TW202118787A

TW202118787A - Anti-4-1bb antibodies, antigen-binding fragments thereof, and bispecific antibodies

Info

Publication number: TW202118787A
Application number: TW109124769A
Authority: TW
Inventors: 張偉; 陳思萌; 王蕾蕾; 姜福偉; 吳建權; 郭新; 楊翠青; 廖成; 林�源; 胡齊悅; 張連山
Original assignee: 大陸商江蘇恆瑞醫藥股份有限公司
Priority date: 2019-07-22
Filing date: 2020-07-22
Publication date: 2021-05-16
Also published as: WO2021013142A1

Abstract

The present disclosure provides anti-4-1BB antibodies, antigen-binding fragments thereof, and bispecific antibodies. Specifically, the present disclosure provides a 4-1BB antibody, antigen-binding fragment thereof, and a recombinant bispecific antibody against both 4-1BB and PD-L1, nucleic acid molecules encoding the antibody, antigen-binding fragment or the bispecific antibody, vectors and host cells containing the nucleic acid molecules, and methods for preparing the antibodies, antigen-binding fragments thereof or the bispecific antibodies. A pharmaceutical composition comprising the antibody, antigen binding fragment thereof or the bispecific antibody and it use for the preparation of a medicament are also provided.

Description

抗4-1BB抗體、其抗原結合片段及雙特異性抗體 Anti-4-1BB antibody, its antigen-binding fragment and bispecific antibody

本公開涉及生物醫藥領域，特別是預防、治療與4-1BB和/或PD-L1相關疾病的領域。具體而言，本公開涉及4-1BB抗體及其抗原結合片段，特異性結合4-1BB和/或PD-L1的雙特異性抗體，其醫藥組成物醫藥組成物和相關製藥用途。 The present disclosure relates to the field of biomedicine, especially the field of prevention and treatment of diseases related to 4-1BB and/or PD-L1. Specifically, the present disclosure relates to 4-1BB antibodies and antigen-binding fragments thereof, bispecific antibodies that specifically bind 4-1BB and/or PD-L1, their medical compositions, medical compositions, and related pharmaceutical applications.

4-1BB(CD137，TNFRSF9)是腫瘤壞死因子受體超家族(TNFRS)的一種跨膜蛋白質。4-1BB在細胞表面以單體或二聚體形式表達，與其配體(4-1BBL)結合後藉由三聚體化以進行信號傳導，是CD8⁺和CD4⁺T細胞、調節性T細胞(Tregs)、NK細胞和NKT細胞、B細胞和中性粒細胞等的共刺激分子。在T細胞上，4-1BB並非組成型表達，而是在T細胞受體(TCR)激活後誘導的，藉由其天然配體4-1BBL或抗體激動劑的刺激，經由TNFR相關因子(TRAF)-2和TRAF-1進行信號傳導。4-1BB的早期信號傳導涉及K-63的多泛素化，激活核因子(NF)-κB和絲裂原活化蛋白激酶(MAPK)途徑，信號傳導導致T細胞的共刺激，細胞增殖，細胞因子產生，成熟和CD8⁺ T細胞存活延長。 4-1BB (CD137, TNFRSF9) is a transmembrane protein of the tumor necrosis factor receptor superfamily (TNFRS). 4-1BB is expressed in the form of monomer or dimer on the cell surface, and after binding to its ligand (4-1BBL), it undergoes signal transduction through trimerization. It is CD8 ⁺ and CD4 ⁺ T cells, regulatory T cells (Tregs), NK cells and NKT cells, B cells and neutrophils and other costimulatory molecules. On T cells, 4-1BB is not constitutively expressed, but is induced after T cell receptor (TCR) activation. It is stimulated by its natural ligand 4-1BBL or antibody agonists, and is stimulated by TNFR-related factors (TRAF). )-2 and TRAF-1 conduct signal transduction. Early signal transduction of 4-1BB involves polyubiquitination of K-63, activation of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) pathways, signal transduction leads to costimulation of T cells, cell proliferation, and cell proliferation. Factor production, maturation and prolonged survival of ^{CD8 + T cells.}

研究顯示，針對4-1BB的抗體激動劑在小鼠中促進T細胞的抗腫瘤控制(Murillo等，Clin Cancer Res.2008；14(21)：6895-906)。激活4-1BB的抗體在許多模型中可以增加共刺激分子的表達，誘導T細胞的存活和增殖，從而增強抗腫瘤免疫應答，引起抗腫瘤功效。現有技術中已有的4-1BB激活抗體包括BMS公司的urelumab，其是一種人IgG4抗體(WO2005035584)；以及Pfizer公司utomilumab，其是一種人IgG2抗體(Fisher等，Cancer Immunol.2012；61：1721-1733)。其他的4-1BB抗體相關專利包括WO2000029445、WO2012032433、WO2003049755、WO2017205745A等。BMS公司和Pfizer公司的4-1BB抗體均已進入I/II期臨床試驗階段，而Pieris公司的同類產品也已在I期臨床試驗之中。 Studies have shown that antibody agonists against 4-1BB promote anti-tumor control of T cells in mice (Murillo et al., Clin Cancer Res. 2008; 14(21): 6895-906). Antibodies that activate 4-1BB can increase the expression of costimulatory molecules in many models and induce the survival and proliferation of T cells, thereby enhancing anti-tumor immune responses and causing anti-tumor effects. Existing 4-1BB activating antibodies in the prior art include urelumab from BMS, which is a human IgG4 antibody (WO2005035584); and utomilumab from Pfizer, which is a human IgG2 antibody (Fisher et al., Cancer Immunol. 2012; 61: 1721). -1733). Other 4-1BB antibody related patents include WO2000029445, WO2012032433, WO2003049755, WO2017205745A and so on. Both BMS and Pfizer’s 4-1BB antibodies have entered phase I/II clinical trials, and similar products from Pieris are also in phase I clinical trials.

程序性死亡分子1(programmed death-1，PD-1)屬CD28家族，主要表達在活化的T細胞、B細胞和髓系細胞上。PD-1有兩個配體，分別為程序性死亡配體1(PD-L1，CD274，B7H1)和程序性死亡配體2(PD-L2)。PD-L2表達較局限，主要表達在抗原呈遞細胞上，如活化的巨噬細胞和樹突狀細胞。PD-L1屬免疫球蛋白(Ig)超家族，含有一個Ig樣C2型結構域和一個Ig樣V型結構域，是一種單次I型跨膜蛋白，經可變剪切後有三種亞型。新鮮分離的T細胞和B細胞表達可忽略量的PD-L1，部分(約16%)CD14⁺單核細胞組成型表達PD-L1。已知干擾素-γ(IFNγ)能上調腫瘤細胞的PD-L1，而PD-L1能阻礙抗腫瘤免疫，方式包括：藉由與活化T細胞上的受體PD-1結合來耐受腫瘤反應性T細胞；使腫瘤細胞對CD8⁺T細胞具有抗性，並藉由腫瘤細胞表達的PD-L1，藉由PD-1信號傳導進行Fas配體介導的裂解；藉由T細胞表達的CD80(B7-1)，反向信號傳導耐受T細胞；以及，促進經誘導的Tregs的發育和維持。 Programmed death-1 (PD-1) belongs to the CD28 family and is mainly expressed on activated T cells, B cells and myeloid cells. PD-1 has two ligands, programmed death ligand 1 (PD-L1, CD274, B7H1) and programmed death ligand 2 (PD-L2). PD-L2 expression is more limited, mainly expressed on antigen-presenting cells, such as activated macrophages and dendritic cells. PD-L1 belongs to the immunoglobulin (Ig) superfamily. It contains an Ig-like C2-type domain and an Ig-like V-type domain. It is a single type I transmembrane protein with three subtypes after variable cleavage. . Freshly isolated T cells and B cells express negligible amounts of PD-L1, and some (about 16%) CD14 ⁺ monocytes express PD-L1 constitutively. It is known that interferon-γ (IFNγ) can upregulate the PD-L1 of tumor cells, and PD-L1 can hinder anti-tumor immunity by binding to the receptor PD-1 on activated T cells to resist tumor response T cells; make tumor cells ^{resistant to CD8 +} T cells, and use PD-L1 expressed by tumor cells to undergo Fas ligand-mediated lysis through PD-1 signaling; CD80 expressed by T cells (B7-1), reverse signal transduction tolerant T cells; and, promote the development and maintenance of induced Tregs.

研究顯示，乳腺癌、肺癌、胃癌、腸癌、腎癌、黑色素瘤、非小細胞肺癌、結腸癌、膀胱癌、卵巢癌、胰腺癌及肝癌等人類腫瘤組織中檢測到高PD-L1蛋白的表達，且PD-L1的表達水平和患者的臨床及預後緊密相關。PD-L1的阻斷型抗體已在幾種已知過度表達PD-L1(包括黑色素瘤，NSCLC)的癌症中顯示出臨床活性。例如，Roche公司針對PD-L1的人源化IgG1單株抗體atezolizumab，在臨床試驗中作為幾種適應症的免疫療法，包括各種類型的實體瘤(參見例如Rittmeyer等，Lancet，2017；389：255-265)，並且被批准用於轉移性非小細胞肺癌和轉移性膀胱癌。另一種PD-L1抗體默沙東(Merck Serono SA)公司的avelumab，已被FDA批准用於治療Merkel細胞癌、膀胱癌、轉移性腎細胞癌，目前正處於非小細胞肺癌、卵巢癌和腎癌等的臨床試驗階段(例如參見Cortellis數據庫)。以及，MedImmune公司的PD-L1抗體Durvalumab，被批准用於轉移性非小細胞肺癌、轉移性膀胱癌，並且正在多種實體瘤和血液癌中進行臨床開發(例如參見Cortellis數據庫)。PD-L1抗體的相關專利包括：WO0139722、WO2013173223、WO2014195852、WO2013181634、WO2015048520、WO2015036511、US2014335093、WO2014100079、WO2014055897、US6803192B1、WO2014022758、US8617546B2和WO2010089411A2。 Studies have shown that human tumors such as breast cancer, lung cancer, stomach cancer, bowel cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer and liver cancer have detected high PD-L1 protein. Expression, and the expression level of PD-L1 is closely related to the clinical and prognosis of the patient. PD-L1 blocking antibodies have shown clinical activity in several cancers known to overexpress PD-L1 (including melanoma, NSCLC). For example, Roche's humanized IgG1 monoclonal antibody atezolizumab against PD-L1 is used in clinical trials as immunotherapy for several indications, including various types of solid tumors (see, for example, Rittmeyer et al., Lancet, 2017; 389:255 -265), and is approved for metastatic non-small cell lung cancer and metastatic bladder cancer. Another PD-L1 antibody, Merck Serono SA's avelumab, has been approved by the FDA for the treatment of Merkel cell carcinoma, bladder cancer, and metastatic renal cell carcinoma. It is currently in non-small cell lung cancer, ovarian cancer, and kidney cancer. The clinical trial phase (see, for example, the Cortellis database). And, MedImmune's PD-L1 antibody Durvalumab is approved for metastatic non-small cell lung cancer and metastatic bladder cancer, and is undergoing clinical development in a variety of solid tumors and blood cancers (see Cortellis database for example). PD-L1 antibody related patents include: WO0139722, WO2013173223, WO2014195852, WO2013181634, WO2015048520, WO2015036511, US2014335093, WO2014100079, WO2014055897, US6803192B1, WO2014022758, US8617546B2 and WO2010089411A2.

現有技術中有4-1BB抗體和PD-L1抗體的聯用的報導，例如4-1BB抗體作為激動劑與中和性PD-L1抗體的組合(Horton等，J Immunother Cancer.2015；3(Suppl 2)：O10)。utomilumab和avelumab的聯合治療也在測試(Chen等，J Clin Oncol 35，2017 suppl；abstr TPS7575，和clinical trial NCT02554812)。雖然兩個抗體聯用有可能增強臨床的抗腫瘤活性，擴大適應症。然而，本領域仍然需要能夠同時結合PD-L1和4-1BB的雙、多特異性抗體，因為雙特異性抗體具有兩個單抗聯用所不具備的活性特點。比如，雙特異性抗體可以使腫瘤部位特異性激活4-1BB，降低傳統激活型單抗在臨床所引起的肝毒性。雙特異性抗體(BsAb)是一類具有雙親嗜性的組合抗體，通常是雙價的(也有四價和六價的)，即有兩條抗原結合臂，具有結合兩種不同特異性抗原的功能。該抗體將同時結合表達PD-L1的抗原呈遞細胞(APC)或腫瘤細胞和表達4-1BB的T細胞，導致細胞毒性T細胞的條件活化。PD-L1與活化T細胞上表達的PD-1結合將導致T細胞抑制。涉及4-1BB抗體和PD-L1抗體的聯用或雙、多特異性抗體的專利包括：WO2019025545、WO2019104716、WO2018114754、WO2019072870、WO2019072869、WO2019072868、WO2019089753、WO2019005639。INHIBRx LLC公司的抗4-1BB/PD-L1雙特異性抗體INBRX-105目前處於非小細胞肺癌、黑色素瘤、頭頸癌、腎細胞癌、胃癌、食管癌、霍奇金病、非霍奇金淋巴瘤、晚期實體瘤等的腫瘤或癌症的I期臨床階段。Genmab公司的抗4-1BB/PD-L1雙特異性抗體GEN-1046目前處於子宮頸腫瘤、子宮內膜樣癌、轉移性非小細胞肺癌、晚期實體瘤等的II期臨床階段。Merus公司的抗4-1BB/PD-L1雙特異性抗體MCLA-145也處於晚期實體瘤、實體瘤的I期臨床階段。 In the prior art, there are reports of the combined use of 4-1BB antibody and PD-L1 antibody. For example, the combination of 4-1BB antibody as an agonist and neutralizing PD-L1 antibody (Horton et al., J Immunother Cancer. 2015; 3(Suppl 2): O10). The combination therapy of utomilumab and avelumab is also being tested (Chen et al., J Clin Oncol 35, 2017 suppl; abstr TPS7575, and clinical trial NCT02554812). Although the combination of the two antibodies may enhance the clinical anti-tumor activity and expand the indications. However, there is still a need in the art for bi- and multi-specific antibodies that can bind PD-L1 and 4-1BB at the same time, because bi-specific antibodies have active characteristics that the combination of two monoclonal antibodies does not possess. For example, bispecific antibodies can specifically activate 4-1BB at the tumor site and reduce the clinical hepatotoxicity caused by traditional activated monoclonal antibodies. Bispecific antibody (BsAb) is a type of combination antibody with parental tropism, usually bivalent (also quadrivalent and hexavalent), that is, it has two antigen-binding arms and has the function of binding two different specific antigens. . The antibody will simultaneously bind to antigen-presenting cells (APC) or tumor cells expressing PD-L1 and T cells expressing 4-1BB, resulting in the conditional activation of cytotoxic T cells. The binding of PD-L1 to PD-1 expressed on activated T cells will result in T cell suppression. Patents related to the combination of 4-1BB antibody and PD-L1 antibody or bi- or multispecific antibodies include: WO2019025545, WO2019104716, WO2018114754, WO2019072870, WO2019072869, WO2019072868, WO2019089753, WO2019005639. INHIBRx LLC’s anti-4-1BB/PD-L1 bispecific antibody INBRX-105 is currently in non-small cell lung cancer, melanoma, head and neck cancer, renal cell carcinoma, gastric cancer, esophageal cancer, Hodgkin’s disease, non-Hodgkin’s Lymphoma, advanced solid tumor and other tumors or cancer phase I clinical stage. Genmab's anti-4-1BB/PD-L1 bispecific antibody GEN-1046 is currently in the phase II clinical stage of cervical tumors, endometrioid carcinoma, metastatic non-small cell lung cancer, advanced solid tumors, etc. Merus's anti-4-1BB/PD-L1 bispecific antibody MCLA-145 is also in the stage I clinical stage of advanced solid tumors and solid tumors.

本領域中對於腫瘤或自身免疫性疾病等疾病仍有未滿足的需求，提供新結構的、更有效的、副作用更小的治療方法和藥物仍然是迫切而必要的。本公開的4-1BB/PD-L1雙特異性抗體保留了正常的Fc段，因此該抗體具有較長的半衰期。另外，本公開的4-1BB/PD-L1雙特異性抗體對人PD-L1蛋白的親和力比對人4-1BB蛋白高約50倍，這極大的保證了該雙抗在腫瘤部位的有效富集，同時，極大的降低了4-1BB端可能引起的毒副作用，提升了藥物的安全性，具有重大臨床價值。 In this field, there is still an unmet need for diseases such as tumors or autoimmune diseases, and it is still urgent and necessary to provide new structural, more effective, and less side effects treatment methods and drugs. The 4-1BB/PD-L1 bispecific antibody of the present disclosure retains the normal Fc segment, so the antibody has a longer half-life. In addition, the affinity of the 4-1BB/PD-L1 bispecific antibody of the present disclosure to human PD-L1 protein is about 50 times higher than that of human 4-1BB protein, which greatly guarantees the effective enrichment of the bispecific antibody at the tumor site. At the same time, it greatly reduces the toxic and side effects that may be caused by the 4-1BB end, improves the safety of the drug, and has great clinical value.

此外，本文以全文引入的方式併入4-1BB抗體和PD-L1抗體的相關申請，包括PCT/CN2019/072484、CN201680027181.4、CN201710341680.7、CN201811248478.0、CN201811396143.3、CN201811526262.6。 In addition, this article incorporates related applications of 4-1BB antibody and PD-L1 antibody by way of full introduction, including PCT/CN2019/072484, CN201680027181.4, CN201710341680.7, CN201811248478.0, CN201811396143.3, CN201811526262.6.

本公開提供了一種抗4-1BB抗體及其抗原結合片段，以及一種能夠結合4-1BB和PD-L1的雙特異性抗體。本公開還提供了編碼該抗體的核酸分子、包含該核酸分子的載體、製備該抗體的方法、包含該抗體的醫藥組成物醫藥組成物、該抗體在製備藥物中的用途、該抗體在治療/診斷與4-1BB和/或PD-L1相關的疾病(例如腫瘤或自身免疫性疾病或傳染病)中的用途或方法、以及包含該抗體的試劑盒。 The present disclosure provides an anti-4-1BB antibody and an antigen-binding fragment thereof, and a bispecific antibody capable of binding 4-1BB and PD-L1. The present disclosure also provides a nucleic acid molecule encoding the antibody, a vector containing the nucleic acid molecule, a method for preparing the antibody, a pharmaceutical composition comprising the antibody, a pharmaceutical composition, the use of the antibody in the preparation of medicines, and the use of the antibody in treatment/ Use or method for diagnosing diseases related to 4-1BB and/or PD-L1 (for example, tumors or autoimmune diseases or infectious diseases), and kits containing the antibodies.

抗4-1BB抗體及其抗原結合片段 Anti-4-1BB antibody and its antigen-binding fragment

本公開提供了一種抗4-1BB抗體及其抗原結合片段，其包含： The present disclosure provides an anti-4-1BB antibody and an antigen-binding fragment thereof, which comprises:

重鏈可變區，其包含至少1個選自如以下序列所示的重鏈互補決定區(HCDR)：SEQ ID NO：1，SEQ ID NO：2，和SEQ ID NO：3；和/或 A heavy chain variable region comprising at least one heavy chain complementarity determining region (HCDR) selected from the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and/or

輕鏈可變區，其包含至少1個選自如以下序列所示的輕鏈互補決定區(LCDR)：SEQ ID NO：4，SEQ ID NO：5，SEQ ID NO：6，和SEQ ID NO：7。 A light chain variable region comprising at least one light chain complementarity determining region (LCDR) selected from the following sequences: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

一些實施方案中，該抗4-1BB抗體及其抗原結合片段包含： In some embodiments, the anti-4-1BB antibody and antigen-binding fragment thereof comprise:

重鏈可變區，其包含分別如SEQ ID NO：1，SEQ ID NO：2和SEQ ID NO：3所示的HCDR1，HCDR2和HCDR3；和/或 A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and/or

輕鏈可變區，其包含分別如SEQ ID NO：4，SEQ ID NO：5和SEQ ID NO：6所示的LCDR1，LCDR2和LCDR3；或該輕鏈可變區包含分別SEQ ID NO：4，SEQ ID NO：7和SEQ ID NO：6所示的LCDR1，LCDR2和LCDR3。 The light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively; or the light chain variable region comprises respectively SEQ ID NO: 4 , LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 7 and SEQ ID NO: 6.

一些實施方案中，該抗4-1BB抗體及其抗原結合片段為鼠源抗體或其片段。在一些實施方案中，該抗4-1BB抗體或片段進一步包含鼠源κ、λ鏈或其變體的輕鏈框架區(FR區)或輕鏈恆定區。在一些實施方案中，該抗4-1BB抗體或片段進一步包含鼠源IgG1、IgG2、IgG3、IgG4或其變體的重鏈框架區(FR區)或重鏈恆定區。 In some embodiments, the anti-4-1BB antibody and antigen-binding fragments thereof are murine antibodies or fragments thereof. In some embodiments, the anti-4-1BB antibody or fragment further comprises a light chain framework region (FR region) or a light chain constant region of a murine kappa, lambda chain or a variant thereof. In some embodiments, the anti-4-1BB antibody or fragment further comprises a heavy chain framework region (FR region) or a heavy chain constant region of murine IgG1, IgG2, IgG3, IgG4, or a variant thereof.

在一些具體的實施方案中，該抗4-1BB抗體及其抗原結合片段為嵌合抗體或其片段。在一些實施方案中，該抗4-1BB抗體或片段進一步包含人源IgG1、IgG2、IgG3或IgG4或其變體的重鏈恆定區。在一些實施方案中，該抗4-1BB抗體或片段進一步包含人源κ、λ鏈或其變體的輕鏈恆定區。 In some specific embodiments, the anti-4-1BB antibody and antigen-binding fragments thereof are chimeric antibodies or fragments thereof. In some embodiments, the anti-4-1BB antibody or fragment further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or variants thereof. In some embodiments, the anti-4-1BB antibody or fragment further comprises a light chain constant region of a human kappa, lambda chain or a variant thereof.

在一些具體的實施方案中，該抗4-1BB抗體及其抗原結合片段為人源化抗體、人抗體或其片段。在一些實施方案中，重鏈FR區來源於人種系重鏈IGHV3-30序列或人種系重鏈IGHV1-46序列。在一些實施方案中，輕鏈FR區來源於人種系輕鏈IGkV3-11或人種系輕鏈IGkV4-1序列。IGHV3-30、IGKV3-11、IGHV1-46、IGKV4-1的胺基酸序列信息可如SEQ ID NO：27-30所示。 In some specific embodiments, the anti-4-1BB antibody and antigen-binding fragments thereof are humanized antibodies, human antibodies or fragments thereof. In some embodiments, the source of the FR region of the heavy chain In the human germline heavy chain IGHV3-30 sequence or the human germline heavy chain IGHV1-46 sequence. In some embodiments, the light chain FR region is derived from the human germline light chain IGkV3-11 or the human germline light chain IGkV4-1 sequence. The amino acid sequence information of IGHV3-30, IGKV3-11, IGHV1-46, and IGKV4-1 can be shown in SEQ ID NO: 27-30.

在一些實施方案中，根據本公開的抗4-1BB抗體及其抗原結合片段包含： In some embodiments, the anti-4-1BB antibodies and antigen-binding fragments thereof according to the present disclosure comprise:

重鏈可變區，其包含分別如SEQ ID NO：1，SEQ ID NO：2和SEQ ID NO：3所示的HCDR1，HCDR2和HCDR3；和輕鏈可變區，其包含分別如SEQ ID NO：4，SEQ ID NO：7和SEQ ID NO：6所示的LCDR1，LCDR2和LCDR3。 The heavy chain variable region, which comprises the HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and the light chain variable region, which comprises the HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: : 4, LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 7 and SEQ ID NO: 6.

在一些實施方案中，根據本公開的抗4-1BB抗體及其抗原結合片段的重鏈可變區包含或由SEQ ID NO：8所示的胺基酸序列組成；和/或，輕鏈可變區包含或由SEQ ID NO：9所示的胺基酸序列組成。 In some embodiments, the heavy chain variable region of the anti-4-1BB antibody and antigen-binding fragment thereof according to the present disclosure comprises or consists of the amino acid sequence shown in SEQ ID NO: 8; and/or, the light chain may The variable region includes or consists of the amino acid sequence shown in SEQ ID NO:9.

在一些實施方案中，根據本公開的抗4-1BB抗體及其抗原結合片段的重鏈包含或由SEQ ID NO：10所示的胺基酸序列組成；和/或，輕鏈包含或由SEQ ID NO：11所示的胺基酸序列組成。 In some embodiments, the heavy chain of the anti-4-1BB antibody and antigen-binding fragment thereof according to the present disclosure comprises or consists of the amino acid sequence shown in SEQ ID NO: 10; and/or, the light chain comprises or consists of SEQ ID NO: 10 ID NO: 11 shows the composition of the amino acid sequence.

在一些實施方案中，根據本公開的抗4-1BB抗體或其抗原結合片段的重鏈可變區有0至10個(1、2、3、4、5、6、7、8、9、10個)胺基酸變化；輕鏈可變區有0至10個(1、2、3、4、5、6、7、8、9、10個)胺基酸變化。在一些具體實施方案中，該胺基酸變化為保守的替換、取代或修飾，和/或不影響功能的缺失、添加。 In some embodiments, the heavy chain variable region of an anti-4-1BB antibody or antigen-binding fragment thereof according to the present disclosure has 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes; 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes in the light chain variable region. In some specific embodiments, the amino acid changes are conservative substitutions, substitutions or modifications, and/or deletions or additions that do not affect the function.

在一些具體實施方案中，根據本公開的抗4-1BB抗體或其抗原結合片段重鏈可變區(VH)的胺基酸序列與SEQ ID NO：8所示的重鏈可變區的胺基酸序列具有至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性；和/或，該抗體的輕鏈可變區(VL)的胺基酸序列與SEQ ID NO：9所示的輕鏈可變區的胺基酸序列具有至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性。 In some specific embodiments, the amino acid sequence of the heavy chain variable region (VH) of the anti-4-1BB antibody or antigen-binding fragment thereof according to the present disclosure is the same as the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 8 The base acid sequence has at least 80%, at least 85%, at least 90%, at least 91%, At least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and/or, the light chain of the antibody may The amino acid sequence of the variable region (VL) and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 9 have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在一些具體實施方案中，根據本公開的抗4-1BB抗體或其抗原結合片段重鏈的胺基酸序列與SEQ ID NO：10所示的序列具有至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性；和/或，該抗體的輕鏈胺基酸序列與SEQ ID NO：11所示的胺基酸序列具有至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性。 In some specific embodiments, the amino acid sequence of the heavy chain of the anti-4-1BB antibody or antigen-binding fragment thereof according to the present disclosure has at least 70%, at least 75%, or at least 80% of the sequence shown in SEQ ID NO: 10 , At least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence Identity; and/or, the light chain amino acid sequence of the antibody and the amino acid sequence shown in SEQ ID NO: 11 have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

雙特異性抗體 Bispecific antibody

本公開提供了一種雙特異性抗體，其結合PD-L1抗原和4-1BB抗原。 The present disclosure provides a bispecific antibody that binds PD-L1 antigen and 4-1BB antigen.

