WO2023072307A1

WO2023072307A1 - Antigen binding fragment targeting cd70, single-chain antibody and chimeric antigen receptor, and use thereof

Info

Publication number: WO2023072307A1
Application number: PCT/CN2022/137448
Authority: WO
Inventors: 齐亚男; 陈军; 赵文旭; 么瑞娜; 沈俊杰; 徐艳敏; 杨智; 代德鹏; 高诗静; 黄霞; 洪娟
Original assignee: 重庆精准生物技术有限公司; 重庆精准生物产业技术研究院有限公司
Priority date: 2021-10-25
Filing date: 2022-12-08
Publication date: 2023-05-04

Abstract

The present invention belongs to the technical field of immunotherapy, and specifically relates to a fully humanized antigen binding fragment targeting CD70 and a single-chain antibody and the use thereof, and a fully humanized chimeric antigen receptor targeting CD70 and the use thereof. The antigen binding fragment and single-chain antibody provided in the present invention have a good affinity, are derived from a natural fully humanized antibody, have a greatly reduced immunogenicity, and can ensure the safety thereof to a certain extent in clinical application. The chimeric antigen receptor and the CAR-T cell provided in the present invention can effectively kill target cells, secrete an effective amount of cytokines and effectively inhibit the growth of tumors in the body, and have a significant effect in terms of resisting tumors expressing CD70 targets.

Description

一种靶向CD70的抗原结合片段、单链抗体和嵌合抗原受体及其应用Antigen-binding fragment, single-chain antibody and chimeric antigen receptor targeting CD70 and application thereof

本申请要求2021年10月25日提交的中国发明专利申请【CN202111239047.X】、名称为“靶向CD70的抗原结合片段和单链抗体及其应用”的优先权，以及2021年10月25日提交的中国发明专利申请【CN202111239065.8】、名称为“一种靶向全人源化CD70的嵌合抗原受体及其应用”的优先权，两个优先权发明专利申请以引用方式全文并入。This application claims the priority of the Chinese invention patent application [CN202111239047.X], entitled "antigen-binding fragments and single-chain antibodies targeting CD70 and their applications" filed on October 25, 2021, and on October 25, 2021 The priority of the Chinese invention patent application [CN202111239065.8], titled "A Chimeric Antigen Receptor Targeting Fully Humanized CD70 and Its Application", was submitted, and the two priority invention patent applications are incorporated by reference in their entirety enter.

技术领域technical field

本发明属于免疫治疗技术领域，具体涉及一种全人源的靶向CD70的抗原结合片段和单链抗体及其应用，和一种靶向全人源化CD70的嵌合抗原受体及其应用。The invention belongs to the technical field of immunotherapy, and specifically relates to a fully human antigen-binding fragment and single-chain antibody targeting CD70 and applications thereof, and a chimeric antigen receptor targeting fully humanized CD70 and applications thereof .

背景技术Background technique

临床上常规的***的方法(例如手术、放疗、化疗等)都具有特异性差、对正常组织危害大、常伴有复发风险等局限，而这些局限促进了新的治疗手段的出现。肿瘤免疫疗法因其安全、有效、不良反应低等优点逐渐脱颖而出，成为继手术、放疗、化疗之外的第四种肿瘤治疗手段。Conventional methods of treating tumors in clinic (such as surgery, radiotherapy, chemotherapy, etc.) have limitations such as poor specificity, great harm to normal tissues, and often accompanied by risk of recurrence, and these limitations have promoted the emergence of new treatment methods. Tumor immunotherapy gradually stands out due to its advantages of safety, effectiveness and low adverse reactions, and has become the fourth tumor treatment method after surgery, radiotherapy and chemotherapy.

CD70，又称CD27配体、TNFSF7，是一种属于肿瘤坏死因子超家族的Ⅱ型跨膜糖蛋白。在正常人组织中，CD70在活化的T细胞、B细胞、自然杀伤细胞及成熟的树突状细胞(DC)上短暂表达，通过与其受体CD27蛋白结合(或相互作用)，向细胞传递活化信号，促进T细胞的活化、增殖、功能成熟及记忆细胞形成，在调控免疫应答过程中发挥重要作用。而在病理状态下，CD70可高表达于多种血液及实体肿瘤中。目前证实的高表达CD70的肿瘤组织包括肾癌、血源性恶性肿瘤、胸腺肿瘤、卵巢癌、成神经胶质细胞瘤、鼻咽癌、胰腺癌和胃肠道癌(例如食道癌、结肠癌和胃癌)等多种肿瘤组织，这表明CD70可能成为肿瘤免疫治疗的一个有效的靶点。目前，以CD70为靶点的免疫治疗药物已经在临床研究中应用，用以治疗急性髓系白血病(AML)、肾细胞癌等。CD70, also known as CD27 ligand and TNFSF7, is a type II transmembrane glycoprotein belonging to the tumor necrosis factor superfamily. In normal human tissues, CD70 is transiently expressed on activated T cells, B cells, natural killer cells and mature dendritic cells (DC), and activates cells through binding (or interacting) with its receptor CD27 protein Signal, promote the activation, proliferation, functional maturation and formation of memory cells of T cells, and play an important role in the regulation of immune response. In pathological conditions, CD70 can be highly expressed in a variety of blood and solid tumors. Tumor tissues with high expression of CD70 confirmed so far include renal cancer, hematogenous malignancies, thymic tumors, ovarian cancer, glioblastoma, nasopharyngeal cancer, pancreatic cancer, and gastrointestinal cancer (such as esophageal cancer, colon cancer, etc.) and gastric cancer) and other tumor tissues, which indicates that CD70 may become an effective target for tumor immunotherapy. At present, immunotherapeutic drugs targeting CD70 have been applied in clinical research to treat acute myeloid leukemia (AML), renal cell carcinoma, etc.

肿瘤免疫疗法中使用的第一代单克隆抗体是鼠源单抗，其会引起人体内的免疫反应，在体内被很快清除，因此给临床应用带来了一定的困难。The first-generation monoclonal antibody used in tumor immunotherapy is a mouse monoclonal antibody, which can cause an immune response in the human body and is quickly cleared in the body, thus bringing certain difficulties to clinical application.

CAR-T全称是嵌合抗原受体T细胞免疫疗法。嵌合抗原受体(chimeric antigen receptor，CAR)是模拟TCR功能的人工受体，包括依次连接的包含ScFv的胞外结构域、铰链区和跨膜区及胞内信号域。胞内信号域通常为CD3ζ链或FcRγ，或与一种或多种共刺激分子相连，如4-1BB，CD28，ICOS(CD278)。CAR分子中包含的ScFv具有能够对肿瘤抗原特异性识别的特点，通过铰链区和跨膜区进行T细胞激活信号传递。目前临床上CAR-T治疗主要应用于血液***的肿瘤治疗。The full name of CAR-T is Chimeric Antigen Receptor T Cell Immunotherapy. Chimeric antigen receptor (chimeric antigen receptor, CAR) is an artificial receptor that mimics the function of TCR, including an extracellular domain containing ScFv, a hinge region, a transmembrane region and an intracellular signaling domain connected in sequence. The intracellular signaling domain is usually CD3ζ chain or FcRγ, or linked to one or more co-stimulatory molecules, such as 4-1BB, CD28, ICOS (CD278). The ScFv contained in the CAR molecule has the characteristics of being able to specifically recognize tumor antigens, and transmits T cell activation signals through the hinge region and the transmembrane region. At present, CAR-T therapy is mainly used in the treatment of hematological tumors clinically.

相较于传统的CAR-T靶点，CD70的适应症更广、安全性高，并且能够覆盖血液***肿瘤和实体瘤，是具有潜力的CAR-T治疗靶点，目前还未有相关靶点药物上市。国外有正在研发中的相关的单抗和ScFv，而国内目前没有。开发靶向CD70的嵌合抗原受体非常有必要，因为开发靶向CD70的嵌合抗原受体及CAR-T细胞制品具有极大的临床应用价值。涉及CAR-T时，所针对的抗原基因序列的选择是关键性的选择。鉴于体内基因表达的复杂性以及各种不可控因素，选择到一个合适的可以用于CAR-T的基因是非常困难的，这是发展CAR-T技术的难点。Compared with traditional CAR-T targets, CD70 has wider indications, higher safety, and can cover blood system tumors and solid tumors. It is a potential CAR-T therapeutic target, and there is no related target yet The drug hits the market. There are related monoclonal antibodies and ScFvs under development in foreign countries, but there are currently no domestic ones. It is very necessary to develop CD70-targeted chimeric antigen receptors, because the development of CD70-targeted chimeric antigen receptors and CAR-T cell products has great clinical application value. When it comes to CAR-T, the selection of the targeted antigen gene sequence is a key choice. In view of the complexity of gene expression in vivo and various uncontrollable factors, it is very difficult to select a suitable gene that can be used for CAR-T, which is the difficulty of developing CAR-T technology.

发明内容Contents of the invention

第一方面，本发明目的之一在于提供一种靶向CD70的抗原结合片段，所述抗原结合片段的可变区为天然全人源抗体来源，降低了免疫原性，提高了在临床应用中的安全性。In the first aspect, one of the objectives of the present invention is to provide an antigen-binding fragment targeting CD70. The variable region of the antigen-binding fragment is derived from a natural fully human antibody, which reduces immunogenicity and improves clinical application. security.

所述抗原结合片段包含重链可变区和轻链可变区；所述轻链可变区包含L-CDR1、L-CDR2和L-CDR3，所述重链可变区包含H-CDR1、H-CDR2和H-CDR3；The antigen-binding fragment comprises a heavy chain variable region and a light chain variable region; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, and the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3;

进一步，所述L-CDR1、所述L-CDR2和所述L-CDR3选自以下氨基酸序列组合中一种：Further, the L-CDR1, the L-CDR2 and the L-CDR3 are selected from one of the following amino acid sequence combinations:

1)QSISSY、AAS、QQSYSTPWT；1) QSISSY, AAS, QQSYSTPWT;

2)QSVSSN、GAS、QQYNNWPLT；2) QSVSSN, GAS, QQYNNWPLT;

3)QSISSS、AAS、QQSYNTPRT；3) QSISSS, AAS, QQSYNTPRT;

4)QSLLSSADDLNY、WAS、QQYYGTPT；4) QSLLSSADDLNY, WAS, QQYYGTPT;

5)QSVSSY、GAS、QQSYTLPLT；5) QSVSSY, GAS, QQSYTLPLT;

6)QTINTY、AAS、QQSYSTLIT；6) QTINTY, AAS, QQSYSTLIT;

7)QSVSSY、GAS、QQSYTLPRT；7) QSVSSY, GAS, QQSYTLPRT;

所述H-CDR1、所述H-CDR2和所述H-CDR3选自以下氨基酸序列组合中一种：The H-CDR1, the H-CDR2 and the H-CDR3 are selected from one of the following amino acid sequence combinations:

1)GYTFTDYY、INPYNGGT、ARSVYDYPFDY；1) GYTFTDYY, INPYNGGT, ARSVYDYPFDY;

2)GDSVSSNSAA、TYYRSKWYN、ARWGPAADGGFDP。2) GDSVSSNSAA, TYYRSKWYN, ARWGPAADGGFDP.

