 30

<400> 30

<210> 31 <210> 31

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 31

<400> 31

<210> 32 <210> 32

<211> 17 <211> 17

<212> PRT <212> PRT

<400> 32

<400> 32

<210> 33 <210> 33

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 33

<400> 33

<210> 34 <210> 34

<211> 11 <211> 11

<212> PRT <212> PRT

<400> 34

<400> 34

<210> 35 <210> 35

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 35

<400> 35

<210> 36 <210> 36

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 36

<400> 36

<210> 37 <210> 37

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 37

<400> 37

<210> 38 <210> 38

<211> 16 <211> 16

<212> PRT <212> PRT

<400> 38

<400> 38

<210> 39 <210> 39

<211> 10 <211> 10

<212> PRT <212> PRT

<400> 39

<400> 39

<210> 40 <210> 40

<211> 17 <211> 17

<212> PRT <212> PRT

<400> 40

<400> 40

<210> 41 <210> 41

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 41

<400> 41

<210> 42 <210> 42

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 42

<400> 42

<210> 43 <210> 43

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 43

<400> 43

<210> 44 <210> 44

<211> 17 <211> 17

<212> PRT <212> PRT

<400> 44

<400> 44

<210> 45 <210> 45

<211> 6 <211> 6

<212> PRT <212> PRT

<400> 45

<400> 45

<210> 46 <210> 46

<211> 10 <211> 10

<212> PRT <212> PRT

<400> 46

<400> 46

<210> 47 <210> 47

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 47

<400> 47

<210> 48 <210> 48

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 48

<400> 48

<210> 49 <210> 49

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 49

<400> 49

<210> 50 <210> 50

<211> 16 <211> 16

<212> PRT <212> PRT

<400> 50

<400> 50

<210> 51 <210> 51

<211> 10 <211> 10

<212> PRT <212> PRT

<400> 51

<400> 51

<210> 52 <210> 52

<211> 17 <211> 17

<212> PRT <212> PRT

<400> 52

<400> 52

<210> 53 <210> 53

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 53

<400> 53

<210> 54 <210> 54

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 54

<400> 54

<210> 55 <210> 55

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 55

<400> 55

<210> 56 <210> 56

<211> 17 <211> 17

<212> PRT <212> PRT

<400> 56

<400> 56

<210> 57 <210> 57

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 57

<400> 57

<210> 58 <210> 58

<211> 11 <211> 11

<212> PRT <212> PRT

<400> 58

<400> 58

<210> 59 <210> 59

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 59

<400> 59

<210> 60 <210> 60

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 60

<400> 60

<210> 61 <210> 61

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 61

<400> 61

<210> 62 <210> 62

<211> 16 <211> 16

<212> PRT <212> PRT

<400> 62

<400> 62

<210> 63 <210> 63

<211> 10 <211> 10

<212> PRT <212> PRT

<400> 63

<400> 63

<210> 64 <210> 64

<211> 17 <211> 17

<212> PRT <212> PRT

<400> 64

<400> 64

<210> 65 <210> 65

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 65

<400> 65

<210> 66 <210> 66

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 66

<400> 66

<210> 67 <210> 67

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 67

<400> 67

<210> 68 <210> 68

<211> 16 <211> 16

<212> PRT <212> PRT

<400> 68

<400> 68

<210> 69 <210> 69

<211> 10 <211> 10

<212> PRT <212> PRT

<400> 69

<400> 69

<210> 70 <210> 70

<211> 17 <211> 17

<212> PRT <212> PRT

<400> 70

<400> 70

<210> 71 <210> 71

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 71

<400> 71

<210> 72 <210> 72

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 72

<400> 72

<210> 73 <210> 73

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 73

<400> 73

<210> 74 <210> 74

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 74

<400> 74

<210> 75 <210> 75

<211> 11 <211> 11

<212> PRT <212> PRT

<400> 75

<400> 75

<210> 76 <210> 76

<211> 6 <211> 6

<212> PRT <212> PRT

<400> 76

<400> 76

<210> 77 <210> 77

<211> 3 <211> 3

<212> PRT <212> PRT

<400> 77

<400> 77

<210> 78 <210> 78

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 78

<400> 78

<210> 79 <210> 79

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 79

<400> 79

<210> 80 <210> 80

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 80

<400> 80

<210> 81 <210> 81

<211> 11 <211> 11

<212> PRT <212> PRT

<400> 81

<400> 81

<210> 82 <210> 82

<211> 6 <211> 6

<212> PRT <212> PRT

<400> 82

<400> 82

<210> 83 <210> 83

<211> 3 <211> 3

<212> PRT <212> PRT

<400> 83

<400> 83

<210> 84 <210> 84

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 84