在一些具體實施方案中，該雙特異性抗體包含： In some specific embodiments, the bispecific antibody comprises:

1)特異性結合第一抗原的第一抗體或其抗原結合片段；和 1) The first antibody or antigen-binding fragment thereof that specifically binds to the first antigen; and

2)特異性結合第二抗原的第二抗體或其抗原結合片段； 2) A second antibody or antigen-binding fragment thereof that specifically binds to the second antigen;

該第一抗原為4-1BB，並且該第二抗原為PD-L1；或，該第一抗原為PD-L1，並且該第二抗原為4-1BB； The first antigen is 4-1BB, and the second antigen is PD-L1; or, the first antigen is PD-L1, and the second antigen is 4-1BB;

當該第一抗原或第二抗原是4-1BB時，該第一抗體、第二抗體或第一抗體、第二抗體的抗原結合片段包含分別如SEQ ID NO：1、2和3所示的HCDR1、HCDR2和HCDR3；和/或，分別如SEQ ID NO：4、7和6所示的LCDR1、LCDR2和LCDR3。 When the first antigen or the second antigen is 4-1BB, the first antibody, the second antibody, or the antigen-binding fragments of the first antibody and the second antibody include those shown in SEQ ID NOs: 1, 2 and 3, respectively. HCDR1, HCDR2, and HCDR3; and/or, LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 4, 7, and 6, respectively.

一些實施方案中，上述雙特異性抗體的第一抗體或其抗原結合片段包含重鏈和輕鏈；和/或第二抗體或其抗原結合片段包含scFv； In some embodiments, the first antibody or antigen-binding fragment thereof of the above-mentioned bispecific antibody comprises a heavy chain and a light chain; and/or the second antibody or antigen-binding fragment thereof comprises an scFv;

其中，該scFv連接於第一抗體或其抗原結合片段的重鏈或輕鏈的N端或C端； Wherein, the scFv is connected to the N-terminus or C-terminus of the heavy chain or light chain of the first antibody or its antigen-binding fragment;

較佳地，該第一抗體或其抗原結合片段包括兩條重鏈和兩條輕鏈，其中該第一抗體或其抗原結合片段的一條重鏈的重鏈可變區與一條輕鏈的輕鏈可變區形成抗原結合部位，另一條重鏈的重鏈可變區與另一條輕鏈的輕鏈可變區形成抗原結合部位。 Preferably, the first antibody or its antigen-binding fragment includes two heavy chains and two light chains, wherein the heavy chain variable region of one heavy chain of the first antibody or its antigen-binding fragment and the light chain of one light chain The chain variable region forms the antigen binding site, and the heavy chain variable region of the other heavy chain and the light chain variable region of the other light chain form the antigen binding site.

一些實施方案中，本公開的雙特異性抗體包含： In some embodiments, the bispecific antibodies of the present disclosure comprise:

1)特異性結合第一抗原的第一抗體或其抗原結合片段，該第一抗體或其抗原結合片段包括重鏈(HC)和輕鏈(LC)；和 1) A first antibody or antigen-binding fragment thereof that specifically binds to a first antigen, the first antibody or antigen-binding fragment thereof including a heavy chain (HC) and a light chain (LC); and

2)特異性結合第二抗原的包含重鏈可變區(VH)和輕鏈可變區(VL)的抗體片段； 2) An antibody fragment containing a heavy chain variable region (VH) and a light chain variable region (VL) that specifically binds to the second antigen;

其中，該抗體片段連接於第一抗體的重鏈或輕鏈的N端或C端； Wherein, the antibody fragment is connected to the N-terminus or C-terminus of the heavy chain or light chain of the first antibody;

該第一抗原為4-1BB，並且該第二抗原為PD-L1；或，該第一抗原為PD-L1，並且該第二抗原為4-1BB。 The first antigen is 4-1BB and the second antigen is PD-L1; or, the first antigen is PD-L1 and the second antigen is 4-1BB.

在一些實施方案中，該抗體片段為Fv、scFv或di-scFv。在一些具體實施方案中，該抗體片段為scFv。 In some embodiments, the antibody fragment is Fv, scFv, or di-scFv. In some specific embodiments, the antibody fragment is a scFv.

在一些實施方案中，該雙特異性抗體分子包含1個該第一抗體或其抗原結合片段和2個該scFv；並且，該第一抗體或其抗原結合片段包括兩條HC和兩條LC，其中該第一抗體或其抗原結合片段的一條HC的重鏈可變區(VH)與一條LC的輕鏈可變區(VL)形成抗原結合部位，另一條HC的重鏈可變區(VH)與另一條LC的輕鏈可變區(VL)形成抗原結合部位。 In some embodiments, the bispecific antibody molecule comprises one of the first antibody or antigen-binding fragment thereof and two of the scFv; and, the first antibody or antigen-binding fragment thereof It includes two HCs and two LCs, where the heavy chain variable region (VH) of one HC of the first antibody or its antigen-binding fragment and the light chain variable region (VL) of one LC form an antigen binding site, and the other The variable region of the heavy chain (VH) of HC and the variable region of the light chain (VL) of another LC form an antigen binding site.

在一些實施方案中，該第一抗體或其抗原結合片段為IgG型抗體，更佳地，為IgG1、IgG2、IgG3、IgG4型抗體，更佳地，為IgG1型抗體。 In some embodiments, the first antibody or antigen-binding fragment thereof is an IgG type antibody, more preferably, an IgG1, IgG2, IgG3, IgG4 type antibody, and more preferably, an IgG1 type antibody.

在一些實施方案中，一個該scFv連接於該第一抗體或其抗原結合片段的一條重鏈或輕鏈的N端，另一個該scFv連接於該第一抗體或其抗原結合片段的另一條重鏈或輕鏈的C端。 In some embodiments, one scFv is connected to the N-terminus of a heavy chain or light chain of the first antibody or antigen-binding fragment thereof, and the other scFv is connected to the other heavy chain of the first antibody or antigen-binding fragment thereof. The C-terminus of the chain or light chain.

在一些實施方案中，兩個該scFv分別連接於該第一抗體或其抗原結合片段的兩條重鏈或兩條輕鏈的N端或C端。在一些具體實施方案中，兩個該scFv分別連接於該第一抗體或其抗原結合片段的兩條重鏈的N端。在一些具體實施方案中，每個該scFv分別連接於該第一抗體或其抗原結合片段的兩條重鏈的C端。 In some embodiments, two of the scFv are respectively linked to the N-terminus or C-terminus of the two heavy chains or the two light chains of the first antibody or antigen-binding fragment thereof. In some specific embodiments, two of the scFv are respectively attached to the N-terminus of the two heavy chains of the first antibody or antigen-binding fragment thereof. In some specific embodiments, each of the scFv is respectively linked to the C-terminus of the two heavy chains of the first antibody or antigen-binding fragment thereof.

在一些具體實施方案中，第一抗原為PD-L1，第二抗原為4-1BB，且該結合第二抗原的scFv連接於該第一抗體或其抗原結合片段的重鏈的N端或C端。在一些具體實施方案中，第一抗原為4-1BB，第二抗原為PD-L1，且該結合第二抗原的scFv連接於該第一抗體或其抗原結合片段的重鏈的N端或C端。 In some specific embodiments, the first antigen is PD-L1, the second antigen is 4-1BB, and the scFv that binds to the second antigen is linked to the N-terminal or C of the heavy chain of the first antibody or antigen-binding fragment thereof. end. In some specific embodiments, the first antigen is 4-1BB, the second antigen is PD-L1, and the scFv that binds the second antigen is connected to the N-terminus or C of the heavy chain of the first antibody or antigen-binding fragment thereof. end.

在一些實施方案中，兩個該scFv分別連接於該第一抗體或其抗原結合片段的兩條輕鏈的N端。在一些具體實施方案中，兩個該scFv分別連接於該第一抗體或其抗原結合片段的兩條輕鏈的C端。在一些具體實施方案中，一個該scFv連接於該第一抗體或其抗原結合片段的一條重鏈或輕鏈的N端，另一個該scFv連接於該第一抗體或其抗原結合片段的另一條重鏈或輕鏈的C端。 In some embodiments, two of the scFv are respectively attached to the N-terminus of the two light chains of the first antibody or antigen-binding fragment thereof. In some specific embodiments, two of the scFv are respectively attached to the C-terminus of the two light chains of the first antibody or antigen-binding fragment thereof. In some embodiments, one of the scFv is linked to a heavy chain of the first antibody or antigen-binding fragment thereof Or the N-terminus of the light chain, and the other scFv is connected to the C-terminus of the other heavy chain or light chain of the first antibody or its antigen-binding fragment.

在一些實施方案中，當該第一抗原或第二抗原是4-1BB時，該第一抗體或scFv包含分別如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3；和/或，分別如SEQ ID NO：4、SEQ ID NO：7和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3。 In some embodiments, when the first antigen or the second antigen is 4-1BB, the first antibody or scFv comprises those shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively. HCDR1, HCDR2, and HCDR3; and/or, LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 6, respectively.

在一些具體的實施方案中，根據本公開的雙特異性抗體包含： In some specific embodiments, the bispecific antibody according to the present disclosure comprises:

1)特異性結合第一抗原的第一抗體或其抗原結合片段，該第一抗體包括重鏈(HC)和輕鏈(LC)；和 1) A first antibody or antigen-binding fragment thereof that specifically binds to a first antigen, the first antibody comprising a heavy chain (HC) and a light chain (LC); and

2)特異性結合第二抗原的scFv；該雙特異性抗體包含1個該第一抗體或其抗原結合片段和2個該scFv；並且，該第一抗體或其抗原結合片段包括兩條HC和兩條LC，其中該第一抗體或其抗原結合片段的一條HC的VH與一條LC的VL形成抗原結合部位，另一條HC的VH與另一條LC的VL形成抗原結合部位；兩個該scFv分別連接於該第一抗體或其抗原結合片段的兩條重鏈的N端；或者，兩個該scFv分別連接於該第一抗體或其抗原結合片段的兩條重鏈的C端；該第一抗原為4-1BB，並且該第二抗原為PD-L1；或者該第一抗原為PD-L1，並且該第二抗原為4-1BB。 2) scFv that specifically binds to the second antigen; the bispecific antibody comprises one of the first antibody or its antigen-binding fragment and two of the scFv; and, the first antibody or its antigen-binding fragment includes two HCs and Two LCs, where the VH of one HC of the first antibody or its antigen-binding fragment and the VL of one LC form the antigen binding site, and the VH of the other HC and the VL of the other LC form the antigen binding site; the two scFvs are respectively Is connected to the N-terminus of the two heavy chains of the first antibody or its antigen-binding fragment; or, two scFv are respectively connected to the C-terminus of the two heavy chains of the first antibody or its antigen-binding fragment; The antigen is 4-1BB and the second antigen is PD-L1; or the first antigen is PD-L1 and the second antigen is 4-1BB.

在一些具體實施方案中，該scFv的VH與VL藉由連接子1連接。在一些具體實施方案中，該兩個scFv藉由連接子2分別與該第一抗體或其抗原結合片段的兩條重鏈的N端或C端連接。在一些具體實施方案中，該scFv的結構為NH₂-VL-連接子1-VH-COOH或NH₂-VH-連接子1-VL-COOH。 In some embodiments, the VH and VL of the scFv are connected by linker 1. In some specific embodiments, the two scFvs are respectively connected to the N-terminus or C-terminus of the two heavy chains of the first antibody or antigen-binding fragment thereof via linker 2. In some specific embodiments, the structure of the scFv is NH ₂ -VL-linker 1-VH-COOH or NH ₂ -VH-linker 1-VL-COOH.

在一些具體實施方案中，該第一抗體或其抗原結合片段的重鏈包含重鏈可變區(VH)和CH1結構域，並且該輕鏈包含輕鏈可變區(VL)和輕鏈恆定區(CL)。該第一抗體的抗原結合片段可以為Fab片段、Fab’片段或F(ab’)2片段。在一些具體實施方案中，該第一抗體或其抗原結合片段的重鏈包含重鏈可變區(VH)和重鏈恆定區(CH)，並且該輕鏈包含輕鏈可變區(VL)和輕鏈恆定區(CL)。該第一抗體可以為全長抗體。 In some specific embodiments, the heavy chain of the first antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a CH1 domain, and the light chain comprises a light chain variable region (VL) and a light chain constant District (CL). The antigen-binding fragment of the first antibody may be a Fab fragment, Fab' fragment or F(ab')2 fragment. In some specific embodiments, the heavy chain of the first antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a heavy chain constant region (CH), and the light chain comprises a light chain variable region (VL) And the light chain constant region (CL). The first antibody may be a full-length antibody.

在一些具體實施方案中，該第一抗體或其抗原結合片段的重鏈為IgG同種型，例如IgG1、IgG2、IgG3或IgG4。在某些實施方案中，該第一抗體或其抗原結合片段的重鏈為人IgG1或IgG2同種型。在一些具體實施方案中，該第一抗體或其抗原結合片段的輕鏈為Kappa同種型。 In some specific embodiments, the heavy chain of the first antibody or antigen-binding fragment thereof is of the IgG isotype, such as IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the heavy chain of the first antibody or antigen-binding fragment thereof is of human IgG1 or IgG2 isotype. In some specific embodiments, the light chain of the first antibody or antigen-binding fragment thereof is of Kappa isotype.

在一些具體實施方案中，該第一抗體或其抗原結合片段的兩條HC包含相同的CDR；和/或，該第一抗體或其抗原結合片段的兩條LC包含相同的CDR。 In some specific embodiments, the two HCs of the first antibody or antigen-binding fragment thereof comprise the same CDR; and/or, the two LCs of the first antibody or the antigen-binding fragment thereof comprise the same CDR.

在一些具體實施方案中，該第一抗體或其抗原結合片段的兩條HC包含相同的VH；和/或，該第一抗體或其抗原結合片段的兩條LC包含相同的VL。 In some specific embodiments, the two HCs of the first antibody or antigen-binding fragment thereof comprise the same VH; and/or, the two LCs of the first antibody or antigen-binding fragment thereof comprise the same VL.

在一些具體實施方案中，該第一抗體或其抗原結合片段的兩條HC具有相同的胺基酸序列；和/或，該第一抗體或其抗原結合片段的兩條LC具有相同的胺基酸序列。 In some specific embodiments, the two HCs of the first antibody or antigen-binding fragment thereof have the same amino acid sequence; and/or, the two LCs of the first antibody or the antigen-binding fragment thereof have the same amino acid sequence. Acid sequence.

在一些具體實施方案中，兩個該scFv具有相同或不相同的胺基酸序列。在一些具體實施方案中，兩個該scFv具有相同的胺基酸序列。 In some specific embodiments, two such scFvs have the same or different amino acid sequences. In some specific embodiments, two such scFvs have the same amino acid sequence.

在一些實施方案中，雙特異性抗體包含兩條第一多肽鏈和兩條第二多肽鏈，其中： In some embodiments, the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, wherein:

a)該兩條第一多肽鏈各自獨立地包含該第一抗體或其抗原結合片段的重鏈(HC)和該scFv；和 a) the two first polypeptide chains each independently comprise the heavy chain (HC) of the first antibody or antigen-binding fragment thereof and the scFv; and

b)該兩條第二多肽鏈各自獨立地包含該第一抗體或其抗原結合片段的輕鏈(LC)； b) the two second polypeptide chains each independently comprise the light chain (LC) of the first antibody or antigen-binding fragment thereof;

其中，該scFv藉由連接子2與該第一抗體或其抗原結合片段的HC的N端或C端相連。 Wherein, the scFv is connected to the N-terminus or C-terminus of the HC of the first antibody or its antigen-binding fragment via linker 2.

在一些具體實施方案中，該雙特異性抗體包含兩條第一多肽鏈和兩條第二多肽鏈，其中： In some specific embodiments, the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, wherein:

i)該兩條第一多肽鏈各自獨立地包含該第一抗體或其抗原結合片段的輕鏈(LC)和該scFv；和 i) the two first polypeptide chains each independently comprise the light chain (LC) of the first antibody or antigen-binding fragment thereof and the scFv; and

ii)該兩條第二多肽鏈各自獨立地包含該第一抗體或其抗原結合片段的重鏈(HC)； ii) The two second polypeptide chains each independently comprise the heavy chain (HC) of the first antibody or antigen-binding fragment thereof;

其中，該scFv藉由連接子2與該第一抗體或其抗原結合片段的LC的N端或C端相連。 Wherein, the scFv is connected to the N-terminus or C-terminus of the LC of the first antibody or its antigen-binding fragment via linker 2.

在一些具體實施方案中，該scFv具有結構：NH₂-VH-連接子1-VL-COOH或NH₂-VL-連接子1-VH-COOH。 In some specific embodiments, the scFv has the structure: NH ₂ -VH-linker 1-VL-COOH or NH ₂ -VL-linker 1-VH-COOH.

在一些具體實施方案中，該連接子1和/或連接子2為肽接頭，例如具有如(G_mS_n)_x所示的胺基酸序列，其中m、n各自獨立地選自1-8的整數(例如，1、2、3、4、5、6、7或8)，x獨立地選自1-20的整數(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)。在一些具體實施方案中，該連接子1和/或連接子2具有如(G₄S)_x所示的胺基酸序列，x獨立地選自1-6的整數。 In some specific embodiments, the linker 1 and/or linker 2 is a peptide linker, for example, having _{an amino acid sequence as shown in (G m} S _n ) _x , wherein m and n are each independently selected from 1- 8 is an integer (e.g., 1, 2, 3, 4, 5, 6, 7 or 8), x is independently selected from an integer of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some specific embodiments, the linker 1 and/or linker 2 has _{an amino acid sequence as shown in (G 4} S) _x , and x is independently selected from an integer of 1-6.

在一些具體實施方案中，該連接子1的胺基酸序列為(G₄S)₃(即“GGGGSGGGGSGGGGS”)，和/或連接子2的胺基酸序列為(G₄S)₄(即“GGGGSGGGGSGGGGSGGGGS”)。 In some specific embodiments, the amino acid sequence of the linker 1 is (G ₄ S) ₃ (ie "GGGGSGGGGSGGGGS"), and/or the amino acid sequence of the linker 2 is (G ₄ S) ₄ (ie "GGGGSGGGGSGGGGSGGGGS").

在一些具體實施方案中，該scFv的VH與VL之間存在二硫鍵。在抗體的VH和VL之間引入二硫鍵的方法是本領域熟知的(例如參見US5747654；Rajagopal等，Prot.Engin.10(1997)1453-1459；Reiter等，Nature Biotechnology.14(1996)1239-1245；Reiter等，Protein Engineering.8(1995)1323-1331；Webber等，Molecular Immunology.32(1995)249-258；Reiter等，Immunity.2(1995)281-287；Reiter等，JBC.269(1994)18327-18331；Reiter等，Inter.J.of Cancer.58(1994)142-149；Reiter等，Cancer Res.54(1994)2714-2718，其藉由全文引用併入本文)。 In some specific embodiments, there is a disulfide bond between the VH and VL of the scFv. The method of introducing disulfide bonds between the VH and VL of an antibody is well known in the art (see, for example, US5747654; Rajagopal et al., Prot. Engin. 10 (1997) 1453-1459; Reiter et al., Nature Biotechnology. 14 (1996) 1239). -1245; Reiter et al., Protein Engineering. 8 (1995) 1323-1331; Webber et al., Molecular Immunology. 32 (1995) 249-258; Reiter et al., Immunity. 2 (1995) 281-287; Reiter et al., JBC.269 (1994) 18327-18331; Reiter et al., Inter. J. of Cancer. 58 (1994) 142-149; Reiter et al., Cancer Res. 54 (1994) 2714-2718, which is incorporated herein by reference in its entirety).

在一些具體的實施方案中，當該第一抗體或scFv結合的是4-1BB時，其包含分別如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3；和/或，分別如SEQ ID NO：4、SEQ ID NO：7和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3。 In some specific embodiments, when the first antibody or scFv binds to 4-1BB, it comprises HCDR1 and HCDR2 as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively. And HCDR3; and/or, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 6, respectively.

在一些實施方案中，該第一抗體或其抗原結合片段特異性結合4-1BB，並且該scFv特異性結合PD-L1，其中： In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds 4-1BB, and the scFv specifically binds PD-L1, wherein:

該第一抗體或其抗原結合片段包含： The first antibody or antigen-binding fragment thereof comprises:

如SEQ ID NO：1、2、3所示的HCDR1、HCDR2、HCDR3；如SEQ ID NO：4所示的LCDR1，如SEQ ID NO：5或7所示的LCDR2；以及，如SEQ ID NO：6所示的LCDR3。 HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 1, 2, 3; LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5 or 7; and, as SEQ ID NO: 6 shown in LCDR3.

在一些實施方案中，該第一抗體或其抗原結合片段特異性結合PD-L1，並且該scFv特異性結合4-1BB，其中，該第一抗體或其抗原結合片段包含： In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds PD-L1, and the scFv specifically binds to 4-1BB, wherein the first antibody or antigen-binding fragment thereof comprises:

如SEQ ID NO：19、20、21所示的HCDR1、HCDR2、HCDR3；如SEQ ID NO：22、23、24所示的LCDR1、LCDR2、LCDR3。 HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 19, 20, 21; LCDR1, LCDR2, LCDR3 shown in SEQ ID NO: 22, 23, 24.

在一些實施方案中，該第一抗體或其抗原結合片段特異性結合4-1BB，並且該scFv特異性結合PD-L1，其中，該scFv包含： In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds 4-1BB, and the scFv specifically binds PD-L1, wherein the scFv comprises:

在一些實施方案中，該第一抗體或其抗原結合片段特異性結合PD-L1，並且該scFv特異性結合4-1BB，其中，該scFv包含： In some embodiments, the first antibody or antigen-binding fragment thereof specifically binds PD-L1, and the scFv specifically binds 4-1BB, wherein the scFv comprises:

如SEQ ID NO：1所示的HCDR1，如SEQ ID NO：2所示的HCDR2，如SEQ ID NO：3所示的HCDR3；如SEQ ID NO：4所示的LCDR1，如SEQ ID NO：5或7所示的LCDR2；以及，如SEQ ID NO：6所示的LCDR3。 The HCDR1 shown in SEQ ID NO: 1, the HCDR2 shown in SEQ ID NO: 2, the HCDR3 shown in SEQ ID NO: 3; the LCDR1 shown in SEQ ID NO: 4, such as SEQ ID NO: 5 Or LCDR2 shown in 7; and LCDR3 shown in SEQ ID NO:6.

在一些具體實施方案中，該第一抗體或其抗原結合片段特異性結合4-1BB，並且該scFv特異性結合PD-L1，其中，該第一抗體或其抗原結合片段包含： In some specific embodiments, the first antibody or antigen-binding fragment thereof specifically binds to 4-1BB, and the scFv specifically binds to PD-L1, wherein the first antibody or antigen-binding fragment thereof comprises:

如SEQ ID NO：1、2、3所示的HCDR1、HCDR2、HCDR3；如SEQ ID NO：4、7、6所示的LCDR1、LCDR2、LCDR3；和，該scFv包含： HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 1, 2, and 3; LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 4, 7, and 6; and, the scFv includes:

在一些具體實施方案中，該第一抗體或其抗原結合片段的重鏈可變區(VH)的胺基酸序列與SEQ ID NO：8所示的重鏈可變區的胺基酸序列具有至少80%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性；並且，該第一抗體或其抗原結合片段的輕鏈可變區(VL)的胺基酸序列與SEQ ID NO：9所示的輕鏈可變區的胺基酸序列具有至少80%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性；和，該scFv的重鏈可變區(VH)的胺基酸序列與SEQ ID NO：25所示的重鏈可變區的胺基酸序列具有至少80%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性；並且，該scFv的的輕鏈可變區(VL)的胺基酸序列與SEQ ID NO：26所示的輕鏈可變區的胺基酸序列具有至少80%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性。 In some specific embodiments, the amino acid sequence of the heavy chain variable region (VH) of the first antibody or antigen-binding fragment thereof is the same as the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 8 The sequence has at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Sequence identity; and, the amino acid sequence of the light chain variable region (VL) of the first antibody or its antigen-binding fragment and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 9 have at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity And, the amino acid sequence of the heavy chain variable region (VH) of the scFv and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 25 have at least 80%, at least 90%, or at least 91% , At least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and, the light chain of the scFv may The amino acid sequence of the variable region (VL) and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 26 have at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在一些實施方案中，該第一抗體或其抗原結合片段的重鏈可變區選自SEQ ID NO：8所示胺基酸序列，和/或，輕鏈可變區選自SEQ ID NO：9所示胺基酸序列。在一些實施方案中，該第一抗體或其抗原結合片段的重鏈可變區選自SEQ ID NO：25所示胺基酸序列，和/或，輕鏈可變區選自SEQ ID NO：26所示胺基酸序列。 In some embodiments, the heavy chain variable region of the first antibody or antigen-binding fragment thereof is selected from the amino acid sequence shown in SEQ ID NO: 8, and/or the light chain variable region is selected from SEQ ID NO: 9 shows the amino acid sequence. In some embodiments, the heavy chain variable region of the first antibody or antigen-binding fragment thereof is selected from the amino acid sequence shown in SEQ ID NO: 25, and/or the light chain variable region is selected from SEQ ID NO: The amino acid sequence shown in 26.

在一些具體實施方案中，該第一抗體或其抗原結合片段的重鏈可變區選自SEQ ID NO：8所示胺基酸序列，輕鏈可變區選自SEQ ID NO：9所示的輕鏈可變區；和，該scFv的重鏈可變區選自SEQ ID NO：25所示胺基酸序列，輕鏈可變區選自SEQ ID NO：26所示的輕鏈可變區。 In some specific embodiments, the heavy chain variable region of the first antibody or antigen-binding fragment thereof is selected from the amino acid sequence shown in SEQ ID NO: 8, and the light chain variable region is selected from the group shown in SEQ ID NO: 9 And, the heavy chain variable region of the scFv is selected from the amino acid sequence shown in SEQ ID NO: 25, and the light chain variable region is selected from the light chain variable shown in SEQ ID NO: 26 Area.