进一步，所述抗原结合片段的轻链可变区包含如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6或SEQ ID NO:7所示的氨基酸序列或其功能性变体；所述重链可变区包含如SEQ ID NO:8、SEQ ID NO:9或SEQ ID NO:10所示的氨基酸序列或其功能性变体。Further, the light chain variable region of the antigen-binding fragment comprises such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 Or the amino acid sequence shown in SEQ ID NO: 7 or its functional variant; The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 or its functional variant.

第二方面，本发明还提供一种靶向CD70的全长抗体，所述全长抗体包含前述的抗原结合片段。In the second aspect, the present invention also provides a full-length antibody targeting CD70, the full-length antibody comprising the aforementioned antigen-binding fragment.

第三方面，本发明目的还在于提供一种靶向CD70的单链抗体。靶向肿瘤的单链抗体(ScFv)药物具有广阔的应用前景，ScFv作为靶向分子，具有以下优点：分子量小，穿透性较强，能实现人源化，易对其进行改造，可大规模生产等。目前国内外已有多种ScFv药物并用于临床。本发明提供的单链抗体可变区为天然全人源抗体来源，序列完全来自于人类抗体基因库，与鼠源抗体、嵌合抗体、人源化抗体相较，其免疫原性大大降低，改善了在临床应用上安全性的问题。In the third aspect, the purpose of the present invention is to provide a single-chain antibody targeting CD70. Tumor-targeting single-chain antibody (ScFv) drugs have broad application prospects. As a targeting molecule, ScFv has the following advantages: small molecular weight, strong penetrability, humanization, easy modification, and large mass production etc. At present, a variety of ScFv drugs have been used in clinic at home and abroad. The variable region of the single-chain antibody provided by the present invention is derived from a natural fully human antibody, and its sequence is completely derived from a human antibody gene library. Compared with murine antibodies, chimeric antibodies, and humanized antibodies, its immunogenicity is greatly reduced. The problem of safety in clinical application is improved.

所述单链抗体包含重链可变区和轻链可变区；所述轻链可变区包含L-CDR1、L-CDR2和L-CDR3，所述重链可变区包含H-CDR1、H-CDR2和H-CDR3；The single-chain antibody comprises a heavy chain variable region and a light chain variable region; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, and the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3;

1)QSISSY、AAS、QQSYSTPWT；1) QSISSY, AAS, QQSYSTPWT;

2)QSVSSN、GAS、QQYNNWPLT；2) QSVSSN, GAS, QQYNNWPLT;

3)QSISSS、AAS、QQSYNTPRT；3) QSISSS, AAS, QQSYNTPRT;

4)QSLLSSADDLNY、WAS、QQYYGTPT；4) QSLLSSADDLNY, WAS, QQYYGTPT;

5)QSVSSY、GAS、QQSYTLPLT；5) QSVSSY, GAS, QQSYTLPLT;

6)QTINTY、AAS、QQSYSTLIT；6) QTINTY, AAS, QQSYSTLIT;

7)QSVSSY、GAS、QQSYTLPRT；7) QSVSSY, GAS, QQSYTLPRT;

1)GYTFTDYY、INPYNGGT、ARSVYDYPFDY；1) GYTFTDYY, INPYNGGT, ARSVYDYPFDY;

进一步，所述轻链可变区包含SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6或SEQ ID NO:7所示的氨基酸序列或其功能性变体；所述重链可变区包含SEQ ID NO:8、SEQ ID NO:9或SEQ ID NO:10所示的氨基酸序列或其功能性变体。Further, the light chain variable region comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO: The amino acid sequence shown in 7 or its functional variant; The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 or its functional variant.

进一步，所述轻链可变区和所述重链可变区通过linker连接，所述linker可选自任一可具有连接作用的多肽，优选为氨基酸序列如SEQ ID NO:11所示的多肽或其功能性变体，编码所述多肽的核酸序列如SEQ ID NO:22所示。Further, the light chain variable region and the heavy chain variable region are connected by a linker, and the linker can be selected from any polypeptide that can have a linking effect, preferably a polypeptide with an amino acid sequence as shown in SEQ ID NO: 11 Or a functional variant thereof, the nucleic acid sequence encoding the polypeptide is shown in SEQ ID NO:22.

进一步，所述单链抗体的氨基酸序列优选为包括以下几组轻链和重链组合的一种：Further, the amino acid sequence of the single-chain antibody preferably includes one of the following combinations of light chains and heavy chains:

1)所述轻链可变区的氨基酸序列如SEQ ID NO:1所示，所述重链可变区的氨基酸序列如SEQ ID NO:8所示；1) The amino acid sequence of the light chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8;

2)所述轻链可变区的氨基酸序列如SEQ ID NO:2所示，所述重链可变区的氨基酸序列如SEQ ID NO:8所示；2) The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8;

3)所述轻链可变区的氨基酸序列如SEQ ID NO:3所示，所述重链可变区的氨基酸序列如SEQ ID NO:8所示；3) The amino acid sequence of the light chain variable region is shown in SEQ ID NO:3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8;

4)所述轻链可变区的氨基酸序列如SEQ ID NO:4所示，所述重链可变区的氨基酸序列如SEQ ID NO:9所示；4) The amino acid sequence of the light chain variable region is shown in SEQ ID NO:4, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:9;

5)所述轻链可变区的氨基酸序列如SEQ ID NO:5所示，所述重链可变区的氨基酸序列如SEQ ID NO:8所示；5) The amino acid sequence of the light chain variable region is shown in SEQ ID NO:5, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8;

6)所述轻链可变区的氨基酸序列如SEQ ID NO:6所示，所述重链可变区的氨基酸序列如SEQ ID NO:10所示；6) The amino acid sequence of the light chain variable region is shown in SEQ ID NO:6, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:10;

7)所述轻链可变区的氨基酸序列如SEQ ID NO:7所示，所述重链可变区的氨基酸序列如SEQ ID NO:8所示。7) The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8.

第四方面，本发明还提供了包含前述任一所述的单链抗体的CAR结构。所述CAR结构还包括铰链区、跨膜区、胞内信号区。所述铰链区序列可以来源于：IgG、CD8、CD7、CD4或其他同等功能蛋白分子；所述跨膜区可以来源于：CD8、CD28、CD3ε、CD4、CD16、CD137、CD80以及CD86或其他同等功能蛋白分子；所述胞内信号区可来源于：CD3、CD137、CD28、CD27、OX40、ICOS、GITR、CD2、CD40、PD-1、PD1L、B7-H3、淋巴细胞功能相关抗原-1(LFA-1)、ICAM-1、CD7、NKG2C、CD83、CD86以及CD127等其他同等功能蛋白分子。In the fourth aspect, the present invention also provides a CAR structure comprising any of the aforementioned single-chain antibodies. The CAR structure also includes a hinge region, a transmembrane region, and an intracellular signal region. The hinge region sequence can be derived from: IgG, CD8, CD7, CD4 or other equivalent functional protein molecules; the transmembrane region can be derived from: CD8, CD28, CD3ε, CD4, CD16, CD137, CD80 and CD86 or other equivalent Functional protein molecules; the intracellular signal region can be derived from: CD3, CD137, CD28, CD27, OX40, ICOS, GITR, CD2, CD40, PD-1, PD1L, B7-H3, lymphocyte function-related antigen-1 ( LFA-1), ICAM-1, CD7, NKG2C, CD83, CD86 and CD127 and other equivalent functional protein molecules.

进一步，所述CAR结构包含天然杀伤细胞受体(NKR)的一种或多种组分，因而形成NKR-CAR。NKR组分可以是来自以下任何天然杀伤细胞受体的跨膜结构域、铰链结构域或胞质结构域：杀伤细胞免疫球蛋白样受体(KIR)，例如KIR2DL1、KIR2DL2/L3、KIR2DL4、KIR2DL5A、KIR2DL5B、KIR2DS1、KIR2DS2、KIR2DS3、KIR2DS4、DIR2DS5、KIR3DL1/S1、KIR3DL2、KIR3DL3、KIR2DP1和KIR3DP1；天然细胞毒性受体(NCR)，例如，NKp30、NKp44、NKp46；免疫细胞受体的信号传导淋巴细胞活化分子(SLAM)家族，例如，CD48、CD229、2B4、CD84、NTB-A、CRA、BLAME和CD2F-10；Fc受体(FcR)，例如，CD16、和CD64；和Ly49受体，例如，LY49A、LY49C。所述的NKR-CAR分子可以与衔接分子或胞内信号结构域(例如，DAP12)相互作用。Further, the CAR structure comprises one or more components of a natural killer cell receptor (NKR), thus forming a NKR-CAR. The NKR component can be a transmembrane domain, hinge domain, or cytoplasmic domain from any of the following natural killer cell receptors: killer cell immunoglobulin-like receptors (KIRs), such as KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A , KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP1; natural cytotoxicity receptors (NCRs), e.g., NKp30, NKp44, NKp46; signaling lymphoid receptors for immune cells Cell Activation Molecule (SLAM) family, e.g., CD48, CD229, 2B4, CD84, NTB-A, CRA, BLAME, and CD2F-10; Fc receptors (FcR), e.g., CD16, and CD64; and Ly49 receptors, e.g. , LY49A, LY49C. The NKR-CAR molecule can interact with an adapter molecule or an intracellular signaling domain (eg, DAP12).

第五方面，本发明还提供一种包含编码前述任一所述的单链抗体的核酸序列，编码所述轻链可变区的核苷酸序列如SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17或SEQ ID NO:18所示；编码所述重链可变区的核苷酸序列如SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21所示。In the fifth aspect, the present invention also provides a nucleic acid sequence encoding any of the aforementioned single-chain antibodies, the nucleotide sequence encoding the light chain variable region is such as SEQ ID NO: 12, SEQ ID NO: 13 , SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18; the nucleotide sequence encoding the heavy chain variable region is as SEQ ID NO Shown in: 19, SEQ ID NO: 20, SEQ ID NO: 21.

第六方面，本发明还提供一种包含前述的核酸序列的重组质粒，所述重组质粒还包括表达载体。表达载体为慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、腺相关病毒表达载体、DNA载体、RNA载体、质粒中的任一种。In the sixth aspect, the present invention also provides a recombinant plasmid comprising the aforementioned nucleic acid sequence, and the recombinant plasmid further includes an expression vector. The expression vector is any one of lentivirus expression vector, retrovirus expression vector, adenovirus expression vector, adeno-associated virus expression vector, DNA vector, RNA vector and plasmid.

在某些实施例中，所述慢病毒载体选自基本上由以下组成的群组：人免疫缺陷病毒1(HIV-1)、人免疫缺陷病毒2(HIV-2)、维斯纳-梅迪病毒(visna-maedi virus，VMV)病毒、山羊关节炎-脑炎病毒(CAEV)、马传染性贫血病毒(EIAV)、猫免疫缺陷病毒(FIV)、牛免疫缺陷病毒(BIV)和猿猴免疫缺陷病毒(SIV)。In certain embodiments, the lentiviral vector is selected from the group consisting essentially of human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), Wiesner-Meyer Visna-maedi virus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) and simian immunization defective virus (SIV).