<400> 84

<210> 85 <210> 85

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 85

<400> 85

<210> 86 <210> 86

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 86

<400> 86

<210> 87 <210> 87

<211> 11 <211> 11

<212> PRT <212> PRT

<400> 87

<400> 87

<210> 88 <210> 88

<211> 6 <211> 6

<212> PRT <212> PRT

<400> 88

<400> 88

<210> 89 <210> 89

<211> 3 <211> 3

<212> PRT <212> PRT

<400> 89

<400> 89

<210> 90 <210> 90

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 90

<400> 90

<210> 91 <210> 91

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 91

<400> 91

<210> 92 <210> 92

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 92

<400> 92

<210> 93 <210> 93

<211> 12 <211> 12

<212> PRT <212> PRT

<400> 93

<400> 93

<210> 94 <210> 94

<211> 12 <211> 12

<212> PRT <212> PRT

<400> 94

<400> 94

<210> 95 <210> 95

<211> 3 <211> 3

<212> PRT <212> PRT

<400> 95

<400> 95

<210> 96 <210> 96

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 96

<400> 96

<210> 97 <210> 97

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 97

<400> 97

<210> 98 <210> 98

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 98

<400> 98

<210> 99 <210> 99

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 99

<400> 99

<210> 100 <210> 100

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 100

<400> 100

<210> 101 <210> 101

<211> 3 <211> 3

<212> PRT <212> PRT

<400> 101

<400> 101

<210> 102 <210> 102

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 102

<400> 102

<210> 103 <210> 103

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 103

<400> 103

<210> 104 <210> 104

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 104

<400> 104

<210> 105 <210> 105

<211> 12 <211> 12

<212> PRT <212> PRT

<400> 105

<400> 105

<210> 106 <210> 106

<211> 12 <211> 12

<212> PRT <212> PRT

<400> 106

<400> 106

<210> 107 <210> 107

<211> 3 <211> 3

<212> PRT <212> PRT

<400> 107

<400> 107

<210> 108 <210> 108

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 108

<400> 108

<210> 109 <210> 109

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 109

<400> 109

<210> 110 <210> 110

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 110

<400> 110

<210> 111 <210> 111

<211> 11 <211> 11

<212> PRT <212> PRT

<400> 111

<400> 111

<210> 112 <210> 112

<211> 6 <211> 6

<212> PRT <212> PRT

<400> 112

<400> 112

<210> 113 <210> 113

<211> 3 <211> 3

<212> PRT <212> PRT

<400> 113

<400> 113

<210> 114 <210> 114

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 114

<400> 114

<210> 115 <210> 115

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 115

<400> 115

<210> 116 <210> 116

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 116

<400> 116

<210> 117 <210> 117

<211> 12 <211> 12

<212> PRT <212> PRT

<400> 117

<400> 117

<210> 118 <210> 118

<211> 12 <211> 12

<212> PRT <212> PRT

<400> 118

<400> 118

<210> 119 <210> 119

<211> 3 <211> 3

<212> PRT <212> PRT

<400> 119

<400> 119

<210> 120 <210> 120

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 120

<400> 120

<210> 121 <210> 121

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 121

<400> 121

<210> 122 <210> 122

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 122

<400> 122

<210> 123 <210> 123

<211> 12 <211> 12

<212> PRT <212> PRT

<400> 123

<400> 123

<210> 124 <210> 124

<211> 12 <211> 12

<212> PRT <212> PRT

<400> 124

<400> 124

<210> 125 <210> 125

<211> 3 <211> 3

<212> PRT <212> PRT

<400> 125

<400> 125

<210> 126 <210> 126

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 126

<400> 126

<210> 127 <210> 127

<211> 114 <211> 114

<212> PRT <212> PRT

<400> 127

<400> 127

<210> 128 <210> 128

<211> 106 <211> 106

<212> PRT <212> PRT

<400> 128

<400> 128

<210> 129 <210> 129

<211> 114 <211> 114

<212> PRT <212> PRT

<400> 129

<400> 129

<210> 130 <210> 130

<211> 106 <211> 106

<212> PRT <212> PRT

<400> 130

<400> 130

<210> 131 <210> 131

<211> 114 <211> 114

<212> PRT <212> PRT

<400> 131

<400> 131

<210> 132 <210> 132

<211> 106 <211> 106

<212> PRT <212> PRT

<400> 132

<400> 132

<210> 133 <210> 133

<211> 114 <211> 114

<212> PRT <212> PRT

<400> 133

<400> 133

<210> 134 <210> 134

<211> 106 <211> 106

<212> PRT <212> PRT

<400> 134

<400> 134

<210> 135 <210> 135

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 135

<400> 135

<210> 136 <210> 136

<211> 17 <211> 17

<212> PRT <212> PRT

<400> 136

<400> 136