在一些具體實施方案中，該第一抗體或其抗原結合片段的重鏈可變區選自SEQ ID NO：25所示胺基酸序列，輕鏈可變區選自SEQ ID NO：26所示的輕鏈可變區；和，該scFv的重鏈可變區選自SEQ ID NO：8所示胺基酸序列，輕鏈可變區選自SEQ ID NO：9所示的輕鏈可變區。 In some specific embodiments, the heavy chain variable region of the first antibody or antigen-binding fragment thereof is selected from the amino acid sequence shown in SEQ ID NO: 25, and the light chain variable region is selected from the group shown in SEQ ID NO: 26 And, the heavy chain variable region of the scFv is selected from the amino acid sequence shown in SEQ ID NO: 8, and the light chain variable region is selected from the light chain variable shown in SEQ ID NO: 9 Area.

在一些具體實施方案中，該雙特異性抗體包含兩條相同的第一多肽鏈和兩條相同的第二多肽鏈。 In some specific embodiments, the bispecific antibody comprises two identical first polypeptide chains and two identical second polypeptide chains.

在一些具體實施方案中，當第一抗體或scFv結合PD-L1時，該第一抗體或scFv可以是現有技術中已有的PD-L1抗體或其scFv片段，其生物仿製藥，及其抗原結合片段。例如，阿特珠單抗(商品名TECENTRIQTM)是針對PD-L1的IgG1同種型的完全人源化的Fc-修飾的單株抗體；MDX-1105，其為結合於PD-L1的完全人單株抗體；阿維魯單抗(MSB0010718C，Merck KGaA，Darmstadt，Germany&Pfizer)，其為同種型IgG1的完全人單株PD-L1抗體；度伐單抗(MedImmune/AstraZeneca)，其為Fc優化的抗PD-L1mAb。 In some specific embodiments, when the first antibody or scFv binds to PD-L1, the first antibody or scFv may be the PD-L1 antibody or its scFv fragments, its biosimilars, and its antigens in the prior art. Combine fragments. For example, atezolizumab (trade name TECENTRIQTM) is a fully humanized Fc-modified monoclonal antibody against the IgG1 isotype of PD-L1; MDX-1105, which is a fully human monoclonal antibody that binds to PD-L1 Strain antibody; Avirulumab (MSB0010718C, Merck KGaA, Darmstadt, Germany & Pfizer), which is a fully human monoclonal PD-L1 antibody of isotype IgG1; Duvazumab (MedImmune/AstraZeneca), which is an Fc-optimized antibody PD-L1mAb.

在一些具體實施方案中，該第一多肽鏈具有選自下列的胺基酸序列：SEQ ID NOs：12-13和15-17中任一項所示的胺基酸序列；和/或，該第二多肽鏈具有選自下列的胺基酸序列：SEQ ID NOs：14和18中任一項所示的胺基酸序列。 In some specific embodiments, the first polypeptide chain has an amino acid sequence selected from the group consisting of: SEQ ID NOs: the amino acid sequence shown in any one of 12-13 and 15-17; and/or, The second polypeptide chain has an amino acid sequence selected from the group consisting of: SEQ ID NOs: the amino acid sequence shown in any one of 14 and 18.

(1)如SEQ ID NO：12所示的第一多肽鏈和如SEQ ID NO：14所示的第二多肽鏈；或 (1) The first polypeptide chain shown in SEQ ID NO: 12 and the second polypeptide chain shown in SEQ ID NO: 14; or

(2)如SEQ ID NO：13所示的第一多肽鏈和如SEQ ID NO：14所示的第二多肽鏈；或 (2) The first polypeptide chain shown in SEQ ID NO: 13 and the second polypeptide chain shown in SEQ ID NO: 14; or

(3)如SEQ ID NO：15所示的第一多肽鏈和如SEQ ID NO：18所示的第二多肽鏈；或 (3) The first polypeptide chain shown in SEQ ID NO: 15 and the second polypeptide chain shown in SEQ ID NO: 18; or

(4)如SEQ ID NO：16所示的第一多肽鏈和如SEQ ID NO：18所示的第二多肽鏈；或 (4) The first polypeptide chain shown in SEQ ID NO: 16 and the second polypeptide chain shown in SEQ ID NO: 18; or

(5)如SEQ ID NO：17所示的第一多肽鏈和如SEQ ID NO：18所示的第二多肽鏈。 (5) The first polypeptide chain as shown in SEQ ID NO: 17 and the second polypeptide chain as shown in SEQ ID NO: 18.

在一些實施方案中，本公開的雙特異性抗體具有抗體依賴性細胞介導的細胞毒性(ADCC)活性，和/或，具有補體依賴的細胞毒性(CDC)活性。在一些具體實施方案中，本公開的雙特異性抗體具有降低的抗體依賴性細胞介導的細胞毒性(ADCC)活性，和/或具有降低的補體依賴性細胞毒性(CDC)活性。在一些具體實施方案中，該降低的ADCC和/或CDC活性是由雙特異性抗體的第一抗體CH發生突變導致。IgG1的Fc突變比如D265A、N297A、L234A/L235A、L234F/L235A、P329G等可以降低ADCC，P331S或附近的突變可降低CDC。IgG2的突變以及IgG2/4的Fc雜交抗體也可以降低ADCC和CDC。IgG4的核心鉸鏈區包含S228P突變，可增強核心鉸鏈區內的二硫鍵連接，從而阻止IgG4 Fab臂交換，大大減少了半分子抗體的形成，進一步引入F234A和L235A突變，此種形式的IgG4突變抗體改變CH2結構域，降低與Fc受體的相互作用而達到降低ADCC活性的效果。 In some embodiments, the bispecific antibodies of the present disclosure have antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and/or have complement-dependent cytotoxicity (CDC) activity. In some embodiments, the bispecific antibodies of the present disclosure have reduced antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and/or have reduced complement-dependent cytotoxicity (CDC) activity. In some embodiments, the reduced ADCC and/or CDC activity is caused by mutations in the first antibody CH of the bispecific antibody. Fc mutations of IgG1 such as D265A, N297A, L234A/L235A, L234F/L235A, P329G, etc. can reduce ADCC, and mutations in or near P331S can reduce CDC. Mutations of IgG2 and Fc hybrid antibodies of IgG2/4 can also reduce ADCC and CDC. The core hinge region of IgG4 contains the S228P mutation, which can strengthen the disulfide bond connection in the core hinge region, thereby preventing IgG4 Fab arm exchange, greatly reducing the formation of half-molecule antibodies, and further introducing F234A and L235A mutations, this form of IgG4 mutation The antibody changes the CH2 domain, reduces the interaction with the Fc receptor and achieves the effect of reducing ADCC activity.

一些實施方案中，本公開的雙特異性抗體對4-1BB和PD-L1具有親和力。在一些具體實施方案中，本公開的雙特異性抗體對4-1BB和PD-L1具有與其各自的親本抗體相比同等親和力。在一些具體實施方案中，本公開的雙特異性抗體對PD-L1具有與其親本抗體相比同等的親和力。在一些具體實施方案中，本公開的雙特異性抗體對4-1BB具有與其親本抗體相比同等的親和力。在一些具體實施方案中，本公開的雙特異性抗體對4-1BB具有與其親本抗體相比更弱的親和力。在一些具體實施方案中，本公開的雙特異性抗體對PD-L1具有與其親本抗體相比同等的親和力，對4-1BB具有與其親本抗體相比更弱的親和力。 In some embodiments, the bispecific antibodies of the present disclosure have affinity for 4-1BB and PD-L1. In some specific embodiments, the bispecific antibodies of the present disclosure have the same affinity for 4-1BB and PD-L1 as compared to their respective parent antibodies. In some specific embodiments, the bispecific antibody of the present disclosure has the same affinity for PD-L1 as compared to its parent antibody. In some specific embodiments, the bispecific antibodies of the present disclosure have affinity for 4-1BB. Compared with this antibody, the affinity is the same. In some specific embodiments, the bispecific antibody of the present disclosure has a weaker affinity for 4-1BB than its parent antibody. In some specific embodiments, the bispecific antibody of the present disclosure has the same affinity for PD-L1 as compared to its parent antibody, and has a weaker affinity for 4-1BB than its parent antibody.

另一方面，本公開的雙特異性抗體具有良好的熱穩定性。在一些具體實施方案中，本公開的雙特異性抗體具有與親本抗體相比基本相同的熱穩定性。 On the other hand, the bispecific antibody of the present disclosure has good thermal stability. In some specific embodiments, the bispecific antibodies of the present disclosure have substantially the same thermal stability as the parent antibody.

抗4-1BB抗體及其抗原結合片段、雙特異性抗體的表達 Expression of anti-4-1BB antibody, its antigen-binding fragment and bispecific antibody

本公開提供了一種分離的核酸分子，其包含編碼本公開的抗4-1BB抗體及其抗原結合片段的核苷酸序列。 The present disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the anti-4-1BB antibody and antigen-binding fragment thereof of the present disclosure.

本公開提供了一種分離的核酸分子，其包含編碼本公開的特異性結合4-1BB和PD-L1的雙特異性抗體的核苷酸序列。 The present disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific antibody of the present disclosure that specifically binds 4-1BB and PD-L1.

在一些具體實施方案中，該分離的核酸分子包含編碼本公開的第一多肽鏈的核苷酸序列，和/或包含編碼本公開的第二多肽鏈的核苷酸序列。在一些具體實施方案中，該分離的核酸分子包含編碼本公開的第一多肽鏈的核苷酸序列和編碼本公開的第二多肽鏈的核苷酸序列。 In some specific embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding the first polypeptide chain of the present disclosure, and/or comprises a nucleotide sequence encoding the second polypeptide chain of the present disclosure. In some specific embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a first polypeptide chain of the present disclosure and a nucleotide sequence encoding a second polypeptide chain of the present disclosure.

在另一個方面，本公開提供了一種載體(例如純株載體或表達載體)，其包含本公開的分離的核酸分子。在一些具體實施方案中，該載體包含編碼本公開的第一多肽鏈和/或第二多肽鏈的核苷酸序列。 In another aspect, the present disclosure provides a vector (e.g., a pure strain vector or an expression vector), which comprises the isolated nucleic acid molecule of the present disclosure. In some specific embodiments, the vector comprises a nucleotide sequence encoding the first polypeptide chain and/or the second polypeptide chain of the present disclosure.

在一些具體實施方案中，本公開的載體是例如質粒、黏粒、噬菌體等。在一些具體實施方案中，該載體能夠在受試者(例如哺乳動物，例如人)體內表達本公開的抗4-1BB抗體及其抗原結合片段、雙特異性抗體、第一多肽鏈或第二多肽鏈。 In some specific embodiments, the vectors of the present disclosure are, for example, plasmids, cosmids, bacteriophages, and the like. In some specific embodiments, the vector is capable of expressing the anti-4-1BB antibody and antigen-binding fragments thereof, bispecific antibody, first polypeptide chain or first polypeptide chain of the present disclosure in a subject (e.g., mammal, e.g., human). Two polypeptide chains.

在另一個方面，本公開提供了一種宿主細胞，其包含本公開的分離的核酸分子或本公開的載體。此類宿主細胞包括但不限於，原核細胞例如大腸桿菌細胞，以及真核細胞例如酵母細胞、昆蟲細胞、植物細胞和動物細胞(如哺乳動物細胞，例如小鼠細胞、人細胞等)。在一些具體實施方案中，本公開的宿主細胞是哺乳動物細胞，例如CHO(例如CHO-K1、CHO-S、CHO DG44)或HEK、HEK293、HEK-293F、Expi293F、PER.C6、NSO細胞或淋巴細胞。 In another aspect, the present disclosure provides a host cell comprising the isolated nucleic acid molecule of the present disclosure or the vector of the present disclosure. Such host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). In some specific embodiments, the host cell of the present disclosure is a mammalian cell, such as CHO (e.g. CHO-K1, CHO-S, CHO DG44) or HEK, HEK293, HEK-293F, Expi293F, PER.C6, NSO cell or Lymphocytes.

在另一個方面，本公開提供了製備本公開的抗4-1BB抗體及其抗原結合片段或本公開的雙特異性抗體的方法，其包括，在允許該抗4-1BB抗體及其抗原結合片段、或本公開的雙特異性抗體表達的條件下，培養本公開的宿主細胞，和從培養的宿主細胞培養物中回收該抗4-1BB抗體及其抗原結合片段、或本公開的雙特異性抗體。 In another aspect, the present disclosure provides a method for preparing the anti-4-1BB antibody and the antigen-binding fragment thereof of the present disclosure, or the bispecific antibody of the present disclosure, which includes allowing the anti-4-1BB antibody and the antigen-binding fragment thereof , Or under the condition that the bispecific antibody of the present disclosure is expressed, culture the host cell of the present disclosure, and recover the anti-4-1BB antibody and its antigen-binding fragment from the cultured host cell culture, or the bispecific antibody of the present disclosure Antibody.

在一些具體實施方案中，抗4-1BB抗體或其抗原結合片段的方法包括：構建包含編碼抗4-1BB抗體及其抗原結合片段的核苷酸序列的表達載體，轉化至宿主細胞並進行培養，從培養物中回收抗體或其抗原結合片段。 In some specific embodiments, the method for an anti-4-1BB antibody or an antigen-binding fragment thereof includes constructing an expression vector containing a nucleotide sequence encoding an anti-4-1BB antibody and an antigen-binding fragment thereof, transforming it into a host cell and culturing it , Recover the antibody or its antigen-binding fragment from the culture.

在一些具體實施方案中，製備本公開的雙特異性抗體方法包括： In some specific embodiments, the method of preparing the bispecific antibody of the present disclosure includes:

(1)構建包含編碼第一多肽鏈的核苷酸序列和編碼第二多肽鏈的核苷酸序列的表達載體； (1) Construct an expression vector comprising a nucleotide sequence encoding the first polypeptide chain and a nucleotide sequence encoding the second polypeptide chain;

(2)將步驟(1)中所述的表達載體轉化至宿主細胞； (2) Transform the expression vector described in step (1) into a host cell;

(3)在允許本公開的雙特異性抗體表達的條件下，培養步驟(2)中所述的宿主細胞；和 (3) Culturing the host cell described in step (2) under conditions that allow the expression of the bispecific antibody of the present disclosure; and

(4)從培養的宿主細胞培養物中回收該雙特異性抗體。 (4) The bispecific antibody is recovered from the culture of the cultured host cell.

可選地，製備本公開的雙特異性抗體方法包括： Optionally, the method for preparing the bispecific antibody of the present disclosure includes:

(1)構建包含編碼第一多肽鏈的核苷酸序列的第一表達載體和包含編碼第二多肽鏈的核苷酸序列的第二表達載體； (1) Constructing a first expression vector containing a nucleotide sequence encoding the first polypeptide chain and a second expression vector containing a nucleotide sequence encoding the second polypeptide chain;

(2)將步驟(1)中所述的第一表達載體和第二表達載體轉化至宿主細胞； (2) Transform the first expression vector and the second expression vector described in step (1) into host cells;

治療方法和醫藥組成物 Treatment method and medical composition

本公開提供一種醫藥組成物，其含有如上所述的抗4-1BB抗體或其抗原結合片段和可藥用的賦形劑、稀釋或載體。 The present disclosure provides a medical composition, which contains the aforementioned anti-4-1BB antibody or antigen-binding fragment thereof and a pharmaceutically acceptable excipient, dilution or carrier.

本公開提供一種治療或預防疾病的方法，包括給予受試者治療或預防有效量的前述抗4-1BB抗體或其抗原結合片段，或醫藥組成物，或抗體藥物偶聯物。在一些實施方案中，該疾病為癌症。在一些具體方案中，該癌症選自：黑色素瘤、乳腺癌、卵巢癌、***癌、胰腺癌、腎癌、肺癌、肝癌、胃癌、結腸直腸癌、膀胱癌、頭頸癌、甲狀腺癌、食管癌、宮頸癌、肉瘤、多發性骨髓瘤、白血病、淋巴癌、膽囊癌和膠質母細胞瘤。 The present disclosure provides a method for treating or preventing diseases, including administering to a subject a therapeutically or preventively effective amount of the aforementioned anti-4-1BB antibody or antigen-binding fragment thereof, or a pharmaceutical composition, or an antibody-drug conjugate. In some embodiments, the disease is cancer. In some specific scenarios, the cancer is selected from: melanoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, bladder cancer, head and neck cancer, thyroid cancer, esophageal cancer , Cervical cancer, sarcoma, multiple myeloma, leukemia, lymphoma, gallbladder cancer and glioblastoma.

另一方面，本公開提供了前述抗4-1BB抗體或其抗原結合片段在製備藥物中的用途；在一些具體的實施方案中，該藥物用於治療癌症。在另一些具體的實施方案中，該藥物用於治療黑色素瘤、乳腺癌、卵巢癌、***癌、胰腺癌、腎癌、肺癌、肝癌、胃癌、結腸直腸癌、膀胱癌、頭頸癌、甲狀腺癌、食管癌、宮頸癌、肉瘤、多發性骨髓瘤、白血病、淋巴癌、膽囊癌和膠質母細胞瘤。 On the other hand, the present disclosure provides the use of the aforementioned anti-4-1BB antibody or antigen-binding fragment thereof in the preparation of a medicament; in some specific embodiments, the medicament is used to treat cancer. In other specific embodiments, the drug is used to treat melanoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, bladder cancer, head and neck cancer, and thyroid cancer , Esophageal cancer, cervical cancer, sarcoma, multiple myeloma, leukemia, lymphoma, gallbladder cancer and glioblastoma.

本公開的雙特異性抗體可用於體外或在受試者體內中抑制4-1BB和PD-L1的活性，阻斷4-1BB和/或PD-L1信號通路，以及用於預防和/或治療與4-1BB和/或PD-L1相關的疾病(例如自身免疫性疾病或腫瘤或傳染病)。 The bispecific antibody of the present disclosure can be used to inhibit the activity of 4-1BB and PD-L1 in vitro or in a subject, block the 4-1BB and/or PD-L1 signaling pathway, and be used for prevention and/or treatment Diseases related to 4-1BB and/or PD-L1 (for example, autoimmune diseases or tumors or infectious diseases).

因此，在另一個方面，本公開提供了一種醫藥組成物，其含有本公開的雙特異性抗體，以及藥學上可接受的載體和/或賦形劑。在某些具體實施方案中，該醫藥組成物還可以包含另外的藥學活性劑。在某些具體實施方案中，該另外的藥學活性劑是用於預防和/或治療與4-1BB和/或PD-L1相關的疾病(例如自身免疫性疾病或腫瘤或傳染病)的藥物。 Therefore, in another aspect, the present disclosure provides a pharmaceutical composition, which contains the bispecific antibody of the present disclosure, and a pharmaceutically acceptable carrier and/or excipient. In certain embodiments, the pharmaceutical composition may also include additional pharmaceutically active agents. In certain specific embodiments, the additional pharmaceutically active agent is a drug for the prevention and/or treatment of diseases related to 4-1BB and/or PD-L1 (for example, autoimmune diseases or tumors or infectious diseases).

在另一個方面，本公開提供了本公開的雙特異性抗體或本公開的醫藥組成物在製備藥物中的用途，該藥物用於在受試者(例如人)中治療與4-1BB和/或PD-L1相關的疾病(如自身免疫性疾病或腫瘤或傳染病)，和/或用於體外或在受試者(例如人)體內抑制4-1BB和/或PD-L1的活性。 In another aspect, the present disclosure provides the use of the bispecific antibody of the present disclosure or the pharmaceutical composition of the present disclosure in the preparation of a medicament for the treatment of 4-1BB and/or in a subject (such as a human) Or PD-L1 related diseases (such as autoimmune diseases or tumors or infectious diseases), and/or used to inhibit the activity of 4-1BB and/or PD-L1 in vitro or in a subject (such as a human).

在另一個方面，本公開提供了一種用於在受試者(例如人)中預防和/或治療與4-1BB和/或PD-L1相關的疾病(如自身免疫性疾病或腫瘤或傳染病)，和/或用於體外或在受試者(例如人)體內抑制4-1BB和/或PD-L1的活性的方法，其中該方法包括，給有此需要的受試者施用有效量的本公開的雙特異性抗體，或者本公開的醫藥組成物。 In another aspect, the present disclosure provides a method for preventing and/or treating diseases related to 4-1BB and/or PD-L1 (such as autoimmune diseases or tumors or infectious diseases) in a subject (such as a human). ), and/or a method for inhibiting the activity of 4-1BB and/or PD-L1 in vitro or in a subject (such as a human), wherein the method includes administering an effective amount of The bispecific antibody of the present disclosure, or the pharmaceutical composition of the present disclosure.

在本公開中，該與4-1BB和/或PD-L1相關的疾病包括但不限於如自身免疫性疾病或腫瘤或傳染病病，例如腫瘤，包括但不限於：腺癌、白血病、淋巴瘤、黑色素瘤、肉瘤，或包括但不限於腎上腺、膽囊、骨、骨髓、腦、乳腺、膽管、胃腸道、心臟、腎臟、肝臟、肺、肌肉、卵巢、胰腺、副甲狀腺、陰莖、***、皮膚、唾液腺、脾臟、睾丸、胸腺、甲狀腺和子宮相關的腫瘤。 In the present disclosure, the diseases related to 4-1BB and/or PD-L1 include, but are not limited to, autoimmune diseases or tumors or infectious diseases, such as tumors, including but not limited to: adenocarcinoma, leukemia, lymphoma , Melanoma, sarcoma, or including but not limited to adrenal gland, gallbladder, bone, bone marrow, brain, breast, bile duct, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, skin , Salivary glands, spleen, testes, thymus, thyroid and uterus related tumors.

本公開的抗4-1BB抗體或其抗原結合片段、雙特異性抗體或者本公開的醫藥組成物可以配製成醫學領域已知的任何劑型，較佳注射劑(包括注射液、注射用無菌粉末與注射用濃溶液)、吸入劑、噴霧劑等。較佳劑型取決於預期的給藥方式和治療用途。本公開的醫藥組成物應當是無菌的並在生產和儲存條件下穩定。一種具體劑型是注射劑。注射劑中可以含有緩衝劑，其濃度可以是1-100mM。此外，可以將無菌注射溶液製備為無菌凍乾粉劑(例如，藉由真空乾燥或冷凍乾燥)以便於儲存和使用。此類無菌凍乾粉劑可在使用前分散於合適的載體中，例如無菌無熱原水。 The anti-4-1BB antibody or its antigen-binding fragment, bispecific antibody or the pharmaceutical composition of the present disclosure can be formulated into any dosage form known in the medical field, preferably injections (including injections, sterile powder for injection, and Concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present disclosure should be sterile and stable under production and storage conditions. One specific dosage form is injection. The injection may contain a buffer, and its concentration may be 1-100 mM. In addition, the sterile injection solution can be prepared as a sterile lyophilized powder (for example, by vacuum drying or freeze drying) for storage and use. Such sterile lyophilized powder can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

此外，本公開的抗4-1BB抗體或其抗原結合片段、雙特異性抗體可以以單位劑量形式存在於醫藥組成物中，以便於施用。在某些實施方案中，該單位劑量可以為0.1-2000mg、1-500mg，較佳1-200mg，在該醫藥組成物液體的情況下，其可以包含濃度為1-500mg，較佳1-200mg的活性成分。在一些具體實施方式中，該醫藥組成物單位計量中可含有0.01至99重量%的本公開的4-1BB抗體、其抗原結合片段或雙特異性抗體。 In addition, the anti-4-1BB antibody or its antigen-binding fragment or bispecific antibody of the present disclosure may be present in a pharmaceutical composition in a unit dosage form for easy administration. In some embodiments, the unit dose may be 0.1-2000mg, 1-500mg, preferably 1-200mg, in the case of the pharmaceutical composition liquid, it may contain a concentration of 1-500mg, preferably 1-200mg Active ingredients. In some embodiments, the unit of the pharmaceutical composition may contain 0.01 to 99% by weight of the 4-1BB antibody, antigen-binding fragment or bispecific antibody of the present disclosure.

本公開的抗4-1BB抗體或其抗原結合片段、雙特異性抗體或醫藥組成物可以藉由本領域已知的任何合適的方法來施用，較佳地給藥途徑/方式是胃腸外給藥(例如靜脈注射，皮下注射，腹膜內注射，肌內注射)。技術人員應理解，給藥途徑和/或方式將根據預期目的而發生變化。在一個具體實施方案中，本公開的雙特異性抗體或醫藥組成物藉由靜脈輸注或注射給予。 The anti-4-1BB antibody or its antigen-binding fragment, bispecific antibody or pharmaceutical composition of the present disclosure can be administered by any suitable method known in the art, and the preferred route/mode of administration is parenteral administration ( Such as intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled person should understand that the route and/or manner of administration will vary according to the intended purpose. In a specific embodiment, the bispecific antibody or pharmaceutical composition of the present disclosure is administered by intravenous infusion or injection.

在本公開中，可調整給藥方案以獲得最佳目的反應(例如治療或預防反應)。例如，可以單次給藥，可以在一段時間內多次給藥，或者可以隨治療情況的緊急程度按比例減少或增加劑量。 In the present disclosure, the dosage regimen can be adjusted to obtain the best targeted response (e.g., therapeutic or preventive response). For example, it can be administered as a single dose, it can be administered multiple times over a period of time, or the dose can be reduced or increased proportionally to the urgency of the treatment situation.

在本公開中，受試者可以為哺乳動物，例如人。 In the present disclosure, the subject may be a mammal, such as a human.

檢測/診斷方法和試劑盒 Testing/diagnostic methods and kits

本公開的雙特異性抗體能夠特異性結合4-1BB和/或PD-L1，從而可用於檢測4-1BB和/或PD-L1在樣品中的存在或其水平，以及診斷受試者是否患有與4-1BB和/或PD-L1相關的疾病(如自身免疫性疾病或腫瘤或傳染病)。 The bispecific antibody of the present disclosure can specifically bind to 4-1BB and/or PD-L1, so that it can be used to detect the presence or level of 4-1BB and/or PD-L1 in a sample, and to diagnose whether a subject is suffering from There are diseases related to 4-1BB and/or PD-L1 (such as autoimmune diseases or tumors or infectious diseases).