在某些实施例中，载体包含左(5')逆转录病毒LTR、Psi(Ψ)包装信号、中心多嘌呤段/DNA瓣(cPPT/FLAP)、逆转录病毒导出元件、可操作地连接到编码本文所涵盖的CAR的多核苷酸的启动子和右(3')逆转录病毒LTR。In certain embodiments, the vector comprises a left (5') retroviral LTR, a Psi (Ψ) packaging signal, a central polypurine tract/DNA flap (cPPT/FLAP), a retroviral export element, operably linked to The promoter and right (3') retroviral LTR of the polynucleotide encoding the CAR contemplated herein.

在某些实施例中，CAR包含乙型肝炎病毒转录后调节元件(HPRE)或土拔鼠转录后调节元件(WPRE)以及优化的土拔鼠转录后调节元件(oPRE)。In certain embodiments, the CAR comprises a hepatitis B virus post-transcriptional regulatory element (HPRE) or a woodchuck post-transcriptional regulatory element (WPRE) and an optimized woodchuck post-transcriptional regulatory element (oPRE).

在某些实施例中，所述5'LTR的启动子经异源启动子置换。In certain embodiments, the promoter of the 5'LTR is replaced with a heterologous promoter.

在某些实施例中，所述异源启动子是巨细胞病毒(CMV)启动子、劳斯肉瘤病毒(Rous Sarcoma Virus，RSV)启动子或猿猴病毒40(SV40)启动子。In certain embodiments, the heterologous promoter is a cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, or a Simian Virus 40 (SV40) promoter.

在某些实施例中，所述5'LTR或3'LTR是慢病毒LTR。In certain embodiments, the 5'LTR or 3'LTR is a lentiviral LTR.

在某些实施例中，所述3'LTR是自我失活(SIN)LTR。In certain embodiments, the 3'LTR is a self-inactivating (SIN) LTR.

第七方面，本发明提供一种CAR-T细胞，所述CAR-T细胞是通过前述的表达载体转染T细胞得到。In the seventh aspect, the present invention provides a CAR-T cell obtained by transfecting T cells with the aforementioned expression vector.

在某些实施例中，所述细胞可以表达其它活性剂，例如，增强CAR表达细胞活性的活性剂。活性剂可以是阻断抑制性分子的活性剂。在一些实施例中，抑制性分子(如PD1)可以降低CAR表达细胞的发动免疫效应子反应的能力。抑制性分子包括PD1、PD-L1、CTLA4、TIM3、LAG3、VISTA、BTLA、TIGIT、LAIR1、CD160、2B4、CEACAM(CEACAM-1、CEACAM-3、CEACAM-5)、LAG3、VISTA、BTLA、TIG、LAIR1、CD160、2B4、CD80、CD86、B7-H3(CD276)、B7-H4(VTCN1)、HVEM(TNFRSF14或CD270)、KIR、A2aR、MHC I类、MHC II类、GAL9、腺苷、TGFR(TGFRβ)和TGFRβ。所述抑制性分子的胞外结构域可以融合到跨膜结构域和胞内信号传导结构域，比如PD1 CAR。In certain embodiments, the cells can express other active agents, eg, agents that enhance the activity of CAR expressing cells. The active agent may be one that blocks an inhibitory molecule. In some embodiments, an inhibitory molecule (eg, PD1) can reduce the ability of a CAR-expressing cell to mount an immune effector response. Inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (CEACAM-1, CEACAM-3, CEACAM-5), LAG3, VISTA, BTLA, TIG , LAIR1, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM(TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, TGFR (TGFRβ) and TGFRβ. The extracellular domain of the inhibitory molecule can be fused to a transmembrane domain and an intracellular signaling domain, such as a PD1 CAR.

第八方面，本发明还提供一种前述的抗原结合片段或前述的全长抗体或前述的单链抗体或前述的CAR结构或前述的核酸序列或前述的重组质粒或前述的CAR-T细胞在制备抗肿瘤药物中的应用。具体地，制备的抗肿瘤药物可应用于针对CD70靶点的血液或实体肿瘤的免疫治疗。In the eighth aspect, the present invention also provides the aforementioned antigen-binding fragment or the aforementioned full-length antibody or the aforementioned single-chain antibody or the aforementioned CAR structure or the aforementioned nucleic acid sequence or the aforementioned recombinant plasmid or the aforementioned CAR-T cell in Application in the preparation of antitumor drugs. Specifically, the prepared antitumor drug can be applied to the immunotherapy of blood or solid tumor targeting CD70.

具体地，根据文献及现有技术查阅，表达CD70的肿瘤类型如下表11所示。Specifically, according to literature review and prior art review, the types of tumors expressing CD70 are shown in Table 11 below.

表11表达CD70的肿瘤类型Table 11 Tumor types expressing CD70

优选地，所述肿瘤包含肾癌、血源性恶性肿瘤、胸腺肿瘤、卵巢癌、成神经胶质细胞瘤、鼻咽癌、胰腺癌和胃肠道癌等多种肿瘤组织。所述胃肠道癌包括食道癌、结肠癌和胃癌中的一种或多种。更优选地，所述肿瘤为急性髓系白血病或肾细胞癌。Preferably, the tumor includes various tumor tissues such as renal carcinoma, hematogenous malignant tumor, thymic tumor, ovarian carcinoma, glioblastoma, nasopharyngeal carcinoma, pancreatic carcinoma and gastrointestinal tract carcinoma. The gastrointestinal cancer includes one or more of esophageal cancer, colon cancer and gastric cancer. More preferably, the tumor is acute myeloid leukemia or renal cell carcinoma.

第九方面，本发明还提供一种前述的抗原结合片段或前述的全长抗体或前述的单链抗体在制备检测试剂/检测试剂盒中的用途，该检测试剂或检测试剂盒可以高效精准识别CD70抗原。In the ninth aspect, the present invention also provides a use of the aforementioned antigen-binding fragment or the aforementioned full-length antibody or the aforementioned single-chain antibody in the preparation of a detection reagent/detection kit, which can efficiently and accurately identify CD70 antigen.

第十方面，本发明目的还在于提供一种前述的单链抗体的制备方法，该制备方法是通过噬菌体展示技术构建全人源的单链抗体库。相对于常规获得ScFv的方法，通过噬菌体展示技术构建全人源的单链抗体库，可以在较短时间内获得低免疫原性的治疗用全人源单链抗体。In the tenth aspect, the purpose of the present invention is to provide a method for preparing the aforementioned single-chain antibody. The preparation method is to construct a fully human single-chain antibody library through phage display technology. Compared with the conventional method of obtaining ScFv, constructing a fully human single-chain antibody library through phage display technology can obtain a fully human single-chain antibody with low immunogenicity for treatment in a short period of time.

目前有四种重组抗体平台用于产生用于治疗的人抗体：(1)小鼠单克隆抗体的“人源化”；(2)含人抗体基因的转基因小鼠免疫；(3)从大量的人类抗体库中进行体外筛选全人源抗体；(4)单个B细胞抗体制备技术。噬菌体展示技术是目前发展最为成熟的单抗药物制备技术。它由美国Missouri大学的George Smith于1985年提出，其基本原理是将外源DNA***到丝状噬菌体的基因组DNA，与噬菌体的外壳蛋白融合表达，一起展示到噬菌体表面。通过适当方法的亲和淘选，富集携带某种具有特异亲和力片段的噬菌体，测序即可获得其基因序列。在某些具体实施例中，为筛选到靶向CD70抗原的人源抗体，构建了全人源的单链抗体库。以正常人的PBMC为原材料，抽提总RNA反转录cDNA，扩增抗体可变区，通过连接肽(Linker)将重链可变区VH与轻链可变区VL连接起来，得到抗体序列。将其构建至噬菌粒载体，电转至展示菌株，构建抗体库，由CD70胞外段抗原对文库进行淘选，对筛选出的克隆进行鉴定，最终获得了多个对CD70抗原具有特异性的ScFv。Four recombinant antibody platforms are currently used to generate human antibodies for therapy: (1) "humanization" of mouse monoclonal antibodies; (2) immunization of transgenic mice containing human antibody genes; (4) Single B cell antibody preparation technology. Phage display technology is currently the most mature monoclonal antibody preparation technology. It was proposed by George Smith of the University of Missouri in the United States in 1985. Its basic principle is to insert foreign DNA into the genomic DNA of filamentous phage, express it in fusion with the coat protein of the phage, and display it on the surface of the phage together. Through affinity panning with appropriate methods, phages carrying certain fragments with specific affinity are enriched, and their gene sequences can be obtained by sequencing. In some specific embodiments, in order to screen for human antibodies targeting CD70 antigen, a fully human single-chain antibody library was constructed. Using normal human PBMC as raw material, extract total RNA and reverse transcribe cDNA, amplify the antibody variable region, connect the heavy chain variable region VH and light chain variable region VL through the linker to obtain the antibody sequence . It was constructed into a phagemid carrier, electroporated to display strains, and an antibody library was constructed. The library was panned by the CD70 extracellular segment antigen, and the screened clones were identified. Finally, multiple CD70 antigen-specific antibodies were obtained. ScFv.

本发明有益效果在于：The beneficial effects of the present invention are:

本发明提供的单链抗体可变区为天然全人源抗体来源，序列完全来自于人类抗体基因库，与鼠源抗体、嵌合抗体(嵌合抗体是利用DNA重组技术，将异源单抗的轻、重链可变区基因***含有人抗体恒定区的表达载体中，转化哺乳动物细胞表达出嵌合抗体，其中轻、重链的V区是异源的，而C区是人源的，换句话说，嵌合抗体仍有近1/3部分是异源的)、人源化抗体相较，其免疫原性大大降低，在临床应用上能够一定程度上保证安全性。The variable region of the single-chain antibody provided by the present invention is derived from a natural fully human antibody, and its sequence is completely derived from a human antibody gene library. Insert the light and heavy chain variable region genes into the expression vector containing the constant region of human antibody, transform mammalian cells to express chimeric antibodies, in which the V regions of the light and heavy chains are heterologous, and the C region is human , In other words, nearly 1/3 of chimeric antibodies are still heterologous), compared with humanized antibodies, their immunogenicity is greatly reduced, and their safety can be guaranteed to a certain extent in clinical applications.

本发明提供的单链抗体可以特异性识别人CD70抗原，可应用于针对CD70靶点的血液或实体肿瘤的免疫治疗。The single-chain antibody provided by the invention can specifically recognize human CD70 antigen, and can be applied to the immunotherapy of blood or solid tumors targeting CD70.

本发明提供的单链抗体亲和力性能良好，均可在流式细胞检测中与CD70阳性细胞结合，分子互作研究中显示可与CD70抗原特异性结合，有潜在的临床诊断和治疗用途。The single-chain antibody provided by the invention has good affinity performance, can be combined with CD70 positive cells in flow cytometry, can specifically bind with CD70 antigen in molecular interaction studies, and has potential clinical diagnosis and treatment applications.

第十一方面，本发明目的之一在于提供一种靶向CD70的嵌合抗原受体，该嵌合抗原受体与运载体结合转染T细胞后，可以精准识别CD70靶点，并对靶细胞进行有效杀伤。In the eleventh aspect, one of the objectives of the present invention is to provide a CD70-targeted chimeric antigen receptor, which can accurately recognize the CD70 target after the chimeric antigen receptor is combined with a carrier and transfected into T cells, and targets the target. Cells are effectively killed.