<210> 137 <210> 137

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 137

<400> 137

<210> 138 <210> 138

<211> 10 <211> 10

<212> PRT <212> PRT

<400> 138

<400> 138

<210> 139 <210> 139

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 139

<400> 139

<210> 140 <210> 140

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 140

<400> 140

<210> 141 <210> 141

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 141

<400> 141

<210> 142 <210> 142

<211> 17 <211> 17

<212> PRT <212> PRT

<400> 142

<400> 142

<210> 143 <210> 143

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 143

<400> 143

<210> 144 <210> 144

<211> 10 <211> 10

<212> PRT <212> PRT

<400> 144

<400> 144

<210> 145 <210> 145

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 145

<400> 145

<210> 146 <210> 146

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 146

<400> 146

<210> 147 <210> 147

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 147

<400> 147

<210> 148 <210> 148

<211> 17 <211> 17

<212> PRT <212> PRT

<400> 148

<400> 148

<210> 149 <210> 149

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 149

<400> 149

<210> 150 <210> 150

<211> 10 <211> 10

<212> PRT <212> PRT

<400> 150

<400> 150

<210> 151 <210> 151

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 151

<400> 151

<210> 152 <210> 152

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 152

<400> 152

<210> 153 <210> 153

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 153

<400> 153

<210> 154 <210> 154

<211> 17 <211> 17

<212> PRT <212> PRT

<400> 154

<400> 154

<210> 155 <210> 155

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 155

<400> 155

<210> 156 <210> 156

<211> 10 <211> 10

<212> PRT <212> PRT

<400> 156

<400> 156

<210> 157 <210> 157

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 157

<400> 157

<210> 158 <210> 158

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 158

<400> 158

<210> 159 <210> 159

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 159

<400> 159

<210> 160 <210> 160

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 160

<400> 160

<210> 161 <210> 161

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 161

<400> 161

<210> 162 <210> 162

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 162

<400> 162

<210> 163 <210> 163

<211> 3 <211> 3

<212> PRT <212> PRT

<400> 163

<400> 163

<210> 164 <210> 164

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 164

<400> 164

<210> 165 <210> 165

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 165

<400> 165

<210> 166 <210> 166

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 166

<400> 166

<210> 167 <210> 167

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 167

<400> 167

<210> 168 <210> 168

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 168

<400> 168

<210> 169 <210> 169

<211> 3 <211> 3

<212> PRT <212> PRT

<400> 169

<400> 169

<210> 170 <210> 170

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 170

<400> 170

<210> 171 <210> 171

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 171

<400> 171

<210> 172 <210> 172

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 172

<400> 172

<210> 173 <210> 173

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 173

<400> 173

<210> 174 <210> 174

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 174

<400> 174

<210> 175 <210> 175

<211> 3 <211> 3

<212> PRT <212> PRT

<400> 175

<400> 175

<210> 176 <210> 176

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 176

<400> 176

<210> 177 <210> 177

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 177

<400> 177

<210> 178 <210> 178

<211> 8 <211> 8

<212> PRT <212> PRT

<400> 178

<400> 178

<210> 179 <210> 179

<211> 7 <211> 7

<212> PRT <212> PRT

<400> 179

<400> 179

<210> 180 <210> 180

<211> 5 <211> 5

<212> PRT <212> PRT

<400> 180

<400> 180

<210> 181 <210> 181

<211> 3 <211> 3

<212> PRT <212> PRT

<400> 181

<400> 181

<210> 182 <210> 182

<211> 9 <211> 9

<212> PRT <212> PRT

<400> 182

<400> 182

Claims

An isolated antibody or antigen-binding fragment specifically binding to human programmed death ligand-1 (PD-L1), characterized in that the antibody or antigen-binding fragment comprises a heavy chain CDRs combination and a light chain CDRs combination: (1 ) The heavy chain CDRs combination comprises: CDR1-VH, CDR2-VH and CDR3-VH; the CDR1-VH, CDR2-VH and CDR3-VH have a sequence combination selected from the following:

And, (2) the light chain CDRs combination comprises: CDR1-VL, CDR2-VL and CDR3-VL, and the CDR1-VL, CDR2-VL and CDR3-VL have a sequence combination selected from the following:

Wherein, the antibody or antigen-binding fragment comprises a combination of heavy chain CDRs and light chain CDRs selected from the following: VH5+VL5, VH14+VL14, VH19+VL19, VH20+VL20, VH21+VL21, VH22+VL22, VH23+ VL23, VH24+VL24, VH25+VL25, or VH26+VL26.

The antibody or antigen-binding fragment according to claim 1, wherein the heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10 respectively; the heavy chain variable region The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 127 and SEQ ID NO: 128 respectively; the variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 129 and SEQ ID NO: 130 respectively The heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO: 131 and SEQ ID NO: 132, respectively; or, the heavy chain variable region and the light chain variable region have SEQ ID NO: 133 and The sequence shown in SEQ ID NO:134.

The antibody or antigen-binding fragment as claimed in claim 1, characterized in that it is combined with The dissociation constant (KD) for human programmed death ligand-1 (PD-L1) binding is not greater than 10nM, and the dissociation constant (KD) for binding to cynomolgus monkey programmed death ligand-1 (PD-L1) is not greater than 100nM .

The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment is: (1) a chimeric antibody or a fragment thereof; (2) a humanized antibody or a fragment thereof; (3) Fully human antibodies or fragments thereof.

The antibody or antigen-binding fragment according to claim 1, wherein the antibody comprises the sequence of any one of the constant regions of human or murine antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

The antibody or antigen-binding fragment according to claim 5, wherein the antibody comprises the sequence of the constant region of human or murine antibody IgG1, IgG2, IgG3 or IgG4.

The antibody or antigen-binding fragment as claimed in claim 1, wherein the antigen-binding fragment is selected from F(ab)2, Fab', Fab, Fv, scFv, bispecific antibody, nanobody and antibody minimum recognition unit one or more of.

An isolated nucleic acid molecule, characterized in that the nucleic acid molecule encodes the antibody, antigen-binding fragment, or any combination thereof as described in any one of claims 1-7.

An expression vector comprising the nucleic acid molecule isolated as described in Claim 8.

An isolated host cell comprising the isolated nucleic acid molecule of claim 8, or the expression vector of claim 9.

The host cell according to claim 10, wherein the host cell is a eukaryotic cell or a prokaryotic cell.

The host cell according to claim 11, wherein the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli and/or Bacillus subtilis.

The host cell as claimed in item 12, wherein the host cell For Chinese hamster ovary cells (CHO).

A method for preparing an antibody or antigen-binding fragment, characterized by culturing the host cell as described in Claim 10 under appropriate conditions, and isolating the antibody or antigen-binding fragment.

A pharmaceutical composition, characterized in that the composition comprises the antibody or antigen-binding fragment as described in any one of claims 1-7, the isolated nucleic acid molecule as described in claim 8, and the isolated nucleic acid molecule as described in claim 9 The expression vector, the cell as described in Claim 10, or the product prepared by the method as described in Claim 14, and a pharmaceutically acceptable carrier.

The pharmaceutical composition according to claim 15, further comprising an additional antineoplastic agent.

The antibody or antigen-binding fragment as described in any one of claims 1-7, the isolated nucleic acid molecule as described in claim 8, the expression vector as described in claim 9, the cell as described in claim 10, such as Use of the product prepared by the method described in Claim 14, or the pharmaceutical composition described in Claim 15, in the preparation of drugs for the prevention and/or treatment of diseases related to abnormal expression of PD-L1.

The use according to claim 17, the disease is a tumor.

The use according to claim 18, the tumor is colorectal cancer.

A kit comprising the antibody or antigen-binding fragment thereof as described in any one of claim items 1-7, the isolated nucleic acid molecule as described in claim item 8, the expression vector as described in claim item 9, and the expression vector as described in claim item 9. The cell described in 10, the product prepared by the method described in claim 14, and instructions for use.