在另一個方面，本公開提供了一種試劑盒，其包括本公開的4-1BB抗體或其抗原結合片段，或雙特異性抗體。在一些實施方案中，4-1BB抗體或其抗原結合片段，或雙特異性抗體帶有可檢測的標記。在一些具體實施方案中，含有前述雙特異性抗體的試劑盒還包括第二抗體，其特異性識別本公開的雙特異性抗體的第一抗體或scFv。一些具體實施方案中，該第二抗體還包括可檢測的標記。該可檢測的標記可以是可藉由螢光、光譜、光化學、生物化學、免疫學、電學、光學或化學手段檢測的任何物質。此類標記能夠適用於免疫學檢測(例如，酶聯免疫測定法、放射免疫測定法、螢光免疫測定法、化學發光免疫測定法等)。這類標記是本領域熟知的，包括但不限於酶(例如，辣根過氧化物酶、鹼性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶等)、放射性核素(例如，3H、125I、35S、14C或32P)、螢光染料(例如，異硫氰酸螢光素(FITC)、螢光素、異硫氰酸四甲基羅丹明(TRITC)、藻紅蛋白(PE)、德克薩斯紅、羅丹明、量子點或花菁染料衍生物(例如Cy7、Alexa 750))、吖啶酯類化合物、磁珠、測熱標記物例如膠體金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳膠等)珠、以及用於結合上述標記物修飾的親和素(例如，鏈黴親和素)的生物素。本公開中涵蓋的標記物可藉由本領域已知的方法檢測。例如，放射性標記可使用攝影膠片或閃爍計算器檢測，螢光標記物可使用光檢測器檢測，以檢測發射的光。酶標記物一般藉由給酶提供受質及檢測藉由酶對受質的作用產生的反應產物來檢測，及測熱標記物藉由簡單可視化著色標記物來檢測。在一些實施方案中，可藉由不同長度的接頭將如上該的可檢測的標記連接至本公開的雙特異性抗體，以降低潛在的位阻。 In another aspect, the present disclosure provides a kit, which includes the 4-1BB antibody of the present disclosure or an antigen-binding fragment thereof, or a bispecific antibody. In some embodiments, the 4-1BB antibody or antigen-binding fragment thereof, or bispecific antibody bears a detectable label. In some specific embodiments, the kit containing the aforementioned bispecific antibody further includes a second antibody, which specifically recognizes the first antibody or scFv of the bispecific antibody of the present disclosure. In some embodiments, the second antibody further includes a detectable label. The detectable label can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels can be applied to immunological detection (for example, enzyme-linked immunoassay, radioimmunoassay, fluoroimmunoassay, chemiluminescence immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (for example, fluorescein isothiocyanate (FITC), luciferin, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE ), Texas red, rhodamine, quantum dots or cyanine dye derivatives (e.g. Cy7, Alexa 750)), acridine ester compounds, magnetic beads, calorimetric markers such as colloidal gold or colored glass or plastic ( For example, polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (for example, streptavidin) modified by the above-mentioned label. The markers covered in this disclosure can be detected by methods known in the art. For example, a radioactive label can be detected using photographic film or a scintillation calculator, and a fluorescent label can be detected using a light detector to detect the emitted light. Enzyme markers generally provide substrates for enzymes and detect the reaction products produced by the action of enzymes on substrates. Detection, and calorimetric markers are detected by simple visualization of colored markers. In some embodiments, the detectable label as described above can be connected to the bispecific antibody of the present disclosure by linkers of different lengths to reduce potential steric hindrance.

在另一個方面，本公開提供了檢測4-1BB和/或PD-L1在樣品中的存在或其水平的方法，其包括使用本公開的4-1BB抗體或其抗原結合片段、雙特異性抗體的步驟。在一個較佳地實施方案中，本公開的4-1BB抗體或其抗原結合片段、雙特異性抗體還帶有可檢測的標記。在另一個較佳地實施方案中，該方法還包括，使用帶有可檢測的標記的試劑來檢測本公開的4-1BB抗體或其抗原結合片段、雙特異性抗體段。該方法可以用於診斷目的，或者非診斷目的(例如，該樣品是細胞樣品，而非來自患者的樣品)。 In another aspect, the present disclosure provides a method for detecting the presence or level of 4-1BB and/or PD-L1 in a sample, which includes using the 4-1BB antibody or antigen-binding fragment thereof, or bispecific antibody of the present disclosure A step of. In a preferred embodiment, the 4-1BB antibody or its antigen-binding fragment or bispecific antibody of the present disclosure also bears a detectable label. In another preferred embodiment, the method further includes using a reagent with a detectable label to detect the 4-1BB antibody or its antigen-binding fragment or bispecific antibody fragment of the present disclosure. The method can be used for diagnostic purposes, or for non-diagnostic purposes (for example, the sample is a cell sample, not a sample from a patient).

在另一個方面，本公開提供了診斷受試者是否患有與4-1BB和/或PD-L1相關的疾病(如自身免疫性疾病或腫瘤或傳染病)的方法，其包括：使用本公開的雙特異性抗體檢測4-1BB和/或PD-L1在來自該受試者的樣品中的存在或其水平。在一個較佳地實施方案中，本公開的雙特異性抗體還帶有可檢測的標記。在另一個較佳地實施方案中，該方法還包括，使用帶有可檢測的標記的試劑來檢測本公開的4-1BB抗體或其抗原結合片段、雙特異性抗體的步驟。 In another aspect, the present disclosure provides a method for diagnosing whether a subject has a disease related to 4-1BB and/or PD-L1 (such as an autoimmune disease or tumor or an infectious disease), which includes: using the present disclosure The bispecific antibody detects the presence or level of 4-1BB and/or PD-L1 in a sample from the subject. In a preferred embodiment, the bispecific antibody of the present disclosure also bears a detectable label. In another preferred embodiment, the method further includes the step of using a reagent with a detectable label to detect the 4-1BB antibody or its antigen-binding fragment or bispecific antibody of the present disclosure.

在另一個方面，提供了本公開的4-1BB抗體或其抗原結合片段、雙特異性抗體在製備試劑盒中的用途，該試劑盒用於檢測4-1BB和/或PD-L1在樣品中的存在或其水平，或用於診斷受試者是否患有與4-1BB和/或PD-L1相關的疾病(如自身免疫性疾病或腫瘤或傳染病)。 In another aspect, there is provided the use of the 4-1BB antibody or its antigen-binding fragment or bispecific antibody of the present disclosure in the preparation of a kit for detecting 4-1BB and/or PD-L1 in a sample The presence or level of, or used to diagnose whether the subject has a disease related to 4-1BB and/or PD-L1 (such as autoimmune disease or tumor or infectious disease).

術語定義 Definition of Terms

為了更容易理解本公開，以下具體定義了某些技術和科學術語。除非在本文中另有明確定義，本文使用的所有其它技術和科學術語都具有本公開所屬領域的一般技術人員通常理解的含義。 To make it easier to understand the present disclosure, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which this disclosure belongs.

本公開所用胺基酸三字母代碼和單字母代碼如J.biol.chem，243，p3558(1968)中所述。 The three-letter codes and one-letter codes of amino acids used in the present disclosure are as described in J. biool. chem, 243, p3558 (1968).

本公開該的“抗體”指免疫球蛋白，通常是由兩條相同的重鏈和兩條相同的輕鏈藉由鏈間二硫鍵連接而成的四肽鏈結構。免疫球蛋白重鏈恆定區的胺基酸組成和排列順序不同，故其抗原性也不同。據此，可將免疫球蛋白分為五類，或稱為免疫球蛋白的同種型，即IgM、IgD、IgG、IgA和IgE，其相應的重鏈分別為μ鏈、δ鏈、γ鏈、α鏈、和ε鏈。同一類Ig根據其鉸鏈區胺基酸組成和重鏈二硫鍵的數目和位置的差別，又可分為不同的亞類，如IgG可分為IgG1、IgG2、IgG3、IgG4。輕鏈藉由恆定區的不同分為κ鏈或λ鏈。五類Ig中每類Ig都可以有κ(kappa)鏈或λ(lambda)鏈。 The "antibody" in the present disclosure refers to an immunoglobulin, which is usually a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are μ chain, δ chain, γ chain, Alpha chain, and epsilon chain. The same type of Ig can be divided into different subclasses according to the amino acid composition of its hinge area and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is classified into a kappa chain or a lambda chain by the difference in the constant region. Each of the five types of Ig can have a κ (kappa) chain or a λ (lambda) chain.

在本公開中，本公開所述的抗體輕鏈可變區可進一步包含輕鏈恆定區，該輕鏈恆定區包含人源或鼠源的κ、λ鏈或其變體。 In the present disclosure, the antibody light chain variable region described in the present disclosure may further comprise a light chain constant region comprising human or murine kappa, lambda chains or variants thereof.

在本公開中，本公開所述的抗體重鏈可變區可進一步包含重鏈恆定區，該重鏈恆定區包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其變體。 In the present disclosure, the antibody heavy chain variable region described in the present disclosure may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.

抗體重鏈和輕鏈靠近N端的約110個胺基酸的序列變化很大，為可變區(Fv區)；靠近C端的其餘胺基酸序列相對穩定，為恆定區。可變區包括3個高變區(HVR)和4個序列相對保守的骨架區(FR)。3個高變區決定抗體的特異性，又稱為互補性決定區(CDR)。每條輕鏈可變區(LCVR)和重鏈可變區(HCVR)由3個CDR區4個FR區組成，從胺基端到羧基端依次排列的順序為：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。輕鏈的3個CDR區指LCDR1、LCDR2、和LCDR3；重鏈的3個CDR區指HCDR1、HCDR2和HCDR3。 The sequence of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions. The variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. 3 high The variable region determines the specificity of the antibody and is also called the complementarity determining region (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions. The sequence from the amino end to the carboxyl end is FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

本公開中，“PD-L1”也被稱為“程序性死亡配體1(Programmed death-ligand 1)”、“程序性細胞死亡配體1(Programmed cell death ligand 1)”、“蛋白質PD-L1”、“PD-L1”、“PDL1”、“PDCDL1”、“hPD-L1”、“hPD-LI”、“CD274”及“B7-H1”，並且可互換的使用。人類，獼猴(食蟹猴)，非洲象，野豬和小鼠PD-L1序列可以藉由Genbank登錄號找到，分別為NP_054862.1、XP_005581836、XP_003413533、XP_005665023和NP_068693。 In the present disclosure, "PD-L1" is also referred to as "Programmed death-ligand 1", "Programmed cell death ligand 1", and "Protein PD-L1". L1", "PD-L1", "PDL1", "PDCDL1", "hPD-L1", "hPD-LI", "CD274" and "B7-H1", and can be used interchangeably. Human, macaque (cynomolgus monkey), African elephant, wild boar and mouse PD-L1 sequences can be found by Genbank accession numbers, which are NP_054862.1, XP_005581836, XP_003413533, XP_005665023 and NP_068693, respectively.

術語“抗體”不受任何特定的產生抗體的方法限制。例如，其包括，重組抗體、單株抗體和多株抗體。抗體可以是不同同種型的抗體，例如，IgG(例如，IgG1、IgG2、IgG3或IgG4亞型)、IgA1、IgA2、IgD、IgE或IgM抗體。 The term "antibody" is not limited by any specific method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and multi-strain antibodies. The antibodies may be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibodies.

本公開的抗體包括鼠源抗體、嵌合抗體、人源化抗體，較佳人源化抗體。 The antibodies of the present disclosure include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.

術語“鼠源抗體”在本公開中為如本領域知識和技能製備的對人PD-L1的單株抗體。製備時用PD-L1抗原注射試驗對象，然後分離表達具有所需序列或功能特性的抗體的融合瘤。在本公開一個較佳地實施方案中，該鼠源PD-L1抗體或其抗原結合片段，可進一步包含鼠源κ、λ鏈或其變體的輕鏈恆定區，或進一步包含鼠源IgG1、IgG2、IgG3或其變體的重鏈恆定區。 The term "murine antibody" in the present disclosure refers to a monoclonal antibody to human PD-L1 prepared according to the knowledge and skills in the art. During the preparation, the test subject is injected with the PD-L1 antigen, and then the fusion tumor expressing the antibody with the desired sequence or functional characteristics is isolated. In a preferred embodiment of the present disclosure, the murine PD-L1 antibody or antigen-binding fragment thereof may further comprise murine κ and λ chains Or the light chain constant region of a variant thereof, or further comprise the heavy chain constant region of murine IgG1, IgG2, IgG3 or a variant thereof.

術語“嵌合抗體(chimeric antibody)”，是將鼠源性抗體的可變區與人抗體的恆定區融合而成的抗體，可以減輕鼠源性抗體誘發的免疫應答反應。建立嵌合抗體，要先建立分泌鼠源性特異性單抗的融合瘤，然後從小鼠融合瘤細胞中選殖可變區基因，再如需要選殖人抗體的恆定區基因，將小鼠可變區基因與人恆定區基因連接成嵌合基因後***人載體中，最後在真核工業***或原核工業***中表達嵌合抗體分子。在一個實施方案中，前述4-1BB/PD-L1的雙特異性抗體包含人源IgG1，或者使用胺基酸突變後無ADCC(antibody-dependent cell-mediated cytotoxicity，抗體依賴的細胞介導的細胞毒作用)毒性的IgG4。 The term "chimeric antibody" is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody. To establish a chimeric antibody, it is necessary to first establish a fusion tumor that secretes a murine specific monoclonal antibody, and then select the variable region gene from the mouse fusion tumor cell, and then if the constant region gene of the human antibody needs to be selected, the mouse can The variable region gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system. In one embodiment, the aforementioned bispecific antibody of 4-1BB/PD-L1 comprises human IgG1, or ADCC (antibody-dependent cell-mediated cytotoxicity) without ADCC (antibody-dependent cell-mediated cytotoxicity) after amino acid mutation is used. Toxicity) Toxic IgG4.

術語“人源化抗體(humanized antibody)”，也稱為CDR移植抗體(CDR-grafted antibody)，是指將小鼠的CDR序列移植到人的抗體可變區框架，即不同類型的人種系抗體構架序列中產生的抗體。可以克服嵌合抗體由於攜帶大量小鼠蛋白成分，從而誘導的强烈的抗體可變抗體反應。此類構架序列可以從包括種系抗體基因序列的公共DNA數據庫或公開的參考文獻獲得。如人重鏈和輕鏈可變區基因的種系DNA序列可以在“VBase”人種系序列數據庫(在因特網www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991 Sequences of Proteins of Immunological Interest，第5版中找到。為避免免疫原性下降的同時，引起的活性下降，可對該人抗體可變區框架序列進行最少反向突變或回復突變，以保持活性。本公開的人源化抗體也包括進一步由噬菌體展示對CDR進行親和力成熟後的人源化抗體。 The term "humanized antibody", also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into the human antibody variable region framework, that is, different types of human germlines The antibody produced in the antibody framework sequence. It can overcome the strong variable antibody response induced by the chimeric antibody due to the large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of the human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/vbase), as well as in Kabat, EA, etc. Human, 1991 Sequences of Proteins of Immunological Interest, found in 5th edition. In order to avoid the decrease of immunogenicity and the resulting decrease in activity, the human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain activity. The humanized antibodies of the present disclosure also include humanized antibodies that have been further subjected to affinity maturation for CDR by phage display.

本公開中所述的“抗原結合片段”，指具有抗原結合活性的Fab、Fv、sFv、F(ab’)2、線性抗體、單鏈抗體、scFv、sdAb、sdFv、奈米抗體、肽抗體peptibody、結構域抗體和多特異性抗體(雙特異性抗體、diabody、triabody和tetrabody、串聯二-scFv、串聯三-scFv)。一些實施方案中，“抗原結合片段”包含本公開所述抗體的選自SEQ ID NO：1、2、3、4、5、6中的一個或多個CDR區；或包含選自SEQ ID NO：1、2、3、4、7、6的一個或多個CDR區；或包含選自SEQ ID NO：19、20、21、22、23、24的一個或多個CDR區。 The "antigen-binding fragments" mentioned in the present disclosure refer to Fab, Fv, sFv, F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nano-antibodies, peptide antibodies that have antigen-binding activity peptibody, domain antibody and multispecific antibody (bispecific antibody, diabody, triabody and tetrabody, tandem two-scFv, tandem three-scFv). In some embodiments, the "antigen-binding fragment" includes one or more CDR regions selected from SEQ ID NO: 1, 2, 3, 4, 5, and 6 of the antibody described in the present disclosure; or includes one or more CDR regions selected from SEQ ID NO : One or more CDR regions of 1, 2, 3, 4, 7, and 6; or comprising one or more CDR regions selected from SEQ ID NO: 19, 20, 21, 22, 23, and 24.

“Fv片段”是指含有抗體重鏈可變區和輕鏈可變區，但沒有恆定區，並具有全部抗原結合位點的最小抗體片段。一般地，Fv抗體還包含在VH和VL結構域之間的多肽接頭，且能夠形成抗原結合所需的結構。也可以用不同的連接物將兩個抗體可變區連接成一條多肽鏈，稱為單鏈抗體(single chain antibody)或單鏈Fv(scFv)。 "Fv fragment" refers to the smallest antibody fragment that contains the variable region of the heavy chain of the antibody and the variable region of the light chain, but does not have the constant region, and has all the antigen binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding. Different linkers can also be used to connect the variable regions of two antibodies into a polypeptide chain, which is called single chain antibody or single chain Fv (scFv).

術語“單鏈抗體”、“單鏈Fv”或“scFv”意指包含藉由接頭連接的抗體重鏈可變結構域(VH)和抗體輕鏈可變結構域(VL)的分子。此類scFv分子可具有一般結構：NH2-VL-接頭-VH-COOH或NH2-VH-接頭-VL-COOH。合適的現有技術接頭由重複的GGGGS胺基酸序列或其變體組成，例如使用1-4個重複的變體(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90：6444-6448)。可用於本公開的其他接頭由Alfthan等人(1995)，Protein Eng.8：725-731，Choi等人(2001)，Eur.J.Immuno 1.31：94-106，Hu等人(1996)，Cancer Res.56：3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293：41-56和Roovers等人(2001)，Cancer Immunol.描述。 The term "single chain antibody", "single chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) connected by a linker. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example using 1-4 repeated variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448 ). Other linkers that can be used in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immuno 1.31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.

術語“與PD-L1結合”，指能與人PD-L1相互作用。術語“與4-1BB結合”，指能與人4-1BB相互作用。本公開的術語“抗原結合位點”指抗原上不連續的，由本公開抗體或其抗原結合片段識別的三維空間位點。 The term "binding to PD-L1" refers to the ability to interact with human PD-L1. The term "binding to 4-1BB" refers to the ability to interact with human 4-1BB. The term "antigen-binding site" in the present disclosure refers to a discrete three-dimensional site on an antigen that is recognized by the antibody or antigen-binding fragment thereof of the present disclosure.

術語“親本抗體”是指，用於製備本公開的雙特異性抗體的抗PD-L1抗體或4-1BB抗體，該抗體所具有的胺基酸序列可藉由例如胺基酸置換或結構改變等方式以用於製備本公開的雙特異性抗體所包含的第一抗體或scFv。 The term "parent antibody" refers to the anti-PD-L1 antibody or 4-1BB antibody used to prepare the bispecific antibody of the present disclosure. The amino acid sequence of the antibody can be replaced by, for example, amino acid substitution or structure. Modifications and other methods are used to prepare the first antibody or scFv contained in the bispecific antibody of the present disclosure.

本公開的雙特異性抗體所包含的CDR、VH、VL、CH、CL、HC、LC還可以來自其他被本領域所知曉的能夠特異性結合PD-L1或4-1BB的抗體或其抗體片段替代，或者與上述已知抗體、其抗體片段或其CDR、VH、VL、CH、CL、HC、LC具有至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性的抗體。 The CDR, VH, VL, CH, CL, HC, and LC contained in the bispecific antibody of the present disclosure can also be derived from other antibodies or antibody fragments thereof known in the art that can specifically bind PD-L1 or 4-1BB Alternative, or with the above known antibody, its antibody fragment or its CDR, VH, VL, CH, CL, HC, LC with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% %, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity antibody.

“連接子”是指，由多個胺基酸殘基藉由肽鍵連接形成的線性多肽。本公開的連接子可以為人工合成的胺基酸序列，或天然存在的多肽序列，例如具有鉸鏈區功能的多肽。此類連接子多肽是本領域眾所周知的(參見例如，Holliger P.等(1993)Proc.Natl.Acad.Sci.USA.90：6444-6448；Poljak R.J.等(1994)Structure 2：1121-1123)。 "Linker" refers to a linear polypeptide formed by connecting multiple amino acid residues by peptide bonds. The linker of the present disclosure may be an artificially synthesized amino acid sequence, or a naturally-occurring polypeptide sequence, such as a polypeptide having a hinge region function. Such linker polypeptides are well known in the art (see, for example, Holliger P. et al. (1993) Proc. Natl. Acad. Sci. USA. 90: 6444-6448; Poljak RJ et al. (1994) Structure 2: 1121-1123) .

“特異性結合”是指，兩分子間的非隨機的結合反應，如抗體和其所針對的抗原之間的反應。在某些實施方式中，特異性結合某抗原的抗體(或對某抗原具有特異性的抗體)是指，抗體以小於大約10^-5M，例如小於大約10^-6M、10^-7M、10^-8M、10^-9M或10^-10M或更小的親和力(K_D) 結合該抗原。在本公開中，術語“K_D”是指特定抗體-抗原相互作用的解離平衡常數，其用於描述抗體與抗原之間的結合親和力。平衡解離常數越小，抗體-抗原結合越緊密，抗體與抗原之間的親和力越高。通常，抗體(例如，本公開的抗體)以小於大約10^-5M，例如小於大約10^-6M、10^-7M、10^-8M、10^-9M或10^-10M或更小的解離平衡常數(K_D)結合抗原(例如，HBsAg)，例如，如使用表面等離子體共振術(SPR)在BIACORE儀中測定的。術語“k_d”(sec^-1)是指特定抗體-抗原相互作用的解離速率常數，也稱為k off值。 "Specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. In some embodiments, an antibody that specifically binds to a certain antigen (or an antibody that is specific to a certain antigen) means that the antibody has a concentration of less than about 10 ^-5 M, for example, less than about 10 ^-6 M, 10 ^-7 M, The affinity (K _D ) of 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M or less binds to the antigen. In the present disclosure, the term "K _D "refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. Generally, an antibody (for example, an antibody of the present disclosure) is less than about 10 ^-5 M, such as less than about 10 ^-6 M, 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M or less The dissociation equilibrium constant (K _D ) binds to an antigen (e.g., HBsAg), for example, as determined in a BIACORE instrument using surface plasmon resonance (SPR). The term "k _d "(sec ^-1 ) refers to the dissociation rate constant of a specific antibody-antigen interaction, also known as the k off value.

“ADCC”即antibody-dependent cell-mediated cytotoxicity，抗體依賴的細胞介導的細胞毒作用，是指表達Fc受體的細胞藉由識別抗體的Fc段直接殺傷被抗體包被的靶細胞。可藉由對IgG上Fc段的修飾，降低或消除抗體的ADCC效應功能。該修飾指在抗體的重鏈恆定區進行突變，如選自IgG1的N297A、L234A、L235A、P329G；IgG2/4chimera，IgG4的F234A/L235A突變。 "ADCC" stands for antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cell-mediated cytotoxicity means that cells expressing Fc receptors directly kill target cells coated with antibodies by recognizing the Fc segment of antibodies. The ADCC effect function of antibodies can be reduced or eliminated by modifying the Fc segment of IgG. This modification refers to mutations in the constant region of the heavy chain of the antibody, such as N297A, L234A, L235A, P329G selected from IgG1; IgG2/4chimera, and F234A/L235A mutations of IgG4.

“CDC”即Complement-dependent cytotoxicity，補體依賴的細胞毒性，是指藉由使補體成分C1q與抗體Fc結合來激活補體級聯的細胞毒性形式。檢測抗體的ADCC、CDC活性的方法是本領域已知的，例如可藉由測定待測抗體與Fc受體(例如C1q)之間的結合活性來評價CDC。 "CDC" stands for Complement-dependent cytotoxicity. Complement-dependent cytotoxicity refers to a form of cytotoxicity that activates the complement cascade by binding complement component C1q to antibody Fc. Methods for detecting ADCC and CDC activities of antibodies are known in the art. For example, CDC can be evaluated by measuring the binding activity between the antibody to be tested and the Fc receptor (for example, C1q).

現有技術中熟知生產和純化抗體和抗原結合片段的方法，如冷泉港的抗體實驗技術指南，5-8章和15章。例如，老鼠可以用人PD-L1、4-1BB或其片段免疫，所得到的抗體能被覆性、純化，並且可以用常規的方法進行胺基酸測序。抗原結合片段同樣可以用常規方法製備。公開所述的抗體或其抗原結合片段用基因工程方法在非人源的CDR區加上一個或多個人源FR區。人FR種系序列可以藉由比對IMGT人類抗體可變區種系基因數據庫和MOE軟體，從ImMunoGeneTics(IMGT)的網站http：//imgt.cines.fr得到，或者從免疫球蛋白雜誌，2001ISBN012441351上獲得。 The methods for producing and purifying antibodies and antigen-binding fragments are well known in the prior art, such as Cold Spring Harbor’s Antibody Experiment Technical Guide, Chapters 5-8 and 15. For example, mice can be immunized with human PD-L1, 4-1BB or fragments thereof, and the obtained antibodies can be covered and purified, and amino acid sequencing can be performed by conventional methods. Antigen-binding fragments can also be prepared by conventional methods. The disclosed antibody or its antigen-binding fragment is genetically engineered to add one or more to the non-human CDR region Multiple human origin FR areas. The human FR germline sequence can be obtained by comparing the IMGT human antibody variable region germline gene database and MOE software, from the website of ImmunoGeneTics (IMGT) http://imgt.cines.fr, or from the Journal of Immunoglobulin, 2001ISBN012441351 obtain.

本公開中，工程化的抗體或其抗原結合片段可用常規方法製備和純化。比如，可以將編碼重鏈和/或輕鏈的cDNA序列，選殖並重組至GS表達載體。重組的免疫球蛋白表達載體可以穩定地轉染CHO細胞。作為一種更推薦的現有技術，哺乳動物類表達系統會導致抗體的糖基化，特別是在Fc區的高度保守N端位點。穩定表達抗體的陽性純株在生物反應器的無血清培養基中擴大培養以生產抗體。分泌了抗體的培養液可以用常規技術純化。比如，用含調整過的緩衝液的A或G Sepharose FF管柱進行純化。洗去非特異性結合的組分。再用PH梯度法洗脫結合的抗體，用SDS-PAGE檢測抗體片段，收集。抗體可用常規方法進行過濾濃縮。可溶的混合物和多聚體，也可以用常規方法去除，比如分子篩、離子交換。得到的產物需立即冷凍，如-70℃，或者凍乾。 In the present disclosure, the engineered antibody or antigen-binding fragment thereof can be prepared and purified by conventional methods. For example, the cDNA sequence encoding the heavy chain and/or light chain can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of antibodies, especially in the highly conserved N-terminal sites of the Fc region. Positive pure strains stably expressing antibodies are expanded in the serum-free medium of the bioreactor to produce antibodies. The culture medium from which the antibody is secreted can be purified by conventional techniques. For example, use A or G Sepharose FF column with adjusted buffer for purification. Wash away non-specifically bound components. Then the bound antibody was eluted by the PH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.