所述靶向CD70的嵌合抗原受体，包括胞外结构域、铰链区和跨膜区及胞内信号域，所述胞外结构域包括如SEQ ID NO:23所示的氨基酸序列或其功能性变体。The chimeric antigen receptor targeting CD70 includes an extracellular domain, a hinge region, a transmembrane region and an intracellular signaling domain, and the extracellular domain includes an amino acid sequence as shown in SEQ ID NO: 23 or its functional variant.

在某些具体实施例中，所述胞外结构域包括如SEQ ID NO:23所示的氨基酸序列或其功能性变体，即靶向CD70的ScFv。其制备方法是通过噬菌体展示技术构建全人源的单链抗体库，通过适当方法的亲和淘选，富集携带某种具有特异亲和力片段的噬菌体，测序即可获得其基因序列。为筛选到靶向CD70抗原的人源抗体，构建了全人源的单链抗体库，以正常人的PBMC为原材料，抽提总RNA反转录cDNA，扩增抗体可变区，通过连接肽(Linker)将重链可变区VH与轻链可变区VL连接起来。将其构建至噬菌粒载体，电转至展示菌株，构建抗体库，由CD70胞外段抗原对文库进行淘选，对筛选出的克隆进行鉴定，最终获得了具有特异性结合CD70抗原能力的ScFv。在进行构建嵌合抗原受体或其他用途时，对ScFv进行PCR人工合成即可得到大量靶向CD70的ScFv，即得到包括如SEQ ID NO:23所示的氨基酸序列或其功能性变体的胞外结构域。In some specific embodiments, the extracellular domain includes the amino acid sequence shown in SEQ ID NO: 23 or a functional variant thereof, namely a ScFv targeting CD70. Its preparation method is to construct a fully human single-chain antibody library through phage display technology, and through appropriate methods of affinity panning to enrich phages carrying certain fragments with specific affinity, and then obtain its gene sequence by sequencing. In order to screen for human antibodies targeting the CD70 antigen, a fully human single-chain antibody library was constructed. Using normal human PBMCs as raw materials, the total RNA was extracted to reverse transcribe cDNA, and the variable region of the antibody was amplified. (Linker) links the heavy chain variable region VH to the light chain variable region VL. It was constructed into a phagemid carrier, electroporated to display strains, and an antibody library was constructed. The library was panned by the CD70 extracellular segment antigen, and the screened clones were identified. Finally, the ScFv with the ability to specifically bind to the CD70 antigen was obtained. . When constructing chimeric antigen receptors or other uses, a large number of ScFvs targeting CD70 can be obtained by PCR artificial synthesis of ScFv, that is, the amino acid sequence shown in SEQ ID NO: 23 or its functional variants can be obtained. extracellular domain.

进一步，所述胞外结构域还包括CD8信号肽，其包含如SEQ ID NO:24所示的氨基酸序列或其功能性变体。CD70抗体是在所述CD8信号肽的引导下定位在细胞膜上，如本领域技术人员所知，所有其他的可用于CAR或者抗体领域的信号肽都可以用于本发明中，胞外段一般包含CD8α或GM-CSFRα信号肽。Further, the extracellular domain also includes a CD8 signal peptide, which comprises the amino acid sequence shown in SEQ ID NO: 24 or a functional variant thereof. The CD70 antibody is positioned on the cell membrane under the guidance of the CD8 signal peptide. As known to those skilled in the art, all other signal peptides that can be used in the field of CAR or antibodies can be used in the present invention. The extracellular segment generally includes CD8α or GM-CSFRα signal peptide.

进一步，所述铰链可以来源于：IgG、CD8、CD7、CD4及其功能性变体；所述铰链区优选包含以下氨基酸序列或其功能性变体中的任意一种：SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28，这样可以使得得到的嵌合抗原受体起到更好的细胞杀伤效果和更好的刺激细胞因子分泌。Further, the hinge can be derived from: IgG, CD8, CD7, CD4 and functional variants thereof; the hinge region preferably comprises any one of the following amino acid sequences or functional variants thereof: SEQ ID NO:25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, which can make the obtained chimeric antigen receptor have better cell killing effect and better stimulate cytokine secretion.

进一步，所述跨膜区可以来源于：CD8、CD28、CD3ε、CD4、CD16、CD137、CD80以及CD86；同理，所述跨膜区优选包含以下氨基酸序列或其功能性变体中的任意一种：SEQ ID NO:29、SEQ ID NO:30。Further, the transmembrane region can be derived from: CD8, CD28, CD3ε, CD4, CD16, CD137, CD80 and CD86; similarly, the transmembrane region preferably comprises any of the following amino acid sequences or functional variants thereof Species: SEQ ID NO:29, SEQ ID NO:30.

进一步，所述胞内信号域包含信号传导区和共刺激结构域，所述胞内信号传导区与共刺激结构域可来源于：CD3、CD137、CD28、CD27、OX40、ICOS、GITR、CD2、 CD40、PD-1、PD1L、B7-H3、淋巴细胞功能相关抗原-1(LFA-1)、ICAM-1、CD7、NKG2C、CD83、CD86以及CD127；所述信号传导区优选包含SEQ ID NO:31或SEQ ID NO:56所示的氨基酸序列或其功能性变体；所述共刺激结构域优选包含以下氨基酸序列或其功能性变体中的一种或多种：SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34。Further, the intracellular signaling domain includes a signal transduction region and a costimulatory domain, and the intracellular signal transduction region and costimulatory domain can be derived from: CD3, CD137, CD28, CD27, OX40, ICOS, GITR, CD2, CD40 , PD-1, PD1L, B7-H3, lymphocyte function-associated antigen-1 (LFA-1), ICAM-1, CD7, NKG2C, CD83, CD86 and CD127; the signal transduction region preferably comprises SEQ ID NO:31 Or the amino acid sequence shown in SEQ ID NO:56 or its functional variant; Said co-stimulatory domain preferably comprises one or more in the following amino acid sequence or its functional variant: SEQ ID NO:32, SEQ ID NO:32, SEQ ID NO: 33, SEQ ID NO: 34.

在某些具体实施例中，所述嵌合抗原受体包括以下组合中的任意一种：In some specific embodiments, said chimeric antigen receptor comprises any one of the following combinations:

组合1)：所述铰链区包含如SEQ ID NO:25所示的氨基酸序列或其功能性变体，所述跨膜区包含如SEQ ID NO:29所示的氨基酸序列或其功能性变体，所述胞内信号域包含SEQ ID NO:56所示的氨基酸序列或其功能性变体和SEQ ID NO:32所示的氨基酸序列或其功能性变体；Combination 1): the hinge region comprises an amino acid sequence as shown in SEQ ID NO:25 or a functional variant thereof, and the transmembrane region comprises an amino acid sequence as shown in SEQ ID NO:29 or a functional variant thereof , the intracellular signaling domain comprises an amino acid sequence shown in SEQ ID NO:56 or a functional variant thereof and an amino acid sequence shown in SEQ ID NO:32 or a functional variant thereof;

组合2)：所述铰链区包含如SEQ ID NO:26所示的氨基酸序列或其功能性变体，所述跨膜区包含如SEQ ID NO:30所示的氨基酸序列或其功能性变体，所述胞内信号域包含SEQ ID NO:56所示的氨基酸序列或其功能性变体和SEQ ID NO:33所示的氨基酸序列或其功能性变体；Combination 2): the hinge region comprises an amino acid sequence as shown in SEQ ID NO:26 or a functional variant thereof, and the transmembrane region comprises an amino acid sequence as shown in SEQ ID NO:30 or a functional variant thereof , the intracellular signaling domain comprises an amino acid sequence shown in SEQ ID NO:56 or a functional variant thereof and an amino acid sequence shown in SEQ ID NO:33 or a functional variant thereof;

组合3)：所述铰链区包含如SEQ ID NO:28所示的氨基酸序列或其功能性变体，所述跨膜区包含如SEQ ID NO:30所示的氨基酸序列或其功能性变体，所述胞内信号域包含如SEQ ID NO:31所示的氨基酸序列或其功能性变体和SEQ ID NO:34所示的氨基酸序列或其功能性变体；Combination 3): the hinge region comprises the amino acid sequence shown in SEQ ID NO: 28 or a functional variant thereof, and the transmembrane region comprises the amino acid sequence shown in SEQ ID NO: 30 or a functional variant thereof , the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO: 31 or a functional variant thereof and the amino acid sequence shown in SEQ ID NO: 34 or a functional variant thereof;

组合4)：所述铰链区包含如SEQ ID NO:27所示的氨基酸序列或其功能性变体，所述跨膜区包含如SEQ ID NO:30所示的氨基酸序列或其功能性变体，所述胞内信号域包含SEQ ID NO:31所示的氨基酸序列或其功能性变体和SEQ ID NO:34所示的氨基酸序列或其功能性变体；Combination 4): the hinge region comprises an amino acid sequence as shown in SEQ ID NO:27 or a functional variant thereof, and the transmembrane region comprises an amino acid sequence as shown in SEQ ID NO:30 or a functional variant thereof , the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO: 31 or a functional variant thereof and the amino acid sequence shown in SEQ ID NO: 34 or a functional variant thereof;

组合5)：所述铰链区包含如SEQ ID NO:28所示的氨基酸序列或其功能性变体，所述跨膜区包含如SEQ ID NO:29所示的氨基酸序列或其功能性变体，所述胞内信号域包含SEQ ID NO:56所示的氨基酸序列或其功能性变体和SEQ ID NO:32所示的氨基酸序列或其功能性变体。Combination 5): the hinge region comprises an amino acid sequence as shown in SEQ ID NO:28 or a functional variant thereof, and the transmembrane region comprises an amino acid sequence as shown in SEQ ID NO:29 or a functional variant thereof , the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:56 or a functional variant thereof and the amino acid sequence shown in SEQ ID NO:32 or a functional variant thereof.

在某些实施例中，所述嵌合抗原受体包含天然杀伤细胞受体(NKR)的一种或多种组分，因而形成NKR-CAR。NKR组分可以是来自以下任何天然杀伤细胞受体的跨膜结构域、铰链结构域或胞质结构域：杀伤细胞免疫球蛋白样受体(KIR)，例如KIR2DL1、KIR2DL2/L3、KIR2DL4、KIR2DL5A、KIR2DL5B、KIR2DS1、KIR2DS2、KIR2DS3、KIR2DS4、DIR2DS5、KIR3DL1/S1、KIR3DL2、KIR3DL3、KIR2DP1和KIR3DP1；天然细胞毒性受体(NCR)，例如，NKp30、NKp44、NKp46；免疫细胞受体的信号传导淋巴细胞活化分子(SLAM)家族，例如，CD48、CD229、2B4、CD84、NTB-A、CRA、BLAME和CD2F-10；Fc受体(FcR)，例如，CD16、和CD64；和Ly49受体，例如，LY49A、LY49C。所述的NKR-CAR分子可以与衔接分子或胞内共刺激结构域(例如，DAP12)相互作用。In certain embodiments, the chimeric antigen receptor comprises one or more components of a natural killer cell receptor (NKR), thereby forming a NKR-CAR. The NKR component can be a transmembrane domain, hinge domain, or cytoplasmic domain from any of the following natural killer cell receptors: killer cell immunoglobulin-like receptors (KIRs), such as KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A , KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP1; natural cytotoxicity receptors (NCRs), e.g., NKp30, NKp44, NKp46; signaling lymphoid receptors for immune cells Cell Activation Molecule (SLAM) family, e.g., CD48, CD229, 2B4, CD84, NTB-A, CRA, BLAME, and CD2F-10; Fc receptors (FcR), e.g., CD16, and CD64; and Ly49 receptors, e.g. , LY49A, LY49C. The NKR-CAR molecule can interact with an adapter molecule or an intracellular co-stimulatory domain (eg, DAP12).