“給予”和“處理”當應用於動物、人、實驗受試者、細胞、組織、器官或生物流體時，是指外源性藥物、治療劑、診斷劑或組合物與動物、人、受試者、細胞、組織、器官或生物流體的接觸。“給予”和“處理”可以指例如治療、藥物代謝動力學、診斷、研究和實驗方法。細胞的處理包括試劑與細胞的接觸，以及試劑與流體的接觸，其中該流體與細胞接觸。“給予”和“處理”還意指藉由試劑、診斷、結合組合物或藉由另一種細胞體外和離體處理例如細胞。“處理”當應用於人、獸醫學或研究受試者時，是指治療處理、預防或預防性措施，研究和診斷應用。 "Administration" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions that interact with animals, humans, and recipients. Contact with subjects, cells, tissues, organs or biological fluids. "Administration" and "treatment" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. The treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells. "Administration" and "treatment" also mean by reagents, diagnostics, binding compositions or by another cell body Ex vivo and ex vivo treatments such as cells. "Treatment" when applied to human, veterinary or research subjects, refers to therapeutic treatment, preventive or preventive measures, research and diagnostic applications.

“治療”意指給予患者內用或外用治療劑，例如包含本公開的任一種結合化合物的組合物，該患者具有一種或多種疾病症狀，而已知該治療劑對這些症狀具有治療作用。通常，在受治療患者或群體中以有效緩解一種或多種疾病症狀的量給予治療劑，以誘導這類症狀退化或抑制這類症狀發展到任何臨床可測量的程度。有效緩解任何具體疾病症狀的治療劑的量(也稱作“治療有效量”)可如多種因素變化，例如患者的疾病狀態、年齡和體重，以及藥物在患者產生需要療效的能力。藉由醫生或其它專業衛生保健人士通常用於評價該症狀的嚴重性或進展狀況的任何臨床檢測方法，可評價疾病症狀是否已被減輕。儘管本公開的實施方案(例如治療方法或製品)在緩解每個目標疾病症狀方面可能無效，但是根據本領域已知的任何統計學檢驗方法如Student t檢驗、卡方檢驗、依據Mann和Whitney的U檢驗、Kruskal-Wallis檢驗(H檢驗)、Jonckheere-Terpstra檢驗和Wilcoxon檢驗確定，其在統計學顯著數目的患者中應當減輕目標疾病症狀。 "Treatment" means administering an internal or external therapeutic agent, such as a composition containing any one of the combination compounds of the present disclosure, to a patient who has one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population, to induce the regression of such symptoms or inhibit the development of such symptoms to any clinically measurable degree. The amount of the therapeutic agent effective to alleviate the symptoms of any particular disease (also referred to as a "therapeutically effective amount") can vary depending on various factors, such as the patient's disease state, age and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. By any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms, it can be evaluated whether the symptoms of the disease have been alleviated. Although the embodiments of the present disclosure (such as treatment methods or products) may not be effective in alleviating the symptoms of each target disease, according to any statistical test methods known in the art such as Student t test, chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients.

“保守修飾”或“保守置換或取代”是指具有類似特徵(例如電荷、側鏈大小、疏水性/親水性、主鏈構象和剛性等)的其它胺基酸置換蛋白中的胺基酸，使得可頻繁進行改變而不改變蛋白的生物學活性。本領域技術人員知曉，一般而言，多肽的非必需區域中的單個胺基酸置換基本上不改變生物學活性(參見例如Watson等(1987)Molecular Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224頁，(第4版))。另外，結構或功能類似的胺基酸的置換不大可能破環生物學活性。 "Conservative modification" or "conservative substitution or substitution" means that other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.) replace the amino acid in the protein, This allows frequent changes without changing the biological activity of the protein. Those skilled in the art know that, generally speaking, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co. ., p. 224, (4th edition)). In addition, the substitution of amino acids with similar structure or function is unlikely to disrupt biological activity.

“有效量”包含足以改善或預防醫學疾病的症狀或病症的量。有效量還意指足以允許或促進診斷的量。用於特定患者或獸醫學受試者的有效量可依據以下因素而變化：例如，待治療的病症、患者的總體健康情況、給藥的方法途徑和劑量以及副作用嚴重性。有效量可以是避免顯著副作用或毒性作用的最大劑量或給藥方案。 An "effective amount" includes an amount sufficient to improve or prevent the symptoms or conditions of a medical disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: for example, the condition to be treated, the patient's general health, the method of administration and dosage, and the severity of side effects. The effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.

“外源性”指根據情況在生物、細胞或人體外產生的物質。“內源性”指根據情況在細胞、生物或人體內產生的物質。 "Exogenous" refers to substances produced outside organisms, cells, or humans according to circumstances. "Endogenous" refers to substances produced in cells, organisms, or human bodies according to circumstances.

“同源性”或“同一性”是指兩個多核苷酸序列之間或兩個多肽之間的序列相似性。當兩個比較序列中的位置均被相同鹼基或胺基酸單體亞基佔據時，例如如果兩個DNA分子的每一個位置都被腺嘌呤佔據時，那麼該分子在該位置是同源的。兩個序列之間的同源性百分率是兩個序列共有的匹配或同源位置數除以比較的位置數×100的函數。例如，在序列最佳比對時，如果兩個序列中的10個位置有6個匹配或同源，那麼兩個序列為60%同源。一般而言，當比對兩個序列而得到最大的同源性百分率時進行比較。 "Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by adenine, then the molecule is homologous at that position of. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, in the optimal sequence alignment, if 6 of the 10 positions in the two sequences match or are homologous, then the two sequences will be 60% homologous. Generally speaking, the comparison is made when two sequences are aligned to obtain the greatest percentage of homology.

本文使用的表述“細胞”、“細胞系”和“細胞培養物”可互換使用，並且所有這類名稱都包括後代。因此，單詞“轉化體”和“轉化細胞”包括原代受試細胞和由其衍生的培養物，而不考慮轉移數目。還應當理解的是，由於故意或非有意的突變，所有後代在DNA含量方面不可能精確相同。包括具有與最初轉化細胞中篩選的相同的功能或生物學活性的突變後代。在意指不同名稱的情況下，其由上下文清楚可見。 As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such names include progeny. Therefore, the words "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell. Where a different name is meant, it is clearly visible from the context.

本文使用的“聚合酶鏈式反應”或“PCR”是指其中微量的特定部分的核酸、RNA和/或DNA如在例如美國專利號4,683,195中所述擴增的程序或技術。一般來說，需要獲得來自目標區域末端或之外的序列信息，使得可以設計寡核苷酸引子；這些引子在序列方面與待擴增模板的對應鏈相同或相似。2個引子的5’末端核苷酸可以與待擴增材料的末端一致。PCR可用於擴增特定的RNA序列、來自總基因組DNA的特定DNA序列和由總細胞RNA轉錄的cDNA、噬菌體或質粒序列等。一般參見Mullis等(1987)Cold Spring Harbor Symp.Ouant.Biol.51：263；Erlich編輯，(1989)PCR TECHNOLOGY(Stockton Press，N.Y.)。本文使用的PCR被視為用於擴增核酸測試樣品的核酸聚合酶反應法的一個實例，但不是唯一的實例，該方法包括使用作為引子的已知核酸和核酸聚合酶，以擴增或產生核酸的特定部分。 As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which a small amount of a specific portion of nucleic acid, RNA, and/or DNA is amplified as described in, for example, US Patent No. 4,683,195. In general, it is necessary to obtain sequence information from the end or outside of the target region so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5'terminal nucleotides of the two primers can be consistent with the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, etc. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). The PCR used herein is regarded as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample. The method includes the use of known nucleic acids and nucleic acid polymerases as primers to amplify or produce A specific part of a nucleic acid.

“視需要”或“視需要地”意味著隨後所描述地事件或環境可以但不必發生，該說明包括該事件或環境發生或不發生的場合。例如，“視需要包含1-3個抗體重鏈可變區”意味著特定序列的抗體重鏈可變區可以但不必須存在。 "As needed" or "as needed" means that the event or environment described later can but does not have to occur, and the description includes occasions where the event or environment occurs or does not occur. For example, "containing 1-3 antibody heavy chain variable regions as necessary" means that the antibody heavy chain variable region of a specific sequence may but does not have to be present.

“藥學上可接受的載體和/或賦形劑”是指在藥理學和/或生理學上與受試者和活性成分相容的載體和/或賦形劑，其是本領域公知的，包括但不限於：pH調節劑、表面活性劑、佐劑、離子強度增強劑、稀釋劑、維持滲透壓的試劑、延遲吸收的試劑、防腐劑。例如，pH調節劑包括但不限於磷酸鹽緩衝液。表面活性劑包括但不限於陽離子，陰離子或者非離子型表面活性劑，例如Tween-80。離子強度增強劑包括但不限於氯化鈉。防腐劑包括但不限於各種抗細菌試劑和抗真菌試劑，例如對羥苯甲酸酯、三氯第三丁醇、苯酚、山梨酸等。維持滲透壓的試劑包括但不限於糖、NaCl及其類似物。延遲吸收的試劑包括但不限於單硬脂酸鹽和明膠。 "Pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which is well known in the art, Including but not limited to: pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffer. Surfactants include but are not limited to cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include but are not limited to sodium chloride. Guard against Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid and the like. Agents for maintaining osmotic pressure include, but are not limited to, sugar, NaCl and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin.

“醫藥組成物”表示含有一種或多種本文所述化合物或其生理學上/可藥用的鹽或前體藥物與其他化學組分的混合物，該其他組分例如生理學/可藥用的載體和賦形劑。醫藥組成物的目的是促進對生物體的給藥，利於活性成分的吸收進而發揮生物活性。 "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiological/pharmaceutically acceptable carriers And excipients. The purpose of the medicinal composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.

本公開中，“受試者”是指哺乳動物，例如靈長類哺乳動物，例如人。在一些實施方式中，該受試者(例如人)患有與4-1BB和/或PD-L1相關的疾病，或者，具有患有上述疾病的風險。通常而言，這類疾病或疾病狀態的特徵是其將受益於4-1BB和/或PD-L1水平的降低或4-1BB和/或PD-L1活性的抑制從而得到緩解或治癒。 In the present disclosure, "subject" refers to a mammal, such as a primate mammal, such as a human. In some embodiments, the subject (e.g., human) suffers from a disease related to 4-1BB and/or PD-L1, or is at risk of suffering from the aforementioned diseases. Generally speaking, this type of disease or disease state is characterized in that it will benefit from a reduction in 4-1BB and/or PD-L1 levels or inhibition of 4-1BB and/or PD-L1 activity to be alleviated or cured.

與現有技術相比，本公開的技術方案至少具有以下有益效果： Compared with the prior art, the technical solution of the present disclosure has at least the following beneficial effects:

本公開中4-1BB抗體B1E7藉由引入N53Q突變，從而得到抗體HR137-07，使抗體親和力降低約2.8倍，降低抗原抗體的親和力，同時H137-07的激活活性增強。 In the present disclosure, the 4-1BB antibody B1E7 introduces the N53Q mutation to obtain the antibody HR137-07, which reduces the affinity of the antibody by about 2.8 times, reduces the affinity of the antigen and the antibody, and enhances the activation activity of H137-07.

此外，本公開的雙特異性抗體不僅能夠特異性識別/結合4-1BB和PD-L1，對4-1BB的親和力比其親本抗體減弱，在體外和受試者體內能夠顯著且同時激活4-1BB和抑制PD-L1的活性，激活4-1BB和阻斷PD-L1信號通路。而且該雙抗對人PD-L1蛋白的親和力比對人4-1BB蛋白高約50倍，這極大地保證了該雙抗在腫瘤部位的有效富集，另外也極大地降低了4-1BB端可能引起的毒副作用。由於9EN-FM具有PD-L1依賴的4-1BB靶點交聯，在細胞水平及體內實驗中，本公開的雙特異性抗體甚至展現出了明顯優於其親本抗體聯合用藥的活性和降低的抗體毒性反應，提升了藥物的安全性。因此，本公開的雙特異性抗體具有用於治療與4-1BB和PD-L1相關的疾病(如自身免疫性疾病或腫瘤或傳染病)的潛力，具有重大的臨床價值。 In addition, the bispecific antibody of the present disclosure can not only specifically recognize/bind 4-1BB and PD-L1, but also has a weaker affinity for 4-1BB than its parent antibody, and can significantly and simultaneously activate 4 in vitro and in the subject. -1BB and inhibit the activity of PD-L1, activate 4-1BB and block the PD-L1 signaling pathway. Moreover, the affinity of the double antibody to human PD-L1 protein is about 50 times higher than that of human 4-1BB protein, which greatly guarantees the effective enrichment of the double antibody at the tumor site, and also greatly It reduces the toxic and side effects that may be caused by the 4-1BB end. Because 9EN-FM has PD-L1-dependent 4-1BB target cross-linking, the bispecific antibody of the present disclosure even exhibits significantly better activity and reduction than its parental antibody combination drug in the cell level and in vivo experiments. The antibody toxicity reaction has improved the safety of the drug. Therefore, the bispecific antibody of the present disclosure has the potential to be used to treat diseases related to 4-1BB and PD-L1 (such as autoimmune diseases or tumors or infectious diseases), and has great clinical value.

圖1為抗體HR137-07的4-1BB/NF-κB信號通路激活實驗結果圖。 Figure 1 shows the results of the 4-1BB/NF-κB signal pathway activation experiment of the antibody HR137-07.

圖2A至圖2D為抗4-1BB/PD-L1雙特異性抗體結構示意圖。圖2A為8E的抗體結構示意圖；圖2B為8EN的抗體結構示意圖；圖2C為9E-FM的抗體結構示意圖；圖2D為9EN-FM和9EN-FM-1的抗體結構示意圖。 Figures 2A to 2D are schematic diagrams of the structure of the anti-4-1BB/PD-L1 bispecific antibody. Figure 2A is a schematic diagram of the antibody structure of 8E; Figure 2B is a schematic diagram of the antibody structure of 8EN; Figure 2C is a schematic diagram of the antibody structure of 9E-FM; Figure 2D is a schematic diagram of the antibody structure of 9EN-FM and 9EN-FM-1.

圖3為雙特異性抗體9EN-FM的SDS-PAGE電泳圖。 Figure 3 shows the SDS-PAGE electrophoresis of the bispecific antibody 9EN-FM.

圖4A為雙特異性抗體9EN-FM與PD-L1蛋白的結合ELISA檢測結果圖；圖4B為雙特異性抗體9EN-FM與4-1BB蛋白的結合ELISA檢測結果圖。 Figure 4A is a graph of the binding ELISA detection result of the bispecific antibody 9EN-FM and PD-L1 protein; Figure 4B is a graph of the binding ELISA detection result of the bispecific antibody 9EN-FM and 4-1BB protein.

圖5A為雙特異性抗體9EN-FM與4-1BB蛋白的親和力測定的SPR檢測結果圖；圖5B為9EN-FM與PD-L1蛋白的親和力測定的SPR檢測結果圖。 Fig. 5A is a graph of the SPR detection result of the affinity determination of the bispecific antibody 9EN-FM and 4-1BB protein; Fig. 5B is a graph of the SPR detection result of the affinity determination of 9EN-FM and PD-L1 protein.

圖6為雙特異性抗體9EN-FM的41BB-NFκB信號通路激活實驗的螢光素酶報告基因系統檢測結果圖。 Figure 6 is a diagram showing the detection results of the luciferase reporter gene system in the 41BB-NFκB signal pathway activation experiment of the bispecific antibody 9EN-FM.

圖7為螢光素酶報告系統檢測雙特異性抗體9EN-FM對PD-1/PD-L1信號通路封閉效果的實驗結果圖。 Figure 7 is a diagram showing the experimental results of the luciferase reporter system detecting the blocking effect of the bispecific antibody 9EN-FM on the PD-1/PD-L1 signaling pathway.

圖8為檢測雙特異性抗體9EM-FM對T淋巴細胞激活的實驗結果圖。 Figure 8 is a graph showing the experimental results of detecting the activation of T lymphocytes by the bispecific antibody 9EM-FM.

圖9為9EN-FM-1在Balbc PD-L1和4-1BB雙人源化小鼠上的抗腫瘤活性結果。 Figure 9 shows the results of anti-tumor activity of 9EN-FM-1 on Balbc PD-L1 and 4-1BB double-derived mice.

以下結合實施例用於進一步描述本公開，但這些實施例並非限制本公開的範圍。 The following examples are used to further describe the present disclosure, but these examples do not limit the scope of the present disclosure.

本公開實施例或測試例中未註明具體條件的實驗方法，通常按照常規條件，或按照原料或商品製造廠商所建議的條件。參見Sambrook等，分子選殖，實驗室手冊，冷泉港實驗室；當代分子生物學方法，Ausubel等著，Greene出版協會，Wiley Interscience，NY。未註明具體來源的試劑，為市場購買的常規試劑。 The experimental methods without specific conditions in the embodiments or test examples of the present disclosure usually follow the conventional conditions or the conditions suggested by the raw material or commodity manufacturers. See Sambrook et al., Molecular Selection, Laboratory Manual, Cold Spring Harbor Laboratory; Contemporary Molecular Biology Methods, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY. The reagents without specific sources are the conventional reagents purchased on the market.

實施例1. 抗4-1BB抗體的製備Example 1. Preparation of anti-4-1BB antibody

採用全基因合成的方法在抗4-1BB抗體B1E7(源自PCT/CN2019/072484)序列的輕鏈的CDR2部分引入N53Q突變，從而得到抗體HR137-07的序列。 The N53Q mutation was introduced into the CDR2 part of the light chain of the anti-4-1BB antibody B1E7 (derived from PCT/CN2019/072484) sequence using the method of full gene synthesis, thereby obtaining the sequence of the antibody HR137-07.

抗體HR137-07的製備過程包括： The preparation process of antibody HR137-07 includes:

重鏈載體設計如下：信號肽+突變的重鏈可變區序列+人的IgG1恆定區序列。輕鏈載體設計如下：信號肽+突變的輕鏈可變區序列+人的Kappa 恆定區序列。分別將上述序列***pCEP4載體。請第三方基因合成公司按照上述設計合成表達載體，得到載體質粒後，送測序驗證。將驗證合格的質粒用PEI轉染至人293F細胞中，連續培養，將293F細胞用無血清培養液(上海奧浦邁生物，OPM-293 CD03)培養至對數生長期，用於細胞轉染。將21.4μg人源化抗體輕鏈質粒和23.6μ人源化抗體重鏈質粒溶解在10mL Opti-MEM® I Reduced Serum Medium(GIBCO，31985-070)中混勻，然後加入200ug PEI，混勻，室溫孵育15min，加入50mL細胞中。細胞培養條件為：5% CO2，37℃，125rpm/min。培養期間，第1天和第3天加補料，直到細胞活率低於70%，收取細胞上清，離心過濾。將離心過濾後的細胞培養液上樣到抗體純化親和管柱，經磷酸緩衝液洗管柱，甘胺酸鹽酸緩衝液(pH2.7 0.1M Gly-HCl)洗脫，1M Tris鹽酸pH9.0中和，以及磷酸緩衝液透析，最終獲得純化的抗體。經SDS-PAGE和SEC-HPLC檢測，確認該抗體為HR137-07。 The heavy chain vector is designed as follows: signal peptide + mutant heavy chain variable region sequence + human IgG1 constant region sequence. The light chain vector is designed as follows: signal peptide + mutant light chain variable region sequence + human Kappa Constant region sequence. The above sequences were inserted into the pCEP4 vector. Ask a third-party gene synthesis company to synthesize the expression vector according to the above design, and send the vector plasmid for sequencing verification. The qualified plasmid was transfected into human 293F cells with PEI and cultured continuously, and the 293F cells were cultured with serum-free medium (Shanghai Optima Biotech, OPM-293 CD03) to the logarithmic growth phase for cell transfection. Dissolve 21.4μg of humanized antibody light chain plasmid and 23.6μ of humanized antibody heavy chain plasmid in 10mL Opti-MEM® I Reduced Serum Medium (GIBCO, 31985-070) and mix well, then add 200ug PEI and mix well. Incubate for 15 min at room temperature and add to 50 mL of cells. The cell culture conditions are: 5% CO2, 37°C, 125rpm/min. During the culture period, supplements were added on the 1st and 3rd day until the cell viability was less than 70%, and the cell supernatant was collected and centrifuged. The cell culture solution after centrifugation was loaded onto the antibody purification affinity column, the column was washed with phosphate buffer, and eluted with glycine acid buffer (pH 2.7 0.1M Gly-HCl), and 1M Tris hydrochloric acid, pH 9. 0 neutralization, and phosphate buffer dialysis, and finally obtain purified antibody. SDS-PAGE and SEC-HPLC test confirmed that the antibody is HR137-07.

B1E7的CDR序列如表1所示： The CDR sequence of B1E7 is shown in Table 1:

其中，HCDR為重鏈CDR，LCDR為輕鏈CDR。其中，CDR胺基酸殘基在數量和位置符合已知的Kabat編號規則(LCDR1-3，HCDR2-3)，或者符合Kabat和chothia的編號規則(HCDR1)，表2中CDR的編號規則與表1相同。 Among them, HCDR is the heavy chain CDR, and LCDR is the light chain CDR. Among them, the number and position of CDR amino acid residues comply with the known Kabat numbering rules (LCDR1-3, HCDR2-3), or the numbering rules of Kabat and Chothia (HCDR1). The numbering rules of CDRs are shown in Table 2. 1 is the same.

HR137-07的CDR序列如表2所示： The CDR sequence of HR137-07 is shown in Table 2:

HR137-07的重鏈可變區序列如下所示(下劃線為CDR)： The heavy chain variable region sequence of HR137-07 is shown below (CDR is underlined):

SEQ ID NO：8

SEQ ID NO: 8

HR137-07的輕鏈可變區序列如下所示(下劃線為CDR)： The light chain variable region sequence of HR137-07 is shown below (CDR is underlined):

SEQ ID NO：9

SEQ ID NO: 9

HR137-07的重鏈序列如下所示： The heavy chain sequence of HR137-07 is shown below:

SEQ ID NO：10

SEQ ID NO: 10

HR137-07的輕鏈序列如下所示： The light chain sequence of HR137-07 is as follows:

SEQ ID NO：11

SEQ ID NO: 11

抗體HR137-07與B1E7的區別僅在於，HR137-07在LCDR2中有N53Q突變。 The only difference between antibody HR137-07 and B1E7 is that HR137-07 has N53Q mutation in LCDR2.

實施例2. 抗體HR137-07的抗原結合親和力檢測Example 2. Antigen binding affinity detection of antibody HR137-07

採用Octet檢測抗4-1BB抗體HR137-07與其抗原人4-1BB蛋白之間的親和力。首先，將親和素探針浸入緩衝液PBS中10分鐘，達到平衡。然後，浸入20μg/mL生物素化的抗原人4-1BB蛋白中360秒，使抗原固定至探針上，浸入緩衝液中360秒，洗去多餘的抗原。將待檢測的抗體分別稀釋到100nM、33nM、10nM、3.3nM和1nM，分別加入到黑色96孔板中，體積為200μL/孔，將96孔板放入Octet儀器中。將探針分別置於含有抗體的各孔中，結合120秒。然後再置於緩衝液中，解離360秒。所得的親和力如表3所示，結果顯示，HR137-07與抗原人4-1BB的親和力為11.4nM。與B1E7相比，HR137-07與4-1BB的親和力降低約2.8倍。 Octet was used to detect the affinity between the anti-4-1BB antibody HR137-07 and its antigen human 4-1BB protein. First, immerse the avidin probe in the buffer PBS for 10 minutes to reach equilibrium. Then, it was immersed in 20 μg/mL biotinylated antigen human 4-1BB protein for 360 seconds to immobilize the antigen on the probe, and then immersed in a buffer solution for 360 seconds to wash away the excess antigen. The antibodies to be tested were diluted to 100nM, 33nM, 10nM, 3.3nM and 1nM, respectively, and added to the black 96-well plate with a volume of 200μL/well, and the 96-well plate was placed in the Octet instrument. Place the probes in each well containing the antibody and bind for 120 seconds. Then put it in the buffer and dissociate for 360 seconds. The obtained affinity is shown in Table 3. The result shows that the affinity of HR137-07 to the antigen human 4-1BB is 11.4 nM. Compared with B1E7, the affinity of HR137-07 with 4-1BB decreased by about 2.8 times.

實施例3. 抗體HR137-07的4-1BB/NF-κB螢光素酶報告基因檢測實驗Example 3. 4-1BB/NF-κB luciferase reporter gene detection experiment of antibody HR137-07

在HEK293細胞株(Thermo fisher scientific)中穩定轉染人4-1BB全長基因(Genbank Accession NO：NM_001561.5)，製成人4-1BB蛋白陽性的HEK293細胞株HEK293F-h4-1BB。在該人4-1BB蛋白陽性的HEK293F細胞株中穩定轉染NF-κB螢光素酶報告基因質粒，製成人4-1BB蛋白陽性的，有NF-κB螢光素酶報告基因的穩定細胞株HEK293F-h4-1BB-NFκB。接種50μL 4-1BB-NFκB HEK293細胞(5×10⁵/mL)於96孔板中。藉由抗人IgG的F(ab)₂(Jackson ImmunoResearch)交聯待測抗體。具體的交聯方法是在抗體加入細胞前與抗人IgG的F(ab)2(Jackson Immuno Research)以1：1.5摩爾濃度比例在37℃孵育30分鐘。並加入細胞中溫育5小時。加入螢光素酶檢測試劑ONE-GLUTM luciferase assay reagent(Promega)，用Envision(PerkinElmer，2150)讀取螢光值。結果如圖1所示。結果顯示，相比B1E7，HR137-07展示出更強的激活活性，說明N53Q降低了抗體與4-1BB的親和力，同時提高了抗體對4-1BB的激動活性。類似親和力降低提高抗體激活活性的現象在同TNFR家族中Fas受體激動劑中也有報道(Cell Death and Differentiation(2012)19,1187-1195)。N53Q提高了抗體對4-1BB的激動活性，能增強抗體對T細胞的激活，增強抗腫瘤效果。 The full-length human 4-1BB gene (Genbank Accession NO: NM_001561.5) was stably transfected into the HEK293 cell line (Thermo fisher scientific) to make the HEK293 cell line HEK293F-h4-1BB positive for the human 4-1BB protein. The NF-κB luciferase reporter gene plasmid was stably transfected into the HEK293F cell line positive for human 4-1BB protein to make a stable cell line with NF-κB luciferase reporter gene positive for human 4-1BB protein HEK293F-h4-1BB-NFκB. Inoculate 50 μL of 4-1BB-NFκB HEK293 cells (5×10 ⁵ /mL) in a 96-well plate. The antibody to be tested was cross-linked by anti-human IgG F(ab) _{2 (Jackson ImmunoResearch).} The specific cross-linking method is to incubate with anti-human IgG F(ab)2 (Jackson Immuno Research) at a molar concentration ratio of 1:1.5 at 37°C for 30 minutes before the antibody is added to the cells. And added to the cells and incubated for 5 hours. Add luciferase assay reagent ONE-GLUTM luciferase assay reagent (Promega), and read the fluorescence value with Envision (PerkinElmer, 2150). The result is shown in Figure 1. The results showed that HR137-07 showed stronger activation activity than B1E7, indicating that N53Q reduced the affinity of the antibody to 4-1BB and at the same time increased the antibody's agonistic activity to 4-1BB. Similar to the phenomenon that reduced affinity improves antibody activation activity has also been reported in Fas receptor agonists in the same TNFR family (Cell Death and Differentiation (2012) 19, 1187-1195). N53Q improves the antibody's agonistic activity to 4-1BB, can enhance the antibody's activation of T cells, and enhance the anti-tumor effect.

實施例4. 抗4-1BB抗體HR137-07在食蟹猴中的血藥濃度Example 4. Blood concentration of anti-4-1BB antibody HR137-07 in cynomolgus monkey

採用食蟹猴3隻(普通級，動物來源：廣西桂東靈長類開發實驗有限公司、廣州相觀生物科技有限公司)，分為兩組。第一組兩隻，一雄一雌，靜脈注射B1E7-IgG1。第二組一隻，雄性，靜脈注射HR137-07。每週給藥一次，劑量設置為5mg/kg，給藥容量為10mL/kg(以0.9%氯化鈉注射液稀釋)。在以下時間點採集血液：第一次給藥給藥後的0h、0.08或2h、4h或6h或8h、24h、48h、96h；第8天的0h，第二次給藥後的2h；第15天的0h，第三次給藥後的2h；第21天的0h，第四次給藥後的0h、0.08h、0.08或2h、4h或6h或8h、24h、48h、96h、168h。對血藥濃度藉由ELISA的方法進行檢測。採用生物素標記的4-1BB包被板子，25℃溫育1小時。300μL PBS清洗三次後，加入待測血清，25℃溫育1小時。300μL PBS清洗三次後，每孔加入100μL羊抗人IgG fab溶液，封板膜封板，25℃溫育1小時。二抗檢測濃度1：30000，稀釋液為1% BSA-PBST+1%猴血清。 Three cynomolgus monkeys (general grade, animal source: Guangxi Guidong Primate Development and Experiment Co., Ltd., Guangzhou Xiangguan Biological Technology Co., Ltd.) were used and divided into two groups. The first group of two, one male and one female, were injected intravenously with B1E7-IgG1. One male in the second group was given HR137-07 intravenously. Administer once a week, the dose is set to 5mg/kg, the administration volume is 10mL/kg (with 0.9% chlorination Sodium injection diluted). Blood was collected at the following time points: 0h, 0.08 or 2h, 4h or 6h or 8h, 24h, 48h, 96h after the first administration; 0h on the 8th day, 2h after the second administration; 15 days at 0h, 2h after the third dose; at 21st day, 0h, 0.08h, 0.08 or 2h, 4h or 6h or 8h, 24h, 48h, 96h, 168h after the fourth dose. The blood drug concentration was detected by ELISA method. The plate was coated with biotin-labeled 4-1BB and incubated at 25°C for 1 hour. After washing with 300 μL PBS for three times, add the test serum and incubate at 25°C for 1 hour. After washing with 300 μL PBS for three times, add 100 μL goat anti-human IgG fab solution to each well, seal the plate with sealing film, and incubate at 25°C for 1 hour. The detection concentration of the secondary antibody is 1:30000, and the diluent is 1% BSA-PBST+1% monkey serum.

結果顯示，在給藥第一週時，B1E7和HR137-07的血藥濃度趨勢基本一致。但是在第四週給藥後，B1E7的血藥濃度隨時間迅速降低，HR137-07的血藥濃度降低緩慢，HR137-07給藥後8h的血藥濃度是B1E7給藥後6h的血藥濃度的43.8-113.8倍，顯示HR137-07的半衰期顯著延長，參見表4。 The results showed that in the first week of administration, the blood concentration trends of B1E7 and HR137-07 were basically the same. However, after the fourth week of administration, the blood concentration of B1E7 decreased rapidly over time, and the blood concentration of HR137-07 decreased slowly. The blood concentration of HR137-07 8h after administration was the blood concentration of B1E7 6h after administration. 43.8-113.8 times of HR137-07, showing a significant prolongation of the half-life of HR137-07, see Table 4.

實施例5. 結合4-1BB/PD-L1的雙特異性抗體的製備Example 5. Preparation of bispecific antibodies that bind 4-1BB/PD-L1

1、結合4-1BB/PD-L1的雙特異性抗體的結構 1. The structure of the bispecific antibody that binds to 4-1BB/PD-L1

以下設計並製備了結合4-1BB/PD-L1的雙特異性抗體8E、8EN、9E-FM、9EN-FM和9EN-FM-1，其均為IgG-scFv型雙抗。 The bispecific antibodies 8E, 8EN, 9E-FM, 9EN-FM and 9EN-FM-1 that bind 4-1BB/PD-L1 were designed and prepared as follows, all of which are IgG-scFv type double antibodies.

雙抗中使用的連接子1(linker-1)和連接子2(linker-2)的序列結構為： The sequence structure of linker-1 (linker-1) and linker-2 (linker-2) used in the double antibody is:

連接子1為3個G4S重複(即，GGGGSGGGGSGGGGS(SEQ ID NO：38))； Linker 1 is three G4S repeats (ie, GGGGSGGGGSGGGGS (SEQ ID NO: 38));

連接子2為4個G4S重複(即，GGGGSGGGGSGGGGSGGGGS(SEQ ID NO：39))。 Linker 2 is 4 G4S repeats (ie, GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 39)).

其中，8E和8EN的Fc部分為野生型人IgG1，8E和8EN抗體的第一多肽鏈序列分別如SEQ ID NO：12和13所示，第二多肽鏈序列如SEQ ID NO：14所示。而9E-FM、9EN-FM和9EN-FM-1的Fc部分均進行了突變以消除Fc介導的細胞殺傷作用。其中，9E-FM和9EN-FM的突變為：L234A，L235A和P329G。9EN-FM-1的突變為：L234A，L235A。9E-FM、9EN-FM、9EN-FM-1的抗體的第一多肽鏈序列分別如SEQ ID NO：15、16和17所示，第二多肽鏈序列如SEQ ID NO：18所示。 Among them, the Fc part of 8E and 8EN is wild-type human IgG1, the first polypeptide chain sequence of 8E and 8EN antibodies are shown in SEQ ID NO: 12 and 13, respectively, and the second polypeptide chain sequence is shown in SEQ ID NO: 14. Show. The Fc part of 9E-FM, 9EN-FM and 9EN-FM-1 were all mutated to eliminate Fc-mediated cell killing. Among them, the mutations of 9E-FM and 9EN-FM are: L234A, L235A and P329G. The mutations of 9EN-FM-1 are: L234A, L235A. The sequences of the first polypeptide chain of the antibodies of 9E-FM, 9EN-FM, and 9EN-FM-1 are shown in SEQ ID NO: 15, 16 and 17, respectively, and the sequence of the second polypeptide chain is shown in SEQ ID NO: 18 .

8E的抗體結構如圖2A所示。其中IgG部分為抗人PD-L1抗體(anti-PD-L1 IgG)，scFv部分由抗人4-1BB抗體的重鏈可變區(VH)和輕鏈可變區(VL)組成(anti-4-1BB scFv)，重鏈可變區(VH)和輕鏈可變區(VL)之間由連接子1連接，抗4-1BB scFv由連接子2連接在抗PD-L1IgG的C末端。 The antibody structure of 8E is shown in Figure 2A. The IgG part is an anti-human PD-L1 antibody (anti-PD-L1 IgG), and the scFv part is composed of the heavy chain variable region (VH) and the light chain variable region (VL) of the anti-human 4-1BB antibody (anti- 4-1BB scFv), the heavy chain variable region (VH) and the light chain variable region (VL) are connected by linker 1, and the anti-4-1BB scFv is connected by linker 2 to the C-terminus of anti-PD-L1IgG.

8EN的抗體結構如圖2B所示。其與8E的區別在於，抗4-1BB scFv是由連接子2連接在抗PD-L1 IgG的N末端。 The antibody structure of 8EN is shown in Figure 2B. The difference from 8E is that the anti-4-1BB scFv is connected to the N-terminus of anti-PD-L1 IgG by linker 2.

抗體8E的第一多肽鏈如下所示(下劃線處為連接子)： The first polypeptide chain of antibody 8E is as follows (the linker is underlined):

SEQ ID NO：12

SEQ ID NO: 12

抗體8EN的第一多肽鏈如下所示： The first polypeptide chain of antibody 8EN is as follows:

SEQ ID NO：13

SEQ ID NO: 13

抗體8E和8EN的第二多肽鏈如下所示： The second polypeptide chains of antibodies 8E and 8EN are as follows:

SEQ ID NO：14

SEQ ID NO: 14

9E-FM的抗體結構如圖2C所示。其中IgG部分為抗人4-1BB抗體(anti-4-1BB IgG)，scFv部分由抗人PD-L1抗體的重鏈可變區(VH)和輕鏈可變區(VL)組成(anti- PD-L1 scFv)，重鏈可變區(VH)和輕鏈可變區(VL)之間由連接子1連接，抗PD-L1 scFv由連接子2連接在抗4-1BB IgG的C末端。 The antibody structure of 9E-FM is shown in Figure 2C. The IgG part is an anti-human 4-1BB antibody (anti-4-1BB IgG), and the scFv part is composed of the heavy chain variable region (VH) and the light chain variable region (VL) of the anti-human PD-L1 antibody (anti- PD-L1 scFv), the heavy chain variable region (VH) and the light chain variable region (VL) are connected by linker 1, and the anti-PD-L1 scFv is connected by linker 2 to the C-terminus of anti-4-1BB IgG .

9EN-FM和9EN-FM-1的抗體結構如圖2D所示。其與9E-FM的區別在於，抗PD-L1 scFv是由連接子2連接在抗4-1BB IgG的N末端。 The antibody structures of 9EN-FM and 9EN-FM-1 are shown in Figure 2D. The difference from 9E-FM is that anti-PD-L1 scFv is connected to the N-terminus of anti-4-1BB IgG by linker 2.

抗體9E-FM的第一多肽鏈如下所示(下劃線處為連接子，灰色處為L234A、L235A和P329G突變)： The first polypeptide chain of antibody 9E-FM is shown below (linker is underlined, and L234A, L235A and P329G mutations are in gray):

SEQ ID NO：15

SEQ ID NO: 15

抗體9EN-FM的第一多肽鏈如下所示(下劃線處為連接子，灰色處為L234A、L235A和P329G突變)： The first polypeptide chain of antibody 9EN-FM is shown below (linker is underlined, and mutations of L234A, L235A and P329G are shown in gray):

SEQ ID NO：16

SEQ ID NO: 16

抗體9EN-FM-1的第一多肽鏈如下所示(下劃線處為連接子，灰色處為L234A、和L235A突變)： The first polypeptide chain of the antibody 9EN-FM-1 is shown below (underlined is the linker, and the gray areas are the L234A and L235A mutations):

SEQ ID NO：17

SEQ ID NO: 17

抗體9E-FM、9EN-FM、9EN-FM-1的第二多肽鏈如下所示： The second polypeptide chains of antibodies 9E-FM, 9EN-FM, and 9EN-FM-1 are as follows:

SEQ ID NO：18

SEQ ID NO: 18

PD-L1抗體(HRP00052)的CDR序列如表5所示： The CDR sequence of PD-L1 antibody (HRP00052) is shown in Table 5:

其中，CDR胺基酸殘基在數量和位置符合已知的Kabat編號規則。 Among them, the number and position of CDR amino acid residues comply with the known Kabat numbering rules.

PD-L1抗體(HRP00052)的重鏈可變區序列(下劃線為CDR)： The heavy chain variable region sequence of the PD-L1 antibody (HRP00052) (CDR is underlined):

SEQ ID NO：25

SEQ ID NO: 25

PD-L1抗體(HRP00052)的輕鏈可變區序列(下劃線為CDR)： Light chain variable region sequence of PD-L1 antibody (HRP00052) (CDR is underlined):

SEQ ID NO：26

SEQ ID NO: 26

2、結合4-1BB/PD-L1的雙特異性抗體的製備 2. Preparation of bispecific antibodies that bind 4-1BB/PD-L1

用生長狀態良好，處於對數生長期的CHO-S細胞(購自Thermo公司，貨號A29133)，離心並按照6×10⁶細胞/mL接種250mL。將溶液2(用培養液9.2mL稀釋800ul轉染試劑，混勻)加入溶液1(用培養液10mL稀釋250ug質粒，混勻)中，總體積為20mL，輕柔混勻後室溫孵育1-5分鐘，不超過5分鐘，將混合轉染液逐滴加入細胞液中，邊搖邊加。然後將培養瓶置於5% CO₂，32℃的搖床培養，18-22個小時加輔料Feed(購自Thermo公司，貨號A29133)16mL，增強劑Enhancer(購自Thermo公司，貨號A29133)0.6mL。第五天加輔料Feed(購自Thermo公司，貨號A29133)16mL，123rpm，5% CO₂，32℃培養，待第12-14天離心收集上清。藉由親和層析法(Protein A)和離子交換兩步法進行重組抗體的純化。純化中使用的介質分別是GE公司生產的MabSelect SuRe column(GE，17-5438)和HiTrap Q HP column(GE，17515601)。製備獲得了PD-L1/4-1BB雙特異性抗體9EN-FM，經10%的SDS-PAGE凝膠電泳檢測，如圖3所示。 Use CHO-S cells (purchased from Thermo Company, Catalog No. A29133) in a good growth state in the logarithmic growth phase, centrifuge and ^{inoculate 250 mL at 6×10 6} cells/mL. Add solution 2 (dilute 800ul transfection reagent with 9.2mL culture solution, mix well) into solution 1 (dilute 250ug plasmid with 10mL culture solution, mix well), the total volume is 20mL, mix gently and incubate at room temperature for 1-5 Minutes, no more than 5 minutes, add the mixed transfection solution dropwise to the cell fluid, and add while shaking. Then place the culture flask in a 5% CO ₂ , 32°C shaker, add 16 mL of supplementary feed (purchased from Thermo Company, product number A29133) for 18-22 hours, and enhancer Enhancer (purchased from Thermo Company, product number A29133) 0.6 mL. On the fifth day, 16 mL of supplementary feed (purchased from Thermo Company, article number A29133) was added, 123 rpm, 5% CO ₂ , and cultured at 32° C. The supernatant was collected by centrifugation on the 12th to 14th days. Recombinant antibodies were purified by a two-step method of affinity chromatography (Protein A) and ion exchange. The media used in the purification are MabSelect SuRe column (GE, 17-5438) and HiTrap Q HP column (GE, 17515601) produced by GE. The PD-L1/4-1BB bispecific antibody 9EN-FM was prepared and detected by 10% SDS-PAGE gel electrophoresis, as shown in Figure 3.

結果顯示，該雙抗分子理論分子量為200kDa，第一多肽鏈為75kDa，第二多肽鏈為25kDa，由SDS-PAGE凝膠電泳的非還原條帶大小和還原條帶大小來判斷，條帶大小符合預期，說明該重組雙特異性抗體能夠正確裝配和表達，而且無明顯聚集降解。 The results show that the theoretical molecular weight of the double antibody molecule is 200kDa, the first polypeptide chain is 75kDa, and the second polypeptide chain is 25kDa. It is judged by the size of the non-reduced band and the size of the reduced band of SDS-PAGE gel electrophoresis. The band size is in line with expectations, indicating that the recombinant bispecific antibody can be assembled and expressed correctly, and there is no obvious aggregation and degradation.

實施例6. 抗PD-L1/4-1BB雙特異抗體的抗原結合活性的檢測Example 6. Detection of antigen binding activity of anti-PD-L1/4-1BB bispecific antibody

用ELISA方法檢測PD-L1/4-1BB雙特異性抗體9EN-FM分別與PD-L1蛋白和4-1BB蛋白的結合活性。實驗開始前將96孔板做好相應的標記，以1ug/mL抗原濃度，每孔50ul，4℃冰箱過夜包被。次日，將前天包被好的抗原板取出，洗板機清洗一次(清洗液：1 x PBST)。清洗後以1 x PBST配置的1% BSA封閉液37℃封閉1小時。1x PBST清洗液洗板3次後，加入不同稀釋濃度的待檢雙抗9EN-FM，37℃溫箱孵育1小時。1 x PBST清洗液洗板3次後，加入100ul 1：5000稀釋的羊抗人IgG二抗，37℃溫箱孵育0.5小時洗板後，取TMB顯色液A液和B液1：1比例混合後顯色。15分鐘用1M鹽酸終止顯色反應。在Spectra Max M5多功能讀板機上，檢測450nm的螢光值。結果如圖4A和圖4B所示。 The binding activity of the PD-L1/4-1BB bispecific antibody 9EN-FM with PD-L1 protein and 4-1BB protein was detected by ELISA method. Before the start of the experiment, the 96-well plate was labeled accordingly, and coated with an antigen concentration of 1ug/mL, 50ul per well, and overnight in a refrigerator at 4°C. The next day, Take out the coated antigen plate the day before, and wash it once with a plate washer (washing solution: 1 x PBST). After washing, block with 1% BSA blocking solution in 1 x PBST at 37°C for 1 hour. After washing the plate 3 times with 1x PBST cleaning solution, add the double antibody 9EN-FM of different dilutions to be tested, and incubate for 1 hour in a 37°C incubator. After washing the plate 3 times with 1 x PBST washing solution, add 100ul of 1:5000 diluted goat anti-human IgG secondary antibody, incubate at 37°C for 0.5 hours. After washing the plate, take TMB chromogenic solution A and B at a ratio of 1:1 Color develops after mixing. The color reaction was terminated with 1M hydrochloric acid in 15 minutes. On the Spectra Max M5 multi-function plate reader, the fluorescence value of 450nm was detected. The results are shown in Figure 4A and Figure 4B.

結果顯示，PD-L1/4-1BB雙特異性抗體9EN-FM分別與PD-L1蛋白和4-1BB蛋白的結合活性均保持了與原來的親本單抗相同的結合活性。 The results showed that the binding activity of the PD-L1/4-1BB bispecific antibody 9EN-FM to the PD-L1 protein and 4-1BB protein, respectively, maintained the same binding activity as the original parental monoclonal antibody.

實施例7. 抗PD-L1/4-1BB雙特異抗體的抗原結合親和力的檢測Example 7. Detection of antigen binding affinity of anti-PD-L1/4-1BB bispecific antibody

採用表面等離子共振技術(surface plasmon resonance，SPR)，檢測PD-L1/4-1BB雙特異性抗體9EN-FM分別與其抗原人PD-L1蛋白和人4-1BB蛋白之間的親和力(儀器Biacore，。將抗原人PD-L1蛋白和人4-1BB蛋白分別固定至CM5芯片。偶聯水平設定在100RU，運用儀器Biacore進行檢測(T200，GE Healthcare，BIAC-B20-03)。運行緩衝液為HBS-EP+(10mM HEPES，150mM NaCl，3mM EDTA，0.05%表面活性劑P20)。將稀釋好的抗體(6.25、12.5、25、50、100nM)在30μl/min的流速下流過實驗通道和對比通道(100nM抗原人PD-L1蛋白/抗原人4-1BB)3分鐘，解離5分鐘。然後用再生緩衝液10mM甘胺酸pH1.5(GE Healthcare，BR-1003-54)在30μl/min的流速下運行30秒。數據用Biacore 8K評估軟體進行分析。結果見圖5A和圖5B。 Surface plasmon resonance (SPR) was used to detect the affinity between the PD-L1/4-1BB bispecific antibody 9EN-FM and its antigen human PD-L1 protein and human 4-1BB protein (instrument Biacore, The antigen human PD-L1 protein and human 4-1BB protein were fixed to the CM5 chip respectively. The coupling level was set at 100RU, and the instrument Biacore was used for detection (T200, GE Healthcare, BIAC-B20-03). The running buffer was HBS -EP+(10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20). The diluted antibody (6.25, 12.5, 25, 50, 100nM) flows through the experimental channel and the contrast channel ( 100nM antigen human PD-L1 protein/antigen human 4-1BB) for 3 minutes, dissociate for 5 minutes. Then use regeneration buffer 10mM glycine pH1.5 (GE Healthcare, BR-1003-54) at a flow rate of 30μl/min Run for 30 seconds. The data was analyzed with Biacore 8K evaluation software. The results are shown in Figure 5A and Figure 5B.

結果顯示，該雙特異性抗體與人4-1BB蛋白的親和力是3.2nM，而與人PD-L1蛋白的親和力是0.07nM，該雙抗對人PD-L1蛋白的親和力比對人4-1BB蛋白高約50倍，這極大地保證了該雙抗在腫瘤部位的有效富集，另外也極大地降低了4-1BB端可能引起的毒副作用。 The results show that the affinity of the bispecific antibody to human 4-1BB protein is 3.2nM, and the affinity to human PD-L1 protein is 0.07nM. The affinity of the bispecific antibody to human PD-L1 protein is comparable to that of human 4-1BB The protein is about 50 times higher, which greatly guarantees the effective enrichment of the double antibody at the tumor site, and also greatly reduces the toxic side effects that may be caused by the 4-1BB end.

實施例8. 抗PD-L1/4-1BB雙特異抗體在NF-κB螢光素酶報告基因實驗中檢測依賴於PD-L1的4-1BB信號激活Example 8. Detection of PD-L1-dependent 4-1BB signal activation by anti-PD-L1/4-1BB bispecific antibody in NF-κB luciferase reporter gene experiment

在實施例3中所使用的人4-1BB蛋白陽性的HEK293細胞株中，穩定轉染NF-κB螢光素酶報告基因質粒(Promega，pGL4.32[luc2P/NF-κB-RE/Hygro]Vector)，製成人4-1BB蛋白陽性的，有NF-κB螢光素酶報告基因的穩定細胞株4-1BB/NF-κB HEK293。將不同濃度待測抗體與該4-1BB/NF-κB HEK293細胞共培養6個小時，加入螢光素酶檢測試劑ONE-GLUTM luciferase assay reagent(Promega)，用Envision(PerkinElmer，2150)讀取螢光值。為了檢測PD-L1介導的4-1BB的激活，將穩定高表達PD-L1的Hep3B的細胞株與4-1BB/NF-κB HEK293共同孵育。9EM-FM在PD-L1陽性細胞存在的情況下對4-1BB信號通路有很強的激活活性，如圖6所示。在沒有PD-L1陽性細胞存在的情況下，9EN-FM對於4-1BB的信號無激活作用。對於雙特異性抗體中使用的4-1BB的單抗HR137-07在藉由外源***聯後可激活4-1BB的信號通路，具體的交聯方法是在抗體加入細胞前與抗人IgG的F(ab)₂(Jackson ImmunoResearch)以1：1.5摩爾濃度比例在37℃孵育30分鐘。Urelumab(BMS-663513，全人IgG4)可以單獨激活4-1BB的信號通路，加入外源***聯後激活程度增強。 In the HEK293 cell line positive for the human 4-1BB protein used in Example 3, the NF-κB luciferase reporter gene plasmid (Promega, pGL4.32[luc2P/NF-κB-RE/Hygro] was stably transfected Vector), a stable cell line 4-1BB/NF-κB HEK293 that is positive for human 4-1BB protein and has an NF-κB luciferase reporter gene. Co-culture the 4-1BB/NF-κB HEK293 cells with different concentrations of the antibody to be tested for 6 hours, add the luciferase detection reagent ONE-GLUTM luciferase assay reagent (Promega), and read the fluorescence with Envision (PerkinElmer, 2150). Light value. In order to detect PD-L1-mediated activation of 4-1BB, the Hep3B cell line stably expressing PD-L1 was incubated with 4-1BB/NF-κB HEK293. 9EM-FM has a strong activation activity on the 4-1BB signaling pathway in the presence of PD-L1 positive cells, as shown in Figure 6. In the absence of PD-L1 positive cells, 9EN-FM has no activation effect on the 4-1BB signal. The 4-1BB monoclonal antibody HR137-07 used in the bispecific antibody can activate the 4-1BB signaling pathway after exogenous cross-linking. The specific cross-linking method is to combine with anti-human IgG before the antibody is added to the cell. F(ab) ₂ (Jackson ImmunoResearch) was incubated at 37°C for 30 minutes at a molar concentration ratio of 1:1.5. Urelumab (BMS-663513, fully human IgG4) can activate the 4-1BB signaling pathway alone, and the degree of activation is enhanced after the addition of exogenous cross-linking.

以上結果表明，9EN-FM在體內應用時，只有在PD-L1高表達的區域如腫瘤組織，才能激活4-1BB的活性。 The above results indicate that when 9EN-FM is used in vivo, the activity of 4-1BB can only be activated in areas where PD-L1 is highly expressed, such as tumor tissues.