第十二方面，本发明目的还在于提供一种编码前述的嵌合抗原受体的核酸序列，该核酸序列作为目的基因与运载体结合一同转染T细胞得到CAR-T细胞。In the twelfth aspect, the purpose of the present invention is to provide a nucleic acid sequence encoding the above-mentioned chimeric antigen receptor, and the nucleic acid sequence is used as a target gene combined with a carrier to transfect T cells together to obtain CAR-T cells.

编码所述嵌合抗原受体中组合1)的核酸序列a，包含如SEQ ID NO:35所示的序列；或编码所述嵌合抗原受体中组合2)的核酸序列b，包含如SEQ ID NO:36所示的序列；或编码所述嵌合抗原受体中组合3)的核酸序列c，包含如SEQ ID NO:37所示的序列；或编码所述嵌合抗原受体中组合4)的核酸序列d，包含如SEQ ID NO:38所示的序列；或编码所述组合5)的核酸序列e，包含如SEQ ID NO:58所示的序列。Combination 1) nucleic acid sequence a of coding described chimeric antigen receptor, comprising the sequence shown in SEQ ID NO:35; Or coding described chimeric antigen receptor combination 2) nucleic acid sequence b, comprising such as SEQ The sequence shown in ID NO:36; Or the nucleic acid sequence c of combination 3) in the coding described chimeric antigen receptor, comprising the sequence shown in SEQ ID NO:37; Or the combination in the coding described chimeric antigen receptor 4) the nucleic acid sequence d, comprising the sequence shown in SEQ ID NO:38; or the nucleic acid sequence e encoding said combination 5), comprising the sequence shown in SEQ ID NO:58.

进一步，所述嵌合抗原受体的核酸序列还包括启动子，所述启动子的核酸序列如SEQ ID NO:40或SEQ ID NO:41所示。Further, the nucleic acid sequence of the chimeric antigen receptor also includes a promoter, and the nucleic acid sequence of the promoter is shown in SEQ ID NO:40 or SEQ ID NO:41.

在某些具体实施例中，所述核酸序列a的5'端连接启动子，所述启动子的核酸序列如SEQ ID NO:40或SEQ ID NO:41所示；或所述核酸序列b的5'端连接启动子，所述启动子的核酸序列如SEQ ID NO:41所示；或所述核酸序列c的5'端连接启动子，所述启动子的核酸序列如SEQ ID NO:40或SEQ ID NO:41所示；或所述核酸序列d的5'端连接启动子，所述启动子的核酸序列如SEQ ID NO:40或SEQ ID NO:41所示；或所述核酸序列e的5'端连接启动子，所述启动子的核酸序列如SEQ ID NO:41所示。In some specific embodiments, the 5' end of the nucleic acid sequence a is connected to a promoter, the nucleic acid sequence of the promoter is shown in SEQ ID NO: 40 or SEQ ID NO: 41; or the nucleic acid sequence b is The 5' end is connected to a promoter, and the nucleotide sequence of the promoter is shown in SEQ ID NO: 41; or the 5' end of the nucleic acid sequence c is connected to a promoter, and the nucleotide sequence of the promoter is shown in SEQ ID NO: 40 Or shown in SEQ ID NO:41; Or the 5' end of described nucleic acid sequence d connects promoter, the nucleic acid sequence of described promoter is shown in SEQ ID NO:40 or SEQ ID NO:41; Or described nucleic acid sequence The 5' end of e is connected to a promoter, and the nucleotide sequence of the promoter is shown in SEQ ID NO:41.

在某些具体实施例中，所述核酸序列a的5'端连接信号肽序列a；或所述核酸序列b的5'端连接信号肽序列b；或所述核酸序列c的5'端连接信号肽序列c；或所述核酸序列d的5'端连接信号肽序列d；或所述核酸序列e的5'端连接信号肽序列e；所述信号肽序列a、所述信号肽序列b、所述信号肽序列c、所述信号肽序列d、所述信号肽序列e的核酸序列均如SEQ ID NO:39所示。所述信号肽序列a的5'端连接启动子，所述启动子的核酸序列如SEQ ID NO:40或SEQ ID NO:41所示；或所述信号肽序列b的5'端连接启动子，所述启动子的核酸序列如SEQ ID NO:41所示；或所述信号肽序列c的5'端连接启动子，所述启动子的核酸序列如SEQ ID NO:40或SEQ ID NO:41所示；或所述信号肽序列d的5'端连接启动子，所述启动子的核酸序列如SEQ ID NO:40或SEQ ID NO:41所示；或所述信号肽序列e的5'端连接启动子，所述启动子的核酸序列如SEQ ID NO:41所示。In some specific embodiments, the 5' end of the nucleic acid sequence a is connected to the signal peptide sequence a; or the 5' end of the nucleic acid sequence b is connected to the signal peptide sequence b; or the 5' end of the nucleic acid sequence c is connected to Signal peptide sequence c; or the 5' end of the nucleic acid sequence d is connected to the signal peptide sequence d; or the 5' end of the nucleic acid sequence e is connected to the signal peptide sequence e; the signal peptide sequence a, the signal peptide sequence b The nucleic acid sequences of the signal peptide sequence c, the signal peptide sequence d, and the signal peptide sequence e are all shown in SEQ ID NO:39. The 5' end of the signal peptide sequence a is connected to a promoter, and the nucleic acid sequence of the promoter is shown in SEQ ID NO: 40 or SEQ ID NO: 41; or the 5' end of the signal peptide sequence b is connected to a promoter , the nucleotide sequence of the promoter is shown in SEQ ID NO: 41; or the 5' end of the signal peptide sequence c is connected to the promoter, the nucleotide sequence of the promoter is such as SEQ ID NO: 40 or SEQ ID NO: 41; or the 5' end of the signal peptide sequence d is connected to a promoter, the nucleic acid sequence of the promoter is shown in SEQ ID NO: 40 or SEQ ID NO: 41; or 5 of the signal peptide sequence e The 'end is connected to a promoter, and the nucleotide sequence of the promoter is shown in SEQ ID NO:41.

CD70是活化的T细胞和NK细胞会表达的分子，如果肿瘤微环境中存在上述这种表达CD70的免疫细胞；或者CAR-T细胞自身表达的CD70由于CAR-T细胞的活化而表达升高，就可能会造成“本底较高”。进一步，为了在本底较高的肿瘤环境中发挥CAR的肿瘤特异性的杀伤能力，可以在本发明中的CAR的结构中加上CD70干扰RNA序列。具体地，编码所述嵌合抗原受体的核酸序列(可以包括或不包括启动子)还可以包括CD70干扰RNA，所述CD70干扰RNA序列如SEQ ID NO:42所示。当然，还可以选择其他适合的手段(例如使用基因编辑方法(如本领域技术人员熟知的Cas9、TALE和锌指酶技术)对CAR-T细胞内源性CD70进行敲除，还可以使用内质网阻滞肽连接抗CD70的抗体，对免疫细胞自体表达的CD70进行阻滞等)，以敲除或者破坏CAR-T细胞自身内源性表达的CD70而降低CAR-T可能的耗竭。CD70 is a molecule expressed by activated T cells and NK cells. If the above-mentioned CD70-expressing immune cells exist in the tumor microenvironment; or the expression of CD70 expressed by CAR-T cells is increased due to the activation of CAR-T cells, It may cause "high background". Further, in order to exert the tumor-specific killing ability of CAR in a tumor environment with a high background, a CD70 interfering RNA sequence can be added to the structure of the CAR in the present invention. Specifically, the nucleic acid sequence (which may or may not include a promoter) encoding the chimeric antigen receptor may also include CD70 interfering RNA, and the CD70 interfering RNA sequence is shown in SEQ ID NO:42. Of course, other suitable means can also be selected (such as using gene editing methods (such as Cas9, TALE and zinc finger enzyme technology well known to those skilled in the art) to knock out the endogenous CD70 of CAR-T cells, and endogenous CD70 can also be used to The anti-CD70 antibody is linked to the network blocking peptide to block the CD70 expressed by the immune cells, etc.) to knock out or destroy the endogenously expressed CD70 of the CAR-T cells themselves to reduce the possible exhaustion of CAR-T.

在某些实施例中，为了克服实体瘤中的肿瘤微环境，可以在本发明的CAR的结构中加上缺氧启动子。In some embodiments, in order to overcome the tumor microenvironment in solid tumors, a hypoxic promoter can be added to the structure of the CAR of the present invention.

在某些具体实施例中，所述核酸序列d或所述信号肽序列d的5'端连接启动子，所述启动子的核酸序列如SEQ ID NO:40或SEQ ID NO:41所示；所述核酸序列d的3'端连接CD70干扰RNA序列，所述CD70干扰RNA序列如SEQ ID NO:42所示。In some specific embodiments, the 5' end of the nucleic acid sequence d or the signal peptide sequence d is connected to a promoter, and the nucleic acid sequence of the promoter is shown in SEQ ID NO:40 or SEQ ID NO:41; The 3' end of the nucleic acid sequence d is connected to a CD70 interfering RNA sequence, and the CD70 interfering RNA sequence is shown in SEQ ID NO:42.

第十三方面，本发明目的还在于提供一种包含前述任一所述的嵌合抗原受体的核酸序列的表达载体。In the thirteenth aspect, the purpose of the present invention is to provide an expression vector comprising the nucleic acid sequence of any one of the aforementioned chimeric antigen receptors.

进一步，所述表达载体选自慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、腺相关病毒表达载体、DNA载体、RNA载体、质粒中的任一种。Further, the expression vector is selected from any one of lentivirus expression vector, retrovirus expression vector, adenovirus expression vector, adeno-associated virus expression vector, DNA vector, RNA vector and plasmid.

在某些实施例中，载体包含左(5')逆转录病毒LTR、Psi(Ψ)包装信号、中心多嘌呤段/DNA瓣(cPPT/FLAP)、逆转录病毒导出元件、可操作地连接到编码本发明所涵盖的CAR的多核苷酸的启动子和右(3')逆转录病毒LTR。In certain embodiments, the vector comprises a left (5') retroviral LTR, a Psi (Ψ) packaging signal, a central polypurine tract/DNA flap (cPPT/FLAP), a retroviral export element, operably linked to The promoter and right (3') retroviral LTR of the polynucleotide encoding the CAR encompassed by the invention.

在某些实施例中，编码前述的嵌合抗原受体的核酸序列包含优化的Kozark序列。In certain embodiments, the nucleic acid sequence encoding the aforementioned chimeric antigen receptor comprises an optimized Kozark sequence.

在某些实施例中，所述基因表达载体可以包含分泌型抗PD-1ScFv。在某些实施例中，所述基因表达载体包含PD-1共轭转导肽(如PD-1-CD28-CD137-CD3信号结构)。在某些实施例中，所述基因表达载体可以包含多个CAR组合，如2个靶向不同抗原或同一抗原的不同识别位点的CAR组合。In certain embodiments, the gene expression vector may contain secreted anti-PD-1 ScFv. In certain embodiments, the gene expression vector comprises a PD-1 conjugated transduction peptide (such as a PD-1-CD28-CD137-CD3 signal structure). In some embodiments, the gene expression vector may contain multiple CAR combinations, such as two CAR combinations targeting different antigens or different recognition sites of the same antigen.

第十四方面，本发明目的还在于提供一种工程化的细胞，所述细胞中转导有前述任一所述的嵌合抗原受体的核酸序列或前述任一所述的表达载体；优选地，所述细胞为T细胞、T细胞前体或NK细胞。In the fourteenth aspect, the purpose of the present invention is to provide an engineered cell transduced with the nucleic acid sequence of any of the aforementioned chimeric antigen receptors or any of the aforementioned expression vectors; preferably Preferably, the cells are T cells, T cell precursors or NK cells.

第十五方面，本发明目的还在于进一步提供一种细胞制品，所述细胞制品包括前述任一所述的工程化的细胞。In the fifteenth aspect, the purpose of the present invention is to further provide a cell product, the cell product comprising any one of the above-mentioned engineered cells.

进一步，所述细胞制品还包括其他可增强CAR表达活性的活性剂。Further, the cell preparation also includes other active agents that can enhance the expression activity of CAR.

在某些具体实施例中，所述活性剂为免疫抑制剂，例如环孢素(cyclosporin)、硫唑嘌呤(azathioprine)、甲氨蝶呤(methotrexate)、霉酚酸酯(mycophenolate)和FK506；抗体或其它免疫清除剂(immunoablativeagents)，例如CAMPATH、抗CD3抗体或其它抗体治疗、环磷酰胺(cytoxan)、氟达拉滨(fludarabine)、环孢素(cyclosporin)、雷帕霉素(rapamycin)、霉酚酸(mycophenolicacid)、类固醇(steroids)、FR901228、细胞因子和辐射。In certain embodiments, the active agent is an immunosuppressant, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506; Antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibody or other antibody therapy, cytoxan, fludarabine, cyclosporin, rapamycin , mycophenolic acid, steroids, FR901228, cytokines and radiation.

在某些具体实施例中，增强CAR表达细胞活性的活性剂可以是阻断抑制性分子的活性剂。在一些实施例中，抑制性分子(例如PD1)可以降低CAR表达细胞的发动免疫效应子反应的能力。抑制性分子包括PD1、PD-L1、CTLA4、TIM3、LAG3、VISTA、BTLA、TIGIT、LAIR1、CD160、2B4、CEACAM(CEACAM-1、CEACAM-3、CEACAM-5)、LAG3、VISTA、BTLA、TIG、LAIR1、CD160、2B4、CD80、CD86、B7-H3(CD276)、B7-H4(VTCN1)、HVEM(TNFRSF14或CD270)、KIR、A2aR、MHC I类、MHC II类、GAL9、腺苷、TGFR(TGFRβ)和TGFRβ。所述抑制性分子的胞外结构域可以融合到跨膜结构域和胞内信号传导结构域，比如PD1 CAR。In certain embodiments, the active agent that enhances the activity of CAR-expressing cells may be an active agent that blocks inhibitory molecules. In some embodiments, an inhibitory molecule (eg, PD1) can reduce the ability of a CAR-expressing cell to mount an immune effector response. Inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (CEACAM-1, CEACAM-3, CEACAM-5), LAG3, VISTA, BTLA, TIG , LAIR1, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM(TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, TGFR (TGFRβ) and TGFRβ. The extracellular domain of the inhibitory molecule can be fused to a transmembrane domain and an intracellular signaling domain, such as a PD1 CAR.

进一步，多种另外的治疗剂可以与本文所述的组合物联合使用。例如，潜在有用的另外的治疗剂包括PD-1抑制剂，例如纳武单抗(nivolumab)、派姆单抗(pembrolizumab)、阿巴伏单抗(Abagovomab)和阿特珠单抗(Atezolizumab)。Further, various additional therapeutic agents can be used in combination with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab, pembrolizumab, abavolumab, and atezolizumab .

具体地，适合于与本发明组合使用的另外的治疗剂包括但不限于：伊布替尼(ibrutinib)、奥法木单抗(ofatumumab)、利妥昔单抗(rituximab)、贝伐单抗(bevacizumab)、曲妥珠单抗(trastuzumab)、trastuzumabemtansine、伊马替尼(imatinib)、西妥昔单抗(cetuximab)、帕尼单抗(panitumumab)、卡妥索单抗(Catumaxomab)、替伊莫单抗(ibritumomab)、奥法木单抗、托西莫单抗(tositumomab)、本妥昔单抗(brentuximab)、阿仑单抗(alemtuzumab)、吉妥珠单抗(gemtuzumab)、厄洛替尼(erlotinib)、吉非替尼(gefitinib)、凡德他尼(vandetanib)、阿法替尼(afatinib)、拉帕替尼(lapatinib)、来那替尼(neratinib)、阿昔替尼(axitinib)、马赛替尼(masitinib)、帕唑帕尼(pazopanib)、舒尼替尼(sunitinib)、索拉非尼(sorafenib)、toceranib、来他替尼(lestaurtinib)、阿昔替尼(axitinib)、西地尼布(cediranib)、乐伐替尼(lenvatinib)、尼达尼布(nintedanib)、帕唑帕尼(pazopanib)、瑞格非尼(regorafenib)、司马沙尼(semaxanib)、索拉非尼(sorafenib)、舒尼替尼(sunitinib)、tivozanib、toceranib、凡德他尼、恩曲替尼(entrectinib)、卡博替尼(cabozantinib)、伊马替尼(imatinib)、达沙替尼(dasatinib)、尼洛替尼(nilotinib)、帕纳替尼(ponatinib)、雷多替尼(radotinib)、博舒替尼(bosutinib)、来他替尼(lestaurtinib)、鲁索替尼(ruxolitinib)、pacritinib、考比替尼(cobimetinib)、司美替尼(selumetinib)、曲美替尼(trametinib)、binimetinib、阿雷替尼(alectinib)、色瑞替尼(ceritinib)、克唑替尼(crizotinib)、阿柏西普(aflibercept)、adipotide、地尼白介素。Specifically, additional therapeutic agents suitable for use in combination with the present invention include, but are not limited to: ibrutinib, ofatumumab, rituximab, bevacizumab (bevacizumab), trastuzumab, trastuzumab emtansine, imatinib, cetuximab, panitumumab, catumaxomab, Ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, ermatizumab Erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib Axitinib, masitinib, pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib (axitinib), cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib , sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, Dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, Russo Ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, Crizotinib, aflibercept, adipotide, denileukin.

进一步，所述细胞制品还可以与一些治疗手段组合使用，所述治疗手段可以是手术、化疗、放疗。Furthermore, the cell product can also be used in combination with some treatment means, such as surgery, chemotherapy, and radiotherapy.

第十六方面，本发明目的还在于进一步提供一种药物组合物，所述药物组合物中包括前述任一所述的嵌合抗原受体的核酸序列或前述任一所述的表达载体或前述任一所述的工程化的细胞。In the sixteenth aspect, the purpose of the present invention is to further provide a pharmaceutical composition, which includes the nucleic acid sequence of any of the aforementioned chimeric antigen receptors or any of the aforementioned expression vectors or the aforementioned Any of the engineered cells described.

第十七方面，本发明目的还在于提供一种前述任一所述的嵌合抗原受体或前述任一所述的嵌合抗原受体的核酸序列或前述任一所述的表达载体或前述任一所述的工程化的细胞或前述任一所述的细胞制品或前述任一所述的药物组合物在制备抗肿瘤药物中的应用。In the seventeenth aspect, the purpose of the present invention is to provide any of the aforementioned chimeric antigen receptors or the nucleic acid sequence of any of the aforementioned chimeric antigen receptors or any of the aforementioned expression vectors or the aforementioned Application of any of the above-mentioned engineered cells or any of the above-mentioned cell products or any of the above-mentioned pharmaceutical compositions in the preparation of anti-tumor drugs.

具体地，所述肿瘤包含肾癌、血源性恶性肿瘤、胸腺肿瘤、卵巢癌、成神经胶质细胞瘤、鼻咽癌、胰腺癌和胃肠道癌的一种或多种。所述胃肠道癌包括食道癌、结肠癌和胃癌中的一种或多种。Specifically, the tumor comprises one or more of renal cancer, hematogenous malignant tumor, thymus tumor, ovarian cancer, glioblastoma, nasopharyngeal cancer, pancreatic cancer and gastrointestinal tract cancer. The gastrointestinal cancer includes one or more of esophageal cancer, colon cancer and gastric cancer.

本发明提供的嵌合抗原受体及CAR-T细胞可以有效杀伤786-O-Luc-GFP靶细胞，以及可以分泌有效量的细胞因子。The chimeric antigen receptor and CAR-T cells provided by the present invention can effectively kill 786-O-Luc-GFP target cells, and can secrete effective amounts of cytokines.

本发明提供的嵌合抗原受体及CAR-T细胞可以有效抑制体内肿瘤的生长，抗表达CD70靶点的肿瘤效果显著。The chimeric antigen receptor and CAR-T cells provided by the present invention can effectively inhibit the growth of tumors in vivo, and have a significant anti-tumor effect on expressing CD70 targets.

本发明中，术语“功能性变体”是指包括与参考氨基酸序列具有基本上相同的功能(例如，可以具备参考氨基酸序列的一种或多种的活性)，且与参考氨基酸序列具有至少85％(例如，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％，或至少100％)序列同一性的氨基酸序列。在某些实施例中，所述功能性变体具有与参考氨基酸序列相同或较低或较高的活性。In the present invention, the term "functional variant" refers to a variant that has substantially the same function as the reference amino acid sequence (for example, may have one or more activities of the reference amino acid sequence), and has at least 85% of the reference amino acid sequence. % (e.g., 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 at least 100%) sequence identity of amino acid sequences. In certain embodiments, the functional variant has the same or lower or higher activity than the reference amino acid sequence.

附图说明Description of drawings

为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍。显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the description of the embodiments or the prior art. Apparently, the drawings in the following description are some embodiments of the present invention, and those skilled in the art can obtain other drawings according to these drawings without any creative effort.

图1为ELISA检测阳性克隆情况。Figure 1 shows the situation of positive clones detected by ELISA.

图2为人源化ScFv A-7-18对K562细胞流式染色。Figure 2 shows the flow cytometric staining of humanized ScFv A-7-18 on K562 cells.

图3为人源化ScFv A-7-18对K562-CD70细胞流式染色。Figure 3 shows the flow cytometric staining of humanized ScFv A-7-18 on K562-CD70 cells.

图4为DK-10-A01对K562细胞流式染色。Figure 4 is the flow cytometric staining of DK-10-A01 on K562 cells.

图5为DK-10-A02对K562细胞流式染色。Figure 5 shows the flow cytometric staining of DK-10-A02 on K562 cells.

图6为DK-10-A03对K562细胞流式染色。Figure 6 shows the flow cytometric staining of DK-10-A03 on K562 cells.

图7为DK-10-A04对K562细胞流式染色。Figure 7 shows the flow cytometric staining of DK-10-A04 on K562 cells.

图8为DK-10-A05对K562细胞流式染色。Figure 8 is the flow cytometric staining of DK-10-A05 on K562 cells.