實施例9. 抗PD-L1/4-1BB雙特異抗體對PD-1/PD-L1信號通路的封閉實驗Example 9. Blocking experiment of PD-1/PD-L1 signaling pathway by anti-PD-L1/4-1BB bispecific antibody

在Jurkat細胞中穩定轉染PD-1和NFAT螢光素酶報告基因質粒(Promega，pGL4.30[luc2P/NFAT-RE/Hygro] Vector)，製成帶有PD-1表達的並帶NFAT螢光素酶報告基因的穩定細胞株。同時加入穩定高表達PD-L1的Hep3B的細胞株。加入不同濃度待測抗體後培養6個小時，加入螢光素酶檢測試劑ONE-GLUTM螢光素酶檢測試劑(Promega)，用Envision(PerkinElmer，2150)讀取螢光值，結果如圖7所示。 Stable transfection of PD-1 and NFAT luciferase reporter gene plasmids (Promega, pGL4.30[luc2P/NFAT-RE/Hygro] Vector) into Jurkat cells to make PD-1 expression and NFAT fluorescent Stable cell line of luciferase reporter gene. At the same time, a Hep3B cell line with stable and high expression of PD-L1 was added. After adding different concentrations of antibody to be tested, incubate for 6 hours, add luciferase detection reagent ONE-GLUTM luciferase detection reagent (Promega), and read the fluorescence value with Envision (PerkinElmer, 2150). The result is shown in Figure 7. Show.

結果顯示，9EN-FM能劑量依賴性的解除PD-1/PD-L1信號通路介導的對NFAT螢光信號的抑制。與9EN-FM抑制活性要略低於雙特異性抗體中使用的PD-L1單抗(HRP00052)。 The results show that 9EN-FM can dose-dependently relieve the inhibition of NFAT fluorescent signal mediated by PD-1/PD-L1 signaling pathway. The inhibitory activity with 9EN-FM is slightly lower than that of the PD-L1 monoclonal antibody (HRP00052) used in the bispecific antibody.

實施例10. T淋巴細胞激活實驗Example 10. T lymphocyte activation experiment

將人外周血單核淋巴細胞(StemExpress)中加入100ng/mL SEB(康博貝擃)，同時加入待測抗體。為了驗證PD-L1介導的4-1BB的激活，在外周血單核淋巴細胞中加入了高表達PD-L1的HEK293細胞。HEK293細胞瞬時轉染PD-L1質粒(Lipofectemine 2000，Invitrogen)，轉染後24小時，用10g/mL絲裂黴素C(TGI)作用1小時。再用PBS清洗三遍後加入用SEB刺激的人外周血單核淋巴細胞。共同培養72小時後，用ELISA的方法檢測上清中的IL-2(human IL-2 kit，cisbio)的分泌。結果如圖8所示。 The human peripheral blood mononuclear lymphocytes (StemExpress) were added with 100ng/mL SEB (Kangbo Beizhan), and the antibody to be tested was added at the same time. In order to verify the PD-L1-mediated activation of 4-1BB, HEK293 cells with high PD-L1 expression were added to peripheral blood mononuclear lymphocytes. HEK293 cells were transiently transfected with PD-L1 plasmid (Lipofectemine 2000, Invitrogen), 24 hours after transfection, 10g/mL mitomycin C (TGI) was used for 1 hour. After washing three times with PBS, human peripheral blood mononuclear lymphocytes stimulated with SEB were added. After 72 hours of co-cultivation, the secretion of IL-2 (human IL-2 kit, cisbio) in the supernatant was detected by ELISA. The result is shown in Figure 8.

結果顯示，在沒有加入PD-L1高表達HEK293細胞時，9EM-FM對於T細胞的激活要強於兩個單抗HR137-07，PD-L1單抗(HRP00052)單獨作用以及兩個單抗聯用。當在SEB實驗中加入PD-L1高表達的HEK-293細胞時，9EN-FM對於T細胞的活性要進一步增強。進一步說明9EN-FM介導了PD-L1依賴的T細胞激活。 The results showed that when HEK293 cells with high PD-L1 expression were not added, 9EM-FM activated T cells stronger than the two monoclonal antibodies HR137-07, PD-L1 monoclonal antibody (HRP00052) alone and two monoclonal antibodies combined use. When HEK-293 cells with high PD-L1 expression were added to the SEB experiment, the activity of 9EN-FM on T cells should be further enhanced. It further shows that 9EN-FM mediates PD-L1-dependent T cell activation.

實施例11. 4-1BB/PD-L1雙抗小鼠體內抗腫瘤活性Example 11. Anti-tumor activity in mice of 4-1BB/PD-L1 double antibody

將MC38-hPD-L1細胞以5×10⁵個/0.1mL濃度接種於Balbc-hPD-L1/h4-1BB雙人源化小鼠(百奧賽圖公司提供)的右側皮下，待腫瘤生長到大約120mm³時按腫瘤體積挑選42隻隨機分組，每組6隻，共7組，分別為：人IgG(7.5mg/kg)、HRP00052(2.25mg/kg)、HR137-07(0.225mg/kg)、HPR00052(2.25mg/kg)+HR137-07(0.225mg/kg)、9EN-FM-1(0.3mg/kg)、9EN-FM-1(3mg/kg)和9EN-FM-1(10mg/kg)。每週給藥2次，給藥和觀察期間每週測量2次小鼠體重和腫瘤體積如圖9所示，並記錄測量值。 MC38-hPD-L1 cells were inoculated subcutaneously on the right side of Balbc-hPD-L1/h4-1BB double-derived mice (provided by Biocyto) at a concentration of ^{5×10 5 cells/0.1mL, and the tumor grew to about 120mm} ^{At 3} o'clock, 42 mice were selected according to the tumor volume and randomly grouped, 6 mice in each group, a total of 7 groups: human IgG (7.5mg/kg), HRP00052 (2.25mg/kg), HR137-07 (0.225mg/kg), HPR00052(2.25mg/kg)+HR137-07(0.225mg/kg), 9EN-FM-1(0.3mg/kg), 9EN-FM-1(3mg/kg) and 9EN-FM-1(10mg/kg) ). The administration was administered twice a week, and the mouse body weight and tumor volume were measured twice a week during the administration and observation period, as shown in Figure 9, and the measured values were recorded.

計算T/C值，公式如下： To calculate the T/C value, the formula is as follows:

T/C%=給藥組腫瘤體積平均值/對照組腫瘤平均體積%； T/C%= average tumor volume in the treatment group/average tumor volume in the control group%;

CR%(腫瘤完全消退比例)=腫瘤完全消退的小鼠數量/入組小鼠數量。 CR% (proportion of complete tumor regression) = number of mice with complete tumor regression/number of mice enrolled in the group.

結果顯示，9EN-FM具有劑量依賴地抗腫瘤活性，0.3mpk，3mpk和10mpk組的腫瘤的T/C%值分別為132.2、69.9以及10.7(見表6)。在高劑量10mpk給藥組的抗腫瘤T/C與兩藥聯用組的T/C相類似分別為10.7和19.6，但是從腫瘤完全消退的比例來說(CR%)，9EN-FM-1 10mpk 組為83.3%。說明9EN-FM-1具有比HR137-07和HRP00052聯用更好的抗腫瘤活性。 The results showed that 9EN-FM has a dose-dependent anti-tumor activity, and the T/C% values of tumors in the 0.3 mpk, 3 mpk and 10 mpk groups were 132.2, 69.9 and 10.7, respectively (see Table 6). The anti-tumor T/C in the high-dose 10mpk administration group was similar to the T/C of the two-drug combination group, 10.7 and 19.6, respectively, but in terms of the rate of complete tumor regression (CR%), 9EN-FM-1 10mpk Group is 83.3%. It shows that 9EN-FM-1 has better anti-tumor activity than HR137-07 and HRP00052 in combination.

實施例12. 4-1BB/PD-L1雙特異性抗體在食蟹猴中的單劑量藥物代謝動力學研究Example 12. Single-dose pharmacokinetic study of 4-1BB/PD-L1 bispecific antibody in cynomolgus monkeys

採用2-4.5歲雄性食蟹猴3隻(普通級，動物來源：廣西桂東靈長類開發實驗有限公司、廣州相觀生物科技有限公司)，靜脈推注射9EN-FM。單次給藥，劑量設置為5mg/kg，給藥容量為10mL/kg(以0.9%氯化鈉注射液稀釋)。在以下時間點採集血液：第一次給藥給藥後的0h、0.25h、2h、8h、24h、48h、96h、168小時、240小時、336小時、504小時、672小時。對血藥濃度藉由ELISA的方法進行檢測。採用生物素標記的4-1BB 包被板子，25℃溫育1小時。300μL PBS清洗三次後，加入待測血清，25℃溫育1小時。300μL PBS清洗三次後，每孔加入100μL羊抗人IgG fab溶液，封板膜封板，25℃溫育1小時。二抗檢測濃度1：30000，稀釋液為1% BSA-PBST+1%猴血清。 Three male cynomolgus monkeys aged 2-4.5 (general grade, animal source: Guangxi Guidong Primate Development and Experiment Co., Ltd., Guangzhou Xiangguan Biotechnology Co., Ltd.) were used to inject 9EN-FM intravenously. For single administration, the dose is set to 5mg/kg, and the administration volume is 10mL/kg (diluted with 0.9% sodium chloride injection). Blood was collected at the following time points: 0h, 0.25h, 2h, 8h, 24h, 48h, 96h, 168 hours, 240 hours, 336 hours, 504 hours, 672 hours after the first administration. The blood drug concentration was detected by ELISA method. 4-1BB labeled with biotin Coat the plate and incubate at 25°C for 1 hour. After washing with 300 μL PBS for three times, add the test serum and incubate at 25°C for 1 hour. After washing with 300 μL PBS for three times, add 100 μL goat anti-human IgG fab solution to each well, seal the plate with sealing film, and incubate at 25°C for 1 hour. The detection concentration of the secondary antibody is 1:30000, and the diluent is 1% BSA-PBST+1% monkey serum.

結果顯示，9EN-FM-1在5mpk的劑量下半衰期為37.3小時，這與HRP00052同劑量下抗體半衰期類似見表7，提示這是主要是在抗體未達到靶點飽和濃度的條件下，PD-L1這一端介導的抗體分佈。 The results show that the half-life of 9EN-FM-1 at a dose of 5mpk is 37.3 hours, which is similar to the antibody half-life at the same dose of HRP00052. See Table 7, indicating that this is mainly due to the condition that the antibody does not reach the saturation concentration of the target. The distribution of antibodies mediated by this end of L1.

其他序列信息如下： Other sequence information is as follows:

人種系重鏈模板IGHV3-30序列： Human germline heavy chain template IGHV3-30 sequence:

SEQ ID NO：27

SEQ ID NO: 27

人種系輕鏈模板IGKV3-11序列： Human germline light chain template IGKV3-11 sequence:

SEQ ID NO：28

SEQ ID NO: 28

人種系重鏈模板IGHV1-46序列： Human germline heavy chain template IGHV1-46 sequence:

SEQ ID NO：29

SEQ ID NO: 29

人種系輕鏈模板IGKV4-1序列： Human germline light chain template IGKV4-1 sequence:

SEQ ID NO：30

SEQ ID NO: 30

抗體8E的第一多肽鏈胺基酸序列的編碼核酸序列： The coding nucleic acid sequence of the amino acid sequence of the first polypeptide chain of antibody 8E:

SEQ ID NO：31

SEQ ID NO: 31

抗體8EN的第一多肽鏈胺基酸序列的編碼核酸序列： The coding nucleic acid sequence of the amino acid sequence of the first polypeptide chain of antibody 8EN:

SEQ ID NO：32

SEQ ID NO: 32

抗體8E、抗體8EN的第二多肽鏈胺基酸序列的編碼核酸序列： The coding nucleic acid sequence of the amino acid sequence of the second polypeptide chain of antibody 8E and antibody 8EN:

SEQ ID NO：33

SEQ ID NO: 33

抗體9E-FM的第一多肽鏈胺基酸序列的編碼核酸序列： The coding nucleic acid sequence of the amino acid sequence of the first polypeptide chain of antibody 9E-FM:

SEQ ID NO：34

SEQ ID NO: 34

抗體9EN-FM的第一多肽鏈胺基酸序列的編碼核酸序列： The coding nucleic acid sequence of the amino acid sequence of the first polypeptide chain of antibody 9EN-FM:

SEQ ID NO：35

SEQ ID NO: 35

抗體9EN-FM-1的第一多肽鏈胺基酸序列的編碼核酸序列： The coding nucleic acid sequence of the amino acid sequence of the first polypeptide chain of antibody 9EN-FM-1:

SEQ ID NO：36

SEQ ID NO: 36

抗體9E、抗體9EN-FM、抗體9EN-FM-1的第二多肽鏈胺基酸序列的編碼核酸序列： The coding nucleic acid sequence of the amino acid sequence of the second polypeptide chain of antibody 9E, antibody 9EN-FM, and antibody 9EN-FM-1:

SEQ ID NO：37

SEQ ID NO: 37

<110> 江蘇恆瑞醫藥股份有限公司 <110> Jiangsu Hengrui Pharmaceutical Co., Ltd.

<120> 抗4-1BB抗體、其抗原結合片段及雙特異性抗體 <120> Anti-4-1BB antibody, its antigen-binding fragment and bispecific antibody

<130> 702073CPCT <130> 702073CPCT

<160> 39 <160> 39

<170> SIPOSequenceListing 1.0 <170> SIPOSequenceListing 1.0

<210> 1 <210> 1

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(10) <222> (1)..(10)

<223> B1E7和HR137-07 HCDR1 <223> B1E7 and HR137-07 HCDR1

<400> 1 <400> 1

<210> 2 <210> 2

<211> 17 <211> 17

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(17) <222> (1)..(17)

<223> B1E7和HR137-07 HCDR2 <223> B1E7 and HR137-07 HCDR2

<400> 2 <400> 2

<210> 3 <210> 3

<211> 11 <211> 11

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(11) <222> (1)..(11)

<223> B1E7和HR137-07 HCDR3 <223> B1E7 and HR137-07 HCDR3

<400> 3 <400> 3

<210> 4 <210> 4

<211> 15 <211> 15

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(15) <222> (1)..(15)

<223> B1E7和HR137-07 LCDR1 <223> B1E7 and HR137-07 LCDR1

<400> 4 <400> 4

<210> 5 <210> 5

<211> 7 <211> 7

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(7) <222> (1)..(7)

<223> B1E7 LCDR2 <223> B1E7 LCDR2

<400> 5 <400> 5

<210> 6 <210> 6

<211> 9 <211> 9

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(9) <222> (1)..(9)

<223> B1E7和HR137-07 LCDR3 <223> B1E7 and HR137-07 LCDR3

<400> 6 <400> 6

<210> 7 <210> 7

<211> 7 <211> 7

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(7) <222> (1)..(7)

<223> HR137-07 LCDR2 <223> HR137-07 LCDR2

<400> 7 <400> 7

<210> 8 <210> 8

<211> 120 <211> 120

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(120) <222> (1)..(120)

<223> HR137-07重鏈可變區 <223> HR137-07 heavy chain variable region

<400> 8 <400> 8

<210> 9 <210> 9

<211> 114 <211> 114

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(114) <222> (1)..(114)

<223> HR137-07輕鏈可變區 <223> HR137-07 light chain variable region

<400> 9 <400> 9

<210> 10 <210> 10

<211> 450 <211> 450

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 鏈 <221> Chain

<222> (1)..(450) <222> (1)..(450)

<223> HR137-07重鏈 <223> HR137-07 heavy chain

<400> 10 <400> 10

<210> 11 <210> 11

<211> 218 <211> 218

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 鏈 <221> Chain

<222> (1)..(218) <222> (1)..(218)

<223> HR137-07輕鏈 <223> HR137-07 light chain

<400> 11 <400> 11

<210> 12 <210> 12

<211> 715 <211> 715

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 鏈 <221> Chain

<222> (1)..(715) <222> (1)..(715)

<223> 8E的第一多肽鏈 <223> The first polypeptide chain of 8E

<400> 12 <400> 12

<210> 13 <210> 13

<211> 715 <211> 715

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 鏈 <221> Chain

<222> (1)..(715) <222> (1)..(715)

<223> 8EN的第一多肽鏈 <223> The first polypeptide chain of 8EN

<400> 13 <400> 13

<210> 14 <210> 14

<211> 218 <211> 218

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 鏈 <221> Chain

<222> (1)..(218) <222> (1)..(218)

<223> 8E和8EN的第二多肽鏈 <223> The second polypeptide chain of 8E and 8EN

<400> 14 <400> 14

<210> 15 <210> 15

<211> 715 <211> 715

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 鏈 <221> Chain

<222> (1)..(715) <222> (1)..(715)

<223> 9E-FM的第一多肽鏈 <223> The first polypeptide chain of 9E-FM

<400> 15 <400> 15

<210> 16 <210> 16

<211> 715 <211> 715

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 鏈 <221> Chain

<222> (1)..(715) <222> (1)..(715)

<223> 抗體9EN-FM的第一多肽鏈 <223> The first polypeptide chain of antibody 9EN-FM

<400> 16 <400> 16

<210> 17 <210> 17

<211> 715 <211> 715

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 鏈 <221> Chain

<222> (1)..(715) <222> (1)..(715)

<223> 9EN-FM-1的第一多肽鏈 <223> The first polypeptide chain of 9EN-FM-1

<400> 17 <400> 17

<210> 18 <210> 18

<211> 218 <211> 218

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 鏈 <221> Chain

<222> (1)..(218) <222> (1)..(218)

<223> 9E-FM、9EN-FM、9EN-FM-1的第二多肽鏈 <223> The second polypeptide chain of 9E-FM, 9EN-FM, 9EN-FM-1

<400> 18 <400> 18

<210> 19 <210> 19

<211> 5 <211> 5

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(5) <222> (1)..(5)

<223> PD-L1抗體(HRP00052)HCDR1 <223> PD-L1 antibody (HRP00052) HCDR1

<400> 19 <400> 19

<210> 20 <210> 20

<211> 17 <211> 17

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(17) <222> (1)..(17)

<223> PD-L1抗體(HRP00052)HCDR2 <223> PD-L1 antibody (HRP00052) HCDR2

<400> 20 <400> 20

<210> 21 <210> 21

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(10) <222> (1)..(10)

<223> PD-L1抗體(HRP00052)HCDR3 <223> PD-L1 antibody (HRP00052) HCDR3

<400> 21 <400> 21

<210> 22 <210> 22

<211> 15 <211> 15

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(15) <222> (1)..(15)

<223> PD-L1抗體(HRP00052)LCDR1 <223> PD-L1 antibody (HRP00052) LCDR1

<400> 22 <400> 22

<210> 23 <210> 23

<211> 7 <211> 7

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(7) <222> (1)..(7)

<223> PD-L1抗體(HRP00052)LCDR2 <223> PD-L1 antibody (HRP00052) LCDR2

<400> 23 <400> 23

<210> 24 <210> 24

<211> 9 <211> 9

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(9) <222> (1)..(9)

<223> PD-L1抗體(HRP00052)LCDR3 <223> PD-L1 antibody (HRP00052) LCDR3

<400> 24 <400> 24

<210> 25 <210> 25

<211> 119 <211> 119

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(119) <222> (1)..(119)

<223> PD-L1抗體(HRP00052)的重鏈可變區 <223> The heavy chain variable region of PD-L1 antibody (HRP00052)

<400> 25 <400> 25

<210> 26 <210> 26

<211> 111 <211> 111

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(111) <222> (1)..(111)

<223> PD-L1抗體(HRP00052)的輕鏈可變區 <223> Light chain variable region of PD-L1 antibody (HRP00052)

<400> 26 <400> 26

<210> 27 <210> 27

<211> 98 <211> 98

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(98) <222> (1)..(98)

<223> 人種系重鏈模板IGHV3-30 <223> Human germline heavy chain template IGHV3-30

<400> 27 <400> 27

<210> 28 <210> 28

<211> 95 <211> 95

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(95) <222> (1)..(95)

<223> 人種系輕鏈模板IGKV3-11 <223> Human germline light chain template IGKV3-11

<400> 28 <400> 28

<210> 29 <210> 29

<211> 98 <211> 98

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(98) <222> (1)..(98)

<223> 人種系重鏈模板IGHV1-46 <223> Human germline heavy chain template IGHV1-46

<400> 29 <400> 29

<210> 30 <210> 30

<211> 101 <211> 101

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 結構域 <221> domain

<222> (1)..(101) <222> (1)..(101)

<223> 人種系輕鏈模板IGKV4-1 <223> Human germline light chain template IGKV4-1

<400> 30 <400> 30

<210> 31 <210> 31

<211> 2148 <211> 2148

<212> DNA/RNA <212> DNA/RNA

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 基因 <221> Gene

<222> (1)..(2148) <222> (1)..(2148)

<223> 抗體8E的第一多肽鏈胺基酸序列的編碼核酸序列 <223> Nucleic acid sequence encoding the amino acid sequence of the first polypeptide chain of antibody 8E

<400> 31 <400> 31

<210> 32 <210> 32

<211> 2148 <211> 2148

<212> DNA/RNA <212> DNA/RNA

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 基因 <221> Gene

<222> (1)..(2148) <222> (1)..(2148)

<223> 抗體8EN的第一多肽鏈胺基酸序列的編碼核酸序列 <223> Nucleic acid sequence encoding the amino acid sequence of the first polypeptide chain of antibody 8EN

<400> 32 <400> 32

<210> 33 <210> 33

<211> 657 <211> 657

<212> DNA/RNA <212> DNA/RNA

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 基因 <221> Gene

<222> (1)..(657) <222> (1)..(657)

<223> 抗體8E、抗體8EN的第二多肽鏈胺基酸序列的編碼核酸序列 <223> The coding nucleic acid sequence of the amino acid sequence of the second polypeptide chain of antibody 8E and antibody 8EN

<400> 33 <400> 33

<210> 34 <210> 34

<211> 2148 <211> 2148

<212> DNA/RNA <212> DNA/RNA

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 基因 <221> Gene

<222> (1)..(2148) <222> (1)..(2148)

<223> 抗體9E-FM的第一多肽鏈胺基酸序列的編碼核酸序列 <223> Nucleic acid sequence encoding the amino acid sequence of the first polypeptide chain of antibody 9E-FM

<400> 34 <400> 34

<210> 35 <210> 35

<211> 2148 <211> 2148

<212> DNA/RNA <212> DNA/RNA

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 基因 <221> Gene

<222> (1)..(2148) <222> (1)..(2148)

<223> 抗體9EN-FM的第一多肽鏈胺基酸序列的編碼核酸序列 <223> Nucleic acid sequence encoding the amino acid sequence of the first polypeptide chain of antibody 9EN-FM

<400> 35 <400> 35

<210> 36 <210> 36

<211> 2148 <211> 2148

<212> DNA/RNA <212> DNA/RNA

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 基因 <221> Gene

<222> (1)..(2148) <222> (1)..(2148)

<223> 抗體9EN-FM-1的第一多肽鏈胺基酸序列的編碼核酸序列 <223> Nucleic acid sequence encoding the amino acid sequence of the first polypeptide chain of antibody 9EN-FM-1

<400> 36 <400> 36

<210> 37 <210> 37

<211> 657 <211> 657

<212> DNA/RNA <212> DNA/RNA

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 基因 <221> Gene

<222> (1)..(657) <222> (1)..(657)

<223> 抗體9E、抗體9EN-FM、抗體9EN-FM-1的第二多肽鏈胺基酸序列的编編碼核酸序列 <223> The coding nucleic acid sequence of the amino acid sequence of the second polypeptide chain of antibody 9E, antibody 9EN-FM, and antibody 9EN-FM-1

<400> 37 <400> 37

<210> 38 <210> 38

<211> 15 <211> 15

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 肽 <221> peptide

<222> (1)..(15) <222> (1)..(15)

<223> 連接子1 <223> Connector 1

<400> 38 <400> 38

<210> 39 <210> 39

<211> 20 <211> 20

<212> PRT <212> PRT

<213> 人工序列(Artificial Sequence) <213> Artificial Sequence

<220> <220>

<221> 肽 <221> peptide

<222> (1)..(20) <222> (1)..(20)

<223> 連接子2 <223> Linker 2

<400> 39 <400> 39

Claims

一種抗4-1BB抗體或其抗原結合片段，包含： An anti-4-1BB antibody or antigen-binding fragment thereof, comprising:

重鏈可變區，其包含分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3；和/或 A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; and/or

輕鏈可變區，其包含分别如SEQ ID NO：4、SEQ ID NO：7和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3。 The light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 6, respectively.

如請求項1所述的抗4-1BB抗體或其抗原結合片段，其為鼠源抗體、嵌合抗體、人抗體、人源化抗體或其片段。 The anti-4-1BB antibody or antigen-binding fragment thereof according to claim 1, which is a murine antibody, a chimeric antibody, a human antibody, a humanized antibody or a fragment thereof.

如請求項1或2所述的抗4-1BB抗體或其抗原結合片段，其中： The anti-4-1BB antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein:

該抗體或其抗原結合片段的重鏈框架區源自SEQ ID NO：27或SEQ ID NO：29所示的胺基酸序列；和/或，輕鏈框架區源自SEQ ID NO：28或SEQ ID NO：30所示的胺基酸序列。 The heavy chain framework region of the antibody or its antigen-binding fragment is derived from the amino acid sequence shown in SEQ ID NO: 27 or SEQ ID NO: 29; and/or, the light chain framework region is derived from SEQ ID NO: 28 or SEQ ID NO: The amino acid sequence shown by 30.

如請求項2所述的抗4-1BB抗體或其抗原結合片段，其中該人源化抗體的重鏈可變區包含人源IgG1、IgG2、IgG3或IgG4或其變體的重鏈框架區； The anti-4-1BB antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain variable region of the humanized antibody comprises the heavy chain framework region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof;

較佳地，包含人源IgG1、IgG2或IgG4的重鏈框架區； Preferably, it comprises a heavy chain framework region of human IgG1, IgG2 or IgG4;

更佳地，包含人源IgG1的重鏈框架區。 More preferably, it comprises the heavy chain framework region of human IgG1.

如請求項1所述的抗4-1BB抗體或其抗原結合片段，其中： The anti-4-1BB antibody or antigen-binding fragment thereof according to claim 1, wherein:

重鏈可變區的胺基酸序列如SEQ ID NO：8所示，和/或輕鏈可變區的胺基酸序列如SEQ ID NO：9所示。 The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8, and/or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 9.