图9为DK-10-A06对K562细胞流式染色。Figure 9 shows the flow cytometric staining of DK-10-A06 on K562 cells.

图10为DK-10-A07对K562细胞流式染色。Figure 10 shows the flow cytometric staining of DK-10-A07 on K562 cells.

图11为DK-10-A01对K562-CD70细胞流式染色。Figure 11 shows the flow cytometric staining of DK-10-A01 on K562-CD70 cells.

图12为DK-10-A02对K562-CD70细胞流式染色。Figure 12 shows the flow cytometric staining of DK-10-A02 on K562-CD70 cells.

图13为DK-10-A03对K562-CD70细胞流式染。Figure 13 shows the flow cytometry staining of K562-CD70 cells by DK-10-A03.

图14为DK-10-A04与CD70抗原的结合动力学检测。Figure 14 shows the detection of binding kinetics between DK-10-A04 and CD70 antigen.

图15为DK-10-A05对K562-CD70细胞流式染色。Figure 15 shows the flow cytometric staining of DK-10-A05 on K562-CD70 cells.

图16为DK-10-A06对K562-CD70细胞流式染色。Figure 16 shows the flow cytometric staining of DK-10-A06 on K562-CD70 cells.

图17为DK-10-A07对K562-CD70细胞流式染色。Figure 17 shows the flow cytometric staining of DK-10-A07 on K562-CD70 cells.

图18为人源化ScFv A-7-18与CD70抗原的结合动力学检测。Figure 18 shows the detection of binding kinetics between humanized ScFv A-7-18 and CD70 antigen.

图19为DK-10-A01与CD70抗原的结合动力学检测。Figure 19 shows the detection of binding kinetics between DK-10-A01 and CD70 antigen.

图20为DK-10-A02与CD70抗原的结合动力学检测。Figure 20 shows the detection of binding kinetics between DK-10-A02 and CD70 antigen.

图21为DK-10-A03与CD70抗原的结合动力学检测。Figure 21 shows the detection of binding kinetics between DK-10-A03 and CD70 antigen.

图22为DK-10-A04与CD70抗原的结合动力学检测。Figure 22 is the detection of binding kinetics between DK-10-A04 and CD70 antigen.

图23为DK-10-A05与CD70抗原的结合动力学检测。Figure 23 is the detection of binding kinetics between DK-10-A05 and CD70 antigen.

图24为DK-10-A07与CD70抗原的结合动力学检测。Figure 24 shows the detection of binding kinetics between DK-10-A07 and CD70 antigen.

图25为不同ScFv CAR-T制剂体外细胞杀伤结果。Figure 25 shows the in vitro cell killing results of different ScFv CAR-T preparations.

图26为不同ScFv CAR-T制剂体外IFN-γ细胞因子分泌结果。Figure 26 shows the results of in vitro IFN-γ cytokine secretion of different ScFv CAR-T preparations.

图27为不同ScFv CAR-T制剂体外IL-2细胞因子分泌结果。Figure 27 shows the secretion results of IL-2 cytokines in vitro by different ScFv CAR-T preparations.

图28为不同ScFv CAR-T制剂活体成像荧光值变化曲线。Figure 28 is a graph showing the variation curves of fluorescence values for in vivo imaging of different ScFv CAR-T preparations.

图29为不同ScFv CAR-T制剂的肿瘤体积生长曲线。Figure 29 is the tumor volume growth curves of different ScFv CAR-T preparations.

图30为不同ScFv CAR-T制剂的活体成像图。Figure 30 is an in vivo imaging diagram of different ScFv CAR-T preparations.

图31为含CD70(1)ScFv的不同CAR-T制剂的体外阳性率检测结果。Figure 31 shows the in vitro positive rate detection results of different CAR-T preparations containing CD70(1) ScFv.

图32为含CD70(1)ScFv的不同CAR-T制剂的体外细胞杀伤结果。Figure 32 shows the in vitro cell killing results of different CAR-T preparations containing CD70(1) ScFv.

图33为含CD70(1)ScFv的不同CAR-T制剂的体外细胞因子分泌结果。Figure 33 is the in vitro cytokine secretion results of different CAR-T preparations containing CD70(1) ScFv.

图34为含CD70(1)ScFv的不同CAR-T制剂的体内CAR阳性率检测结果。Fig. 34 is the in vivo CAR positive rate detection results of different CAR-T preparations containing CD70(1) ScFv.

图35为含CD70(1)ScFv的不同CAR-T制剂的活体成像荧光值变化曲线。Fig. 35 is a graph showing the change in fluorescence value of in vivo imaging of different CAR-T preparations containing CD70(1) ScFv.

图36为含CD70(1)ScFv的不同CAR-T制剂的体内肿瘤体积生长曲线。Figure 36 is the in vivo tumor volume growth curves of different CAR-T preparations containing CD70(1) ScFv.

图37为含CD70(1)ScFv的不同CAR-T制剂的活体成像图。Figure 37 is an in vivo imaging diagram of different CAR-T preparations containing CD70(1) ScFv.

图38为CAR-8和CAR-9的体外阳性率检测结果。Figure 38 shows the in vitro positive rate detection results of CAR-8 and CAR-9.

图39为CAR-8和CAR-9的体外细胞杀伤结果。Figure 39 shows the in vitro cell killing results of CAR-8 and CAR-9.

图40为CAR-8和CAR-9的体外细胞因子分泌结果。Figure 40 is the in vitro cytokine secretion results of CAR-8 and CAR-9.

具体实施方式Detailed ways

为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Apparently, the described embodiments are some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

需要说明的是，在本文中，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者装置不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者装置所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括该要素的过程、方法、物品或者装置中还存在另外的相同要素。It should be noted that, in this document, the term "comprising", "comprising" or any other variation thereof is intended to cover a non-exclusive inclusion such that a process, method, article or apparatus comprising a set of elements includes not only those elements, It also includes other elements not expressly listed, or elements inherent in the process, method, article, or device. Without further limitations, an element defined by the phrase "comprising a ..." does not preclude the presence of additional identical elements in the process, method, article, or apparatus comprising that element.

如在本说明书中使用的，术语“大约”，典型地表示为所述值的+/-5％，更典型的是所述值的+/-4％，更典型的是所述值的+/-3％，更典型的是所述值的+/-2％，甚至更典型的是所述值的+/-1％，甚至更典型的是所述值的+/-0.5％。As used in this specification, the term "about" typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 4% of the stated value /-3%, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, even more typically +/-0.5% of the stated value.

在本说明书中，某些实施方式可能以一种处于某个范围的格式公开。应该理解，这种“处于某个范围”的描述仅仅是为了方便和简洁，且不应该被解释为对所公开范围的僵化限制。因此，范围的描述应该被认为是已经具体地公开了所有可能的子范围以及在此范围内的独立数字值。例如，范围1～6的描述应该被看作已经具体地公开了子范围如从1到3，从1到4，从1到5，从2到4，从2到6，从3到6等，以及此范围内的单独数字，例如1，2，3，4，5和6。无论该范围的广度如何，均适用以上规则。In this specification, certain embodiments may be disclosed in a range of formats. It should be understood that this description "within a certain range" is merely for convenience and brevity, and should not be construed as an inflexible limitation on the disclosed scope. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, a description of a range 1 to 6 should be read as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc. , and individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the scope.

所举实施例是为了更好地对本发明进行说明，但并不是本发明的内容仅局限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。The examples given are for better description of the present invention, but the content of the present invention is not limited to the examples given. Therefore, non-essential improvements and adjustments to the implementation by those skilled in the art based on the content of the invention above still fall within the protection scope of the present invention.

实施例一：靶向CD70的抗原结合片段和单链抗体及其应用Example 1: Antigen-binding fragments and single-chain antibodies targeting CD70 and their applications

本发明实施例中，抗原淘选的具体实施过程如下：In the embodiment of the present invention, the specific implementation process of antigen panning is as follows:

(1)封闭：5％脱脂奶粉以PBS溶解，过滤后作为封闭液，以适量封闭液分别重悬噬菌体与CD70-proteinG偶联磁珠，滚转混匀；(1) Blocking: Dissolve 5% skimmed milk powder in PBS, filter and use as blocking solution, resuspend phage and CD70-proteinG coupled magnetic beads with appropriate amount of blocking solution, and roll to mix;

(2)共孵育：将CD70-proteinG偶联磁珠置于磁力架，弃上清后以噬菌体将磁珠重悬，滚转共孵育；(2) Co-incubation: place CD70-proteinG coupled magnetic beads on a magnetic stand, discard the supernatant, resuspend the magnetic beads with phage, and roll and co-incubate;

(3)清洗：将磁珠噬菌体混合液置于磁力架，弃上清后加入适量体积的清洗液(例如Tween-80 PBST清洗液)对磁珠进行清洗，根据不同的淘选轮数进行清洗次数的确定；(3) Cleaning: Put the magnetic bead phage mixture on the magnetic stand, discard the supernatant and add an appropriate volume of cleaning solution (such as Tween-80 PBST cleaning solution) to clean the magnetic beads, and clean according to the number of rounds of panning determination of the number of times;

(4)洗脱：将清洗后的磁珠置于磁力架，吸干上清，加入pH为2.2的Gly-HCl，混匀后室温孵育7min后，加入Tris-HCl调节pH接近中性。最终将混合物置于磁力架，将噬菌体上清转移至新的1.5mL EP管完成一轮淘选；(4) Elution: Place the washed magnetic beads on a magnetic stand, blot dry the supernatant, add Gly-HCl with a pH of 2.2, mix well and incubate at room temperature for 7 minutes, then add Tris-HCl to adjust the pH to near neutral. Finally, the mixture was placed on the magnetic stand, and the phage supernatant was transferred to a new 1.5mL EP tube to complete a round of panning;

(5)富集：将噬菌体接入到TGI菌液中进行感染，37℃静置后离心，保留200μL培养基重悬沉淀，涂布于2YTAG板，倒置过夜培养；(5) Enrichment: Infect the bacteriophage into the TGI bacterial solution, let stand at 37°C and then centrifuge, retain 200 μL of medium to resuspend the pellet, spread it on a 2YTAG plate, and culture it upside down overnight;

(6)洗板：将过夜培养的平板由培养基洗下菌斑，作为下一轮文库包装的种子菌液。(6) Plate washing: Wash the plate cultured overnight to remove plaques from the culture medium, which will be used as the seed bacterial solution for the next round of library packaging.

本发明实施例中，ELISA检测的具体实施过程如下：In the embodiment of the present invention, the specific implementation process of ELISA detection is as follows:

(1)包被：用碳酸盐包被缓冲液稀释CD70抗原至1μg/mL，100μL/孔加入至96孔板，4℃封盖过夜；(1) Coating: Dilute the CD70 antigen to 1 μg/mL with carbonate coating buffer, add 100 μL/well to a 96-well plate, and cover overnight at 4°C;

(2)噬菌体样品的获得：将过夜培养的噬菌体单克隆重组菌液4℃、8000rpm离心10min，取上清作为检测样品；(2) Acquisition of phage samples: Centrifuge the overnight cultured phage monoclonal recombinant bacterial solution at 4°C and 8000rpm for 10min, and take the supernatant as a test sample;

(3)封闭：将抗原包被板在洗板机上以PBS洗板3次后，每孔加入5％脱脂奶粉封闭液，37℃封闭；(3) Blocking: wash the antigen-coated plate 3 times with PBS on a plate washer, add 5% skimmed milk powder blocking solution to each well, and block at 37°C;

(4)抗体孵育：将洗板机以PBS洗板3次后，每孔加入100μL待检噬菌体单克隆重组菌液，37℃孵育1h；(4) Antibody incubation: After washing the plate 3 times with PBS in a plate washer, add 100 μL of monoclonal recombinant bacteriophage solution to be tested to each well, and incubate at 37°C for 1 hour;

(5)加二抗：将洗板机以PBS洗板3次，每孔加入1：5000Anti-M13-HRP二抗100μL，37℃孵育1h；(5) Add secondary antibody: Wash the plate 3 times with PBS in a plate washer, add 100 μL of 1:5000 Anti-M13-HRP secondary antibody to each well, and incubate at 37°C for 1 hour;

(6)显色：将洗板机洗板6次，每孔加入TMB显色液100μL，室温避光放置25min显色；(6) Color development: Wash the plate 6 times with a plate washer, add 100 μL of TMB color development solution to each well, and place it at room temperature in the dark for 25 minutes to develop color;

(7)终止：每孔加入50μL 2M H2SO4终止反应；(7) Termination: Add 50 μL 2M H2SO4 to each well to terminate the reaction;

(8)检测：将检测板置于酶标仪中检测OD ₄₅₀吸光度，高于阴性对照2.5倍的噬菌体克隆即为阳性克隆。 (8) Detection: Place the detection plate in a microplate reader to detect the OD ₄₅₀ absorbance, and the phage clones that are 2.5 times higher than the negative control are positive clones.

1.1全人源单链抗体文库的构建1.1 Construction of fully human single-chain antibody library

采用Ficoll分离液进行PBMC分离：将Ficoll分离液缓慢加入采集的正常人血液，使得Ficoll分离液与正常人血液保持清晰的分离界面。将装有所述血液和分离液的50mL离心管于15℃左右离心20min。离心之后，整个液面分为四层，上层为血浆混合物，下层为红细胞和粒细胞，中层为Ficoll液体，其中在上、中层交界处有以PBMC为主的白色云雾层狭窄带，即PBMC细胞层。用无菌巴氏吸管小心地吸去上层的血浆混合物，然后再用新的无菌巴氏吸管吸取PBMC，获得分离的PBMC。Use Ficoll separation solution for PBMC separation: slowly add Ficoll separation solution to the collected normal human blood, so that the Ficoll separation solution and normal human blood maintain a clear separation interface. Centrifuge the 50mL centrifuge tube containing the blood and separation solution at about 15°C for 20min. After centrifugation, the entire liquid surface is divided into four layers, the upper layer is plasma mixture, the lower layer is red blood cells and granulocytes, and the middle layer is Ficoll liquid, in which there is a narrow band of white cloud layer mainly composed of PBMC at the junction of the upper layer and the middle layer, that is, PBMC cells layer. Use a sterile Pasteur pipette to carefully suck off the upper plasma mixture, and then use a new sterile Pasteur pipette to draw PBMCs to obtain isolated PBMCs.

通过常规方法提取总RNA并反转录成cDNA。基于重链和轻链种系基因序列的相似度，分别在可变区两端设计简并引物(李晓琳，大容量非免疫人源性Fab噬菌体抗体库的构建及初步筛选，《中国协和医科大学》硕士学位论文，2007年6月)；Sblattero,D.,Bradbury,A.,(1998)A definitive set of oligonucleotide primers foramplifying human V regions.Immunotechnology 3,271-278)。其中，抗体结构中VL(轻链)排在前面或者后面，相应地VH(重链)排在后面或者前面，且VL和VH之间由柔性Linker进行连接。 PCR得到抗体的重链可变区基因片段和轻链可变区基因片段，通过常规的重叠PCR的方法扩增得到ScFv核酸片段(PCR方法参考《分子克隆实验指南(Molecular Cloning:A Laboratory Manual)》(第三版)，美国，Joe Sambrook，David Russell.科学出版社)。将ScFv核酸片段与噬菌粒载体pComb3xss进行连接，通过电转仪将产物转化至TGI菌株中，得到全人源单链抗体库。Total RNA was extracted by conventional methods and reverse transcribed into cDNA. Based on the similarity of the heavy chain and light chain germline gene sequences, degenerate primers were designed at both ends of the variable region (Li Xiaolin, Construction and preliminary screening of a large-capacity non-immune human Fab phage antibody library, Peking Union Medical College "Master's Thesis, June 2007); Sblattero, D., Bradbury, A., (1998) A definitive set of oligonucleotide primers foramplifying human V regions. Immunotechnology 3, 271-278). Among them, in the antibody structure, the VL (light chain) is arranged in front or behind, and the VH (heavy chain) is arranged in the back or in front accordingly, and VL and VH are connected by a flexible Linker. The heavy chain variable region gene fragment and the light chain variable region gene fragment of the antibody were obtained by PCR, and the ScFv nucleic acid fragment was amplified by conventional overlapping PCR method (for the PCR method, refer to Molecular Cloning: A Laboratory Manual) (Third Edition), USA, Joe Sambrook, David Russell. Science Press). The ScFv nucleic acid fragment was connected to the phagemid vector pComb3xss, and the product was transformed into a TGI strain by electroporation to obtain a fully human single-chain antibody library.

1.2噬菌体展示全人源单链抗体文库制备1.2 Phage display fully human scFv library preparation

将文库菌液加入到新鲜的LB液体培养基中，进行复苏培养。再以VCSM13:菌＝50:1的感染复数添加VSCM13辅助噬菌体，充分混匀，静置后在摇床中继续培养。将培养物离心弃上清，沉淀则以氨苄青霉素、卡那霉素双抗性SOB培养基重悬，培养过夜。将菌液于4℃、以8000rpm离心20min，收集上清并加入1/5体积的20％PEG 8000 2.5mmol/L NaCl溶液，冰上孵育1小时。再于4℃、以12000rpm离心30min，将沉淀的噬菌体用PBS重悬并用0.22μm滤膜过滤。Add the library bacteria solution to fresh LB liquid medium for recovery culture. Then add VSCM13 helper phage at a multiplicity of infection of VCSM13:bacteria=50:1, mix thoroughly, and continue culturing in a shaker after standing still. The culture was centrifuged to discard the supernatant, and the precipitate was resuspended in ampicillin and kanamycin double-resistant SOB medium, and cultured overnight. Centrifuge the bacterial solution at 4°C and 8000rpm for 20min, collect the supernatant and add 1/5 volume of 20% PEG 8000 2.5mmol/L NaCl solution, and incubate on ice for 1 hour. Centrifuge at 12000 rpm for 30 min at 4° C., resuspend the precipitated phage in PBS and filter with a 0.22 μm filter membrane.

1.3抗原淘选1.3 Antigen panning

将带有Fc标签的CD70蛋白与proteinG磁珠共孵育，制备为CD70-proteinG偶联磁珠。将偶联磁珠抽入到制备得到的全人源单链抗体文库噬菌体淘选中。经历3-4轮的共孵育、清洗和洗脱的淘选过程，即可富集得到针对CD70抗原的特异性单克隆抗体。CD70 protein with Fc tag was co-incubated with proteinG magnetic beads to prepare CD70-proteinG coupled magnetic beads. The coupled magnetic beads were pumped into the phage panning of the prepared fully human single chain antibody library. After 3-4 rounds of co-incubation, washing and elution panning process, the specific monoclonal antibody against CD70 antigen can be enriched.

1.4阳性克隆的筛选1.4 Screening of positive clones

淘选结束后，挑取最终出库的单克隆菌斑进行ELISA检测筛选。检测结果如图1所示，得到与CD70抗原有结合的噬菌体克隆，淘选得到以下7株ScFv，均具备与人CD70抗原的特异性结合能力。After panning, the final monoclonal plaques were picked for ELISA detection and screening. The test results are shown in Figure 1. Phage clones binding to the CD70 antigen were obtained, and the following seven ScFv strains were obtained by panning, all of which had specific binding ability to the human CD70 antigen.

DK-10-A01 ScFv为：轻链可变区氨基酸序列如SEQ ID NO:1所示，重链可变区的氨基酸序列如SEQ ID NO:8所示，linker的氨基酸序列如SEQ ID NO:11所示；DK-10-A01 ScFv is: the amino acid sequence of the light chain variable region is shown in SEQ ID NO:1, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the linker is shown in SEQ ID NO: as shown in 11;

DK-10-A02 ScFv为：轻链可变区的氨基酸序列如SEQ ID NO:2所示，重链可变区的氨基酸序列如SEQ ID NO:8所示，linker的氨基酸序列如SEQ ID NO:11所示；DK-10-A02 ScFv is: the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the linker is shown in SEQ ID NO :11 shown;

DK-10-A03 ScFv为：轻链可变区的氨基酸序列如SEQ ID NO:3所示，重链可变区的氨基酸序列如SEQ ID NO:8所示；linker的氨基酸序列如SEQ ID NO:11所示；DK-10-A03 ScFv is: the amino acid sequence of the light chain variable region is shown in SEQ ID NO:3, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8; the amino acid sequence of the linker is shown in SEQ ID NO :11 shown;

DK-10-A04 ScFv为：轻链可变区的氨基酸序列如SEQ ID NO:4所示，重链可变区的氨基酸序列如SEQ ID NO:9所示；linker的氨基酸序列如SEQ ID NO:11所示；DK-10-A04 ScFv is: the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:9; the amino acid sequence of the linker is shown in SEQ ID NO :11 shown;

DK-10-A05 ScFv为：轻链可变区的氨基酸序列如SEQ ID NO:5所示，重链可变区的氨基酸序列如SEQ ID NO:8所示；linker的氨基酸序列如SEQ ID NO:11所示；DK-10-A05 ScFv is: the amino acid sequence of the light chain variable region is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8; the amino acid sequence of the linker is shown in SEQ ID NO :11 shown;

DK-10-A06 ScFv为：轻链可变区的氨基酸序列如SEQ ID NO:6所示，重链可变区的氨基酸序列如SEQ ID NO:10所示；linker的氨基酸序列如SEQ ID NO:11所示；DK-10-A06 ScFv is: the amino acid sequence of the light chain variable region is shown in SEQ ID NO:6, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:10; the amino acid sequence of the linker is shown in SEQ ID NO :11 shown;

DK-10-A07 ScFv为：轻链可变区的氨基酸序列如SEQ ID NO:7所示，重链可变区的氨基酸序列如SEQ ID NO:8所示；linker的氨基酸序列如SEQ ID NO:11所示。DK-10-A07 ScFv is: the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8; the amino acid sequence of the linker is shown in SEQ ID NO :11.

1.5 ScFv的表达与纯化1.5 Expression and purification of ScFv

将100ng阳性噬菌体克隆的pComb3xss质粒与100μL感受态Rosetta gami(DE3)细菌混匀并经冰浴5min、42℃热激90s、冰浴3min后，涂布至含有氨苄抗性的LB平板上，置于37℃恒温培养箱中过夜培养。挑取形成的单克隆菌斑置于4ml的LB培养基中，于37℃、250rpm摇床培养8h后，将菌液转接200mL LB培