如請求項1至5中任一項所述的抗4-1BB抗體或其抗原結合片段，其中： The anti-4-1BB antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein:

I)該抗4-1BB抗體或其抗原結合片段包含如SEQ ID NO：10所示的重鏈，和/或如SEQ ID NO：11所示的輕鏈； 1) The anti-4-1BB antibody or antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID NO: 10, and/or a light chain as shown in SEQ ID NO: 11;

II)該抗4-1BB抗體或其抗原結合片段包含如SEQ ID NO：10的變體所示的重鏈，和/或如SEQ ID NO：11的變體所示的輕鏈；較佳地，該SEQ ID NO：10的變體是指在如SEQ ID NO：8所示的重鏈可變區包含1至10個胺基酸變化，和/或，該SEQ ID NO：11的變體是指在如SEQ ID NO：9所示的輕鏈可變區包含1至10個胺基酸變化，該胺基酸變化為保守性的修饰、置换或取代； II) The anti-4-1BB antibody or antigen-binding fragment thereof comprises a heavy chain as shown in a variant of SEQ ID NO: 10, and/or a light chain as shown in a variant of SEQ ID NO: 11; preferably The variant of SEQ ID NO: 10 refers to the variable region of the heavy chain as shown in SEQ ID NO: 8 containing 1 to 10 amino acid changes, and/or, the variant of SEQ ID NO: 11 It means that the light chain variable region as shown in SEQ ID NO: 9 contains 1 to 10 amino acid changes, and the amino acid changes are conservative modifications, substitutions or substitutions;

III)該抗4-1BB抗體或其抗原結合片段包含與SEQ ID NO：10所示的序列具有至少90%序列同一性的重鏈，和/或，與SEQ ID NO：11所示的序列具有至少90%的序列同一性的輕鏈； III) The anti-4-1BB antibody or antigen-binding fragment thereof comprises a heavy chain having at least 90% sequence identity with the sequence shown in SEQ ID NO: 10, and/or, having the sequence shown in SEQ ID NO: 11 A light chain with at least 90% sequence identity;

IV)該抗4-1BB抗體或其抗原結合片段具有降低或消除的ADCC效應；較佳地，該抗體是Fc區具有D265A、N297A、L234A或F/L235A、P329G中的一項或幾項突變的IgG1型抗體，或是IgG2/4的Fc區雜交抗體，或是具有S128P、F234A/L235A中的一項或幾項突變的IgG4型抗體；或者 IV) The anti-4-1BB antibody or its antigen-binding fragment has a reduced or eliminated ADCC effect; preferably, the antibody has one or more mutations in the Fc region of D265A, N297A, L234A or F/L235A, P329G IgG1 type antibody, or IgG2/4 Fc region hybrid antibody, or IgG4 type antibody with one or more mutations of S128P, F234A/L235A; or

V)該抗4-1BB抗體具有降低或消除的CDC效應；較佳地，該抗體在IgG1的Fc具有P331S的突變，或是IgG2/4的Fc區雜交抗體。 V) The anti-4-1BB antibody has a reduced or eliminated CDC effect; preferably, the antibody has a P331S mutation in the Fc of IgG1, or a hybrid antibody to the Fc region of IgG2/4.

一種抗4-1BB抗體或其抗原結合片段，其與請求項1至6中任一項所述的抗4-1BB抗體或其抗原結合片段競爭結合相同的表位。 An anti-4-1BB antibody or antigen-binding fragment thereof, which competes with the anti-4-1BB antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 for binding to the same epitope.

一種多核苷酸，其編碼請求項1至7中任一項所述的抗4-1BB抗體或其抗原結合片段。 A polynucleotide encoding the anti-4-1BB antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

一種表達載體，其含有請求項8所述的多核苷酸。 An expression vector containing the polynucleotide according to claim 8.

一種宿主細胞，其轉化有請求項9所述的表達載體；較佳地，該宿主細胞選自細菌、酵母和哺乳動物細胞；更佳地，該宿主細胞為大腸桿菌、畢赤酵母、中國倉鼠卵巢細胞或人胚腎293細胞。 A host cell transformed with the expression vector of claim 9; preferably, the host cell is selected from bacteria, yeast and mammalian cells; more preferably, the host cell is Escherichia coli, Pichia pastoris, and Chinese hamster Ovarian cells or human embryonic kidney 293 cells.

一種用於製備抗4-1BB抗體或其抗原結合片段的方法，包括步驟： A method for preparing an anti-4-1BB antibody or an antigen-binding fragment thereof, including the steps:

在請求項10所述的宿主細胞中表達請求項9所述的表達載體，以及從該宿主細胞中分離表達的抗4-1BB抗體或其抗原結合片段。 The expression vector of claim 9 is expressed in the host cell of claim 10, and the anti-4-1BB antibody or antigen-binding fragment thereof isolated and expressed from the host cell.

一種醫藥組成物，其含有請求項1至7中任一項所述的抗4-1BB抗體或其抗原結合片段，以及可藥用的賦形劑、稀釋劑或載體。 A pharmaceutical composition comprising the anti-4-1BB antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, and a pharmaceutically acceptable excipient, diluent or carrier.

一種如請求項1至7中任一項所述的抗4-1BB抗體或其抗原結合片段在製備藥物或醫藥組成物中的用途，該藥物或醫藥組成物用於治療疾病； A use of the anti-4-1BB antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 in the preparation of a medicine or a pharmaceutical composition for the treatment of diseases;

較佳地，該疾病為癌症； Preferably, the disease is cancer;

更佳地，該癌症選自：黑色素瘤、乳腺癌、卵巢癌、***癌、胰腺癌、腎癌、肺癌、肝癌、胃癌、結腸直腸癌、膀胱癌、頭頸癌、甲狀腺癌、食管癌、宫頸癌、肉瘤、多發性骨髓瘤、白血病、淋巴癌、膽囊癌和膠質母細胞瘤。 More preferably, the cancer is selected from: melanoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, bladder cancer, head and neck cancer, thyroid cancer, esophageal cancer, cervical cancer Carcinoma, sarcoma, multiple myeloma, leukemia, lymphoma, gallbladder cancer and glioblastoma.

一種治療疾病的方法，該方法包括： A method of treating diseases, the method includes:

向受試者施用治療有效量的請求項1至7中任一項所述的抗4-1BB抗體或其抗原結合片段，或請求項12所述的醫藥組成物； Administering to a subject a therapeutically effective amount of the anti-4-1BB antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or the pharmaceutical composition according to claim 12;

較佳地，該疾病為癌症； Preferably, the disease is cancer;

更佳地，該癌症選自：黑色素瘤、乳腺癌、卵巢癌、***癌、胰腺癌、腎癌、肺癌、肝癌、胃癌、結腸直腸癌、膀胱癌、頭頸癌、甲狀腺癌、食管癌、宫頸癌、肉瘤、多發性骨髓瘤、白血病、淋巴癌、膽囊癌和膠質母細胞瘤。 More preferably, the cancer is selected from: melanoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, bladder cancer, head and neck cancer, thyroid cancer, Esophageal cancer, cervical cancer, sarcoma, multiple myeloma, leukemia, lymphoma, gallbladder cancer and glioblastoma.

一種雙特異性抗體，其包含： A bispecific antibody comprising:

當該第一抗原或第二抗原是4-1BB時，該第一抗體、第二抗體或第一抗體、第二抗體的抗原結合片段包含分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3；和/或，分别如SEQ ID NO：4、SEQ ID NO：7和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3。 When the first antigen or the second antigen is 4-1BB, the first antibody, the second antibody or the antigen-binding fragments of the first antibody and the second antibody comprise SEQ ID NO: 1, SEQ ID NO: 2 and HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 3; and/or LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 6, respectively.

如請求項15所述的雙特異性抗體，其中： The bispecific antibody according to claim 15, wherein:

第一抗體或其抗原結合片段包含重鏈和輕鏈；和 The first antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain; and

第二抗體或其抗原結合片段包含scFv； The second antibody or antigen-binding fragment thereof comprises scFv;

如請求項15或16所述的雙特異性抗體，其中每個雙特異性抗體分子包含1個該第一抗體或其抗原結合片段和2個該scFv； The bispecific antibody according to claim 15 or 16, wherein each bispecific antibody molecule comprises one of the first antibody or antigen-binding fragment thereof and two of the scFv;

並且，一個該scFv連接於該第一抗體或其抗原結合片段的重鏈或輕鏈的N端，另一個該scFv連接於該第一抗體或其抗原結合片段的重鏈或輕鏈的C端；或者，兩個該scFv分别連接於該第一抗體或其抗原結合片段的兩條重鏈或兩條輕鏈的N端；或者，兩個該scFv分别連接於該第一抗體或其抗原結合片段的兩條重鏈或兩條輕鏈的C端。 In addition, one scFv is connected to the N-terminus of the heavy or light chain of the first antibody or its antigen-binding fragment, and the other scFv is connected to the C-terminus of the heavy or light chain of the first antibody or its antigen-binding fragment Or, two of the scFv are respectively connected to the N-terminus of the two heavy chains or two light chains of the first antibody or its antigen-binding fragment; or, the two scFv are respectively connected to the first antibody or its antigen binding The C-terminus of the two heavy chains or the two light chains of the fragment.

如請求項15至17中任一項所述的雙特異性抗體，其中： The bispecific antibody according to any one of claims 15 to 17, wherein:

該scFv的重鏈可變區與輕鏈可變區藉由連接子1連接，和/或該兩個scFv藉由連接子2與分別該第一抗體或其抗原結合片段的兩條重鏈的N端或C端連接； The variable region of the heavy chain and the variable region of the light chain of the scFv are connected by linker 1, and/or the two scFvs are connected to the two heavy chains of the first antibody or its antigen-binding fragment by linker 2. N-terminal or C-terminal connection;

更佳地，該scFv的結構為NH2-VL-連接子1-VH-COOH或NH2-VH-連接子1-VL-COOH，該連接子1和連接子2可以相同或不同。 More preferably, the structure of the scFv is NH2-VL-linker 1-VH-COOH or NH2-VH-linker 1-VL-COOH, and the linker 1 and the linker 2 may be the same or different.

如請求項15至18中任一項所述的雙特異性抗體，其中所述第一抗體或其抗原結合片段為IgG同種型，例如IgG1、IgG2、IgG3或IgG4，較佳地，為IgG1同種型；和/或，該第一抗體或其抗原結合片段的輕鏈為Kappa同種型。 The bispecific antibody according to any one of claims 15 to 18, wherein the first antibody or antigen-binding fragment thereof is of IgG isotype, such as IgG1, IgG2, IgG3 or IgG4, preferably, of IgG1 isotype Type; and/or, the light chain of the first antibody or antigen-binding fragment thereof is of Kappa isotype.

如請求項15至19中任一項所述的雙特異性抗體，其中該雙特異性抗體包含兩條第一多肽鏈和兩條第二多肽鏈，其中： The bispecific antibody according to any one of claims 15 to 19, wherein the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, wherein:

a)該兩條第一多肽鏈各自獨立地包含該第一抗體或其抗原結合片段的重鏈和該scFv；和 a) the two first polypeptide chains each independently comprise the heavy chain of the first antibody or antigen-binding fragment thereof and the scFv; and

b)該兩條第二多肽鏈各自獨立地包含該第一抗體或其抗原結合片段的輕鏈； b) each of the two second polypeptide chains independently comprises the light chain of the first antibody or antigen-binding fragment thereof;

其中，該scFv藉由連接子2與該第一抗體或其抗原結合片段的重鏈的N端或C端相連； Wherein, the scFv is connected to the N-terminus or C-terminus of the heavy chain of the first antibody or its antigen-binding fragment via linker 2;

或者， or,

i)該兩條第一多肽鏈各自獨立地包含該第一抗體或其抗原結合片段的輕鏈和該scFv；和 i) the two first polypeptide chains each independently comprise the light chain of the first antibody or antigen-binding fragment thereof and the scFv; and

ii)該兩條第二多肽鏈各自獨立地包含該第一抗體或其抗原結合片段的重鏈； ii) Each of the two second polypeptide chains independently comprises the heavy chain of the first antibody or antigen-binding fragment thereof;

其中，該scFv藉由連接子2與該第一抗體或其抗原結合片段的輕鏈的N端或C端相連； Wherein, the scFv is connected to the N-terminus or C-terminus of the light chain of the first antibody or its antigen-binding fragment via linker 2;

較佳地，該scFv具有結構：NH2-VH-連接子1-VL-COOH或NH2-VL-連接子1-VH-COOH； Preferably, the scFv has the structure: NH2-VH-linker 1-VL-COOH or NH2-VL-linker 1-VH-COOH;

較佳地，該雙特異性抗體包含兩條相同的第一多肽鏈和兩條相同的第二多肽鏈，該連接子1和連接子2可以相同或不同。 Preferably, the bispecific antibody comprises two identical first polypeptide chains and two identical second polypeptide chains, and the linker 1 and the linker 2 may be the same or different.

如請求項15至20中任一項所述的雙特異性抗體，具有與該第一抗體或其抗原結合片段的親本抗體相比同等或更弱的結合第一抗原的活性； The bispecific antibody according to any one of claims 15 to 20, which has the same or weaker activity of binding to the first antigen than the parent antibody of the first antibody or antigen-binding fragment thereof;

較佳地，該雙特異性抗體，具有與該scFv的親本抗體相比同等或更弱的結合第二抗原的活性； Preferably, the bispecific antibody has the same or weaker activity of binding to the second antigen than the parent antibody of the scFv;

更佳地，該雙特異性抗體，具有與該第一抗體或其抗原結合片段的親本抗體相比同等或更弱的結合第一抗原的活性，且具有與該scFv的親本抗體相比同等或更弱的結合第二抗原的活性； More preferably, the bispecific antibody has the same or weaker activity of binding to the first antigen than that of the parent antibody of the first antibody or antigen-binding fragment thereof, and has the same or weaker activity of binding to the first antigen than that of the parent antibody of the scFv The same or weaker binding activity to the second antigen;

更佳地，該雙特異性抗體，具有與結合PD-L1的親本抗體相比同等的結合活性； More preferably, the bispecific antibody has the same binding activity as that of the parent antibody that binds PD-L1;

更佳地，該雙特異性抗體，具有與結合4-1BB的親本抗體相比更弱的結合活性； More preferably, the bispecific antibody has weaker binding activity than the parent antibody that binds to 4-1BB;

更較佳地，該雙特異性抗體，具有與結合PD-L1的親本抗體相比同等的結合活性，且具有與結合4-1BB的親本抗體相比更弱的結合活性。 More preferably, the bispecific antibody has the same binding activity as that of the parent antibody that binds to PD-L1, and has a weaker binding activity than the parent antibody that binds to 4-1BB.

如請求項15至21中任一項所述的雙特異性抗體，其中該連接子1和/或連接子2為肽接頭，較佳地，該連接子1和/或連接子2具有如(G_mS_n)_x所示的胺基酸序列，其中m、n各自獨立地為選自1-8的整数，x為選自1-20的整数； The bispecific antibody according to any one of claims 15 to 21, wherein the linker 1 and/or the linker 2 is a peptide linker, preferably, the linker 1 and/or the linker 2 has a linker such as ( G _m S _n ) _{an amino acid sequence represented by x} , wherein m and n are each independently an integer selected from 1-8, and x is an integer selected from 1-20;

較佳地，該連接子1和/或連接子2具有如(G₄S)_x所示的胺基酸序列，x為選自1-6的整数； Preferably, the linker 1 and/or the linker 2 has _{an amino acid sequence as shown in (G 4} S) _x , where x is an integer selected from 1-6;

較佳地，該連接子1和/或連接子2為(G₄S)₃或(G₄S)₄； Preferably, the linker 1 and/or the linker 2 is (G ₄ S) ₃ or (G ₄ S) ₄ ;

較佳地，該連接子1為(G₄S)₃，連接子2為(G₄S)₄。 Preferably, the linker 1 is (G ₄ S) ₃ and the linker 2 is (G ₄ S) ₄ .

如請求項15至22中任一項所述的雙特異性抗體，其中該scFv的VH與VL之間存在二硫键。 The bispecific antibody according to any one of claims 15 to 22, wherein there is a disulfide bond between VH and VL of the scFv.

如請求項15至23中任一項所述的雙特異性抗體，其中該第一抗體或其抗原結合片段特異性結合4-1BB，並且該scFv特異性結合PD-L1，其中，該第一抗體或其抗原結合片段包含：如SEQ ID NO：1、2、3所示的HCDR1、HCDR2、HCDR3；和/或，如SEQ ID NO：4、7、6所示的LCDR1、LCDR2、LCDR3；並且，該scFv包含：分别如SEQ ID NO：19、20、21所示的HCDR、HCDR2、HCDR3，和/或，分别如SEQ ID NO：22、23、24所示的LCDR1、LCDR2、LCDR3。 The bispecific antibody according to any one of claims 15 to 23, wherein the first antibody or antigen-binding fragment thereof specifically binds 4-1BB, and the scFv specifically binds PD-L1, wherein the first The antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 1, 2, 3; and/or LCDR1, LCDR2, LCDR3 as shown in SEQ ID NO: 4, 7, and 6; In addition, the scFv includes: HCDR, HCDR2, HCDR3 shown in SEQ ID NO: 19, 20, 21, and/or LCDR1, LCDR2, LCDR3 shown in SEQ ID NO: 22, 23, 24, respectively.

如請求項15至23中任一項所述的雙特異性抗體，其中該第一抗體或其抗原結合片段特異性結合PD-L1，並且該scFv特異性結合4-1BB，其中 The bispecific antibody according to any one of claims 15 to 23, wherein the first antibody or antigen-binding fragment thereof specifically binds PD-L1, and the scFv specifically binds 4-1BB, wherein

該第一抗體或其抗原結合片段包含：分别如SEQ ID NO：19、20、21所示的HCDR、HCDR2、HCDR3，和/或，分别如SEQ ID NO：22、23、24所示的LCDR1、LCDR2、LCDR3；並且，該scFv包含如SEQ ID NO： 1、2、3所示的HCDR1、HCDR2、HCDR3；和/或，如SEQ ID NO：4、7、6所示的LCDR1、LCDR2、LCDR3。 The first antibody or antigen-binding fragment thereof comprises: HCDR, HCDR2, HCDR3 shown in SEQ ID NO: 19, 20, 21, and/or LCDR1 shown in SEQ ID NO: 22, 23, 24, respectively , LCDR2, LCDR3; and, the scFv includes SEQ ID NO: HCDR1, HCDR2, HCDR3 shown in 1, 2, and 3; and/or LCDR1, LCDR2, LCDR3 shown in SEQ ID NO: 4, 7, and 6.

如請求項15至23中任一項所述的雙特異性抗體，其中，該scFv包含： The bispecific antibody according to any one of claims 15 to 23, wherein the scFv comprises:

I)如SEQ ID NO：25所示的VH，和/或，如SEQ ID NO：26所示的VL；或 I) VH as shown in SEQ ID NO: 25, and/or VL as shown in SEQ ID NO: 26; or

II)如SEQ ID NO：8所示的VH，和/或，如SEQ ID NO：9所示的VL。 II) VH as shown in SEQ ID NO: 8, and/or VL as shown in SEQ ID NO: 9.

如請求項15至23中任一項所述的雙特異性抗體，其中： The bispecific antibody according to any one of claims 15 to 23, wherein:

I)該第一抗體或其抗原結合片段包含如SEQ ID NO：8所示的VH和如SEQ ID NO：9所示的VL；並且，該scFv包含如SEQ ID NO：25所示的VH和如SEQ ID NO：26所示的VL；或 I) The first antibody or antigen-binding fragment thereof comprises VH as shown in SEQ ID NO: 8 and VL as shown in SEQ ID NO: 9; and, the scFv comprises VH as shown in SEQ ID NO: 25 and VL as shown in SEQ ID NO: 26; or

II)該第一抗體或其抗原結合片段包含如SEQ ID NO：25所示的VH和如SEQ ID NO：26所示的VL；並且，該scFv包含如SEQ ID NO：8所示的VH和如SEQ ID NO：9所示的VL。 II) The first antibody or antigen-binding fragment thereof comprises VH as shown in SEQ ID NO: 25 and VL as shown in SEQ ID NO: 26; and, the scFv comprises VH as shown in SEQ ID NO: 8 and Such as the VL shown in SEQ ID NO:9.

如請求項15至27中任一項所述的雙特異性抗體，具有降低的ADCC活性，和/或降低的CDC活性。 The bispecific antibody according to any one of claims 15 to 27, which has reduced ADCC activity, and/or reduced CDC activity.

如請求項15至28中任一項所述的雙特異性抗體，其中該第一抗體的Fc區含有突變； The bispecific antibody according to any one of claims 15 to 28, wherein the Fc region of the first antibody contains mutations;

較佳地，第一抗體或其抗原結合片段的Fc區具有以下的一個或多個突變：在第265位突變為A、在第297位突變為A、在第234位突變為A或F、在第235位突變為A或E、在第329位突變為G； Preferably, the Fc region of the first antibody or antigen-binding fragment thereof has one or more of the following mutations: mutation at position 265 to A, mutation at position 297 to A, mutation at position 234 to A or F, Mutation to A or E at position 235 and G to position 329;

更佳地，第一抗體或其抗原結合片段的Fc區具有以下的一個或多個突變：D265A、N297A、L234A或L234F、L235A或L235E、P329G； More preferably, the Fc region of the first antibody or antigen-binding fragment thereof has one or more of the following mutations: D265A, N297A, L234A or L234F, L235A or L235E, P329G;

更佳地，第一抗體或其抗原結合片段的Fc區具有以下突變：L234A/L235A。 More preferably, the Fc region of the first antibody or antigen-binding fragment thereof has the following mutations: L234A/L235A.

如請求項15至29中任一項所述的雙特異性抗體，其中該雙特異性抗體包含： The bispecific antibody according to any one of claims 15 to 29, wherein the bispecific antibody comprises:

I)如SEQ ID NO：12所示的第一多肽鏈和如SEQ ID NO：14所示的第二多肽鏈；或 1) The first polypeptide chain as shown in SEQ ID NO: 12 and the second polypeptide chain as shown in SEQ ID NO: 14; or

II)如SEQ ID NO：13所示的第一多肽鏈和如SEQ ID NO：14所示的第二多肽鏈；或 II) The first polypeptide chain as shown in SEQ ID NO: 13 and the second polypeptide chain as shown in SEQ ID NO: 14; or

III)如SEQ ID NO：15所示的第一多肽鏈和如SEQ ID NO：18所示的第二多肽鏈；或 III) The first polypeptide chain as shown in SEQ ID NO: 15 and the second polypeptide chain as shown in SEQ ID NO: 18; or

IV)如SEQ ID NO：16所示的第一多肽鏈和如SEQ ID NO：18所示的第二多肽鏈；或 IV) The first polypeptide chain as shown in SEQ ID NO: 16 and the second polypeptide chain as shown in SEQ ID NO: 18; or

V)如SEQ ID NO：17所示的第一多肽鏈和如SEQ ID NO：18所示的第二多肽鏈。 V) The first polypeptide chain as shown in SEQ ID NO: 17 and the second polypeptide chain as shown in SEQ ID NO: 18.

一種分離的核酸分子，其包含編碼請求項15至30中任一項所述的雙特異性抗體的核苷酸序列； An isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific antibody according to any one of claims 15 to 30;

較佳地，該分離的核酸分子包含編碼請求項20至30中任一項所述的雙特異性抗體的第一多肽鏈的核苷酸序列，和/或編碼請求項20至30中任一項所述的雙特異性抗體的第二多肽鏈的核苷酸序列；更佳地，該分離的核酸分子包含SEQ ID NO：31-32、34-36所示的序列；和/或SEQ ID NO：33、37所示的序列。 Preferably, the isolated nucleic acid molecule comprises a nucleotide sequence encoding the first polypeptide chain of the bispecific antibody according to any one of claims 20 to 30, and/or encoding any one of claims 20 to 30. The nucleotide sequence of the second polypeptide chain of the bispecific antibody as described in one item; more preferably, the isolated nucleic acid molecule comprises the sequence shown in SEQ ID NO: 31-32, 34-36; and/or SEQ ID NOs: 33 and 37.

一種載體，其包含請求項31所述的分離的核酸分子。 A vector comprising the isolated nucleic acid molecule described in claim 31.

一種宿主細胞，其包含請求項31所述的分離的核酸分子或請求項32所述的載體。 A host cell comprising the isolated nucleic acid molecule according to claim 31 or the vector according to claim 32.

一種製備請求項15至30中任一項所述的雙特異性抗體的方法，其包括，在允許該雙特異性抗體表達的條件下，培養請求項33所述的宿主細胞，和從培養的宿主細胞培養物中回收該雙特異性抗體。 A method for preparing the bispecific antibody according to any one of claims 15 to 30, which comprises culturing the host cell according to claim 33 under conditions allowing the expression of the bispecific antibody, and from the cultured The bispecific antibody is recovered from the host cell culture.

一種醫藥組成物，其含有請求項15至30中任一項所述的雙特異性抗體，以及藥學上可接受的載體和/或賦形劑。 A pharmaceutical composition comprising the bispecific antibody according to any one of claims 15 to 30, and a pharmaceutically acceptable carrier and/or excipient.

一種如請求項15至30中任一項所述的雙特異性抗體或請求項35所述的醫藥組成物在製備藥物中的用途，該藥物用於在受試者中治療疾病； A use of the bispecific antibody according to any one of claims 15 to 30 or the pharmaceutical composition according to claim 35 in the preparation of a medicament for the treatment of diseases in a subject;

較佳地，該疾病為自身免疫性疾病或腫瘤或傳染病，例如腫瘤，包括但不限於實體瘤或血液癌症，或包括但不限於腺癌、白血病、淋巴瘤、黑色素瘤、肉瘤，或包括但不限於腎上腺、膽囊、骨、骨髓、腦、乳腺、膽管、胃腸道、心臟、腎臟、肝臟、肺、肌肉、卵巢、胰腺、副甲狀腺、陰莖、***、皮膚、唾液腺、脾臟、睾丸、胸腺、甲狀腺和子宫相關的腫瘤、傳染病包括但不限於B型肝炎、A型肝炎、HIV； Preferably, the disease is an autoimmune disease or tumor or infectious disease, such as tumor, including but not limited to solid tumor or blood cancer, or including but not limited to adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma, or including But not limited to adrenal gland, gallbladder, bone, bone marrow, brain, breast, bile duct, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, skin, salivary gland, spleen, testis, thymus , Thyroid and uterine related tumors, infectious diseases including but not limited to hepatitis B, hepatitis A, HIV;

較佳地，該受試者可以為哺乳動物，例如人。 Preferably, the subject may be a mammal, such as a human.

一種用於在受試者中治療疾病的方法，其中該方法包括，給有此需要的受試者施用有效量的請求項15至30中任一項所述雙特異性抗體，或者請求項35所述的醫藥組成物； A method for treating a disease in a subject, wherein the method comprises administering an effective amount of the bispecific antibody according to any one of claims 15 to 30 to a subject in need thereof, or claim 35 Said medical composition;

較佳地，該受試者可以為哺乳動物，更佳地，該受試者為人。 Preferably, the subject may be a mammal, and more preferably, the subject is a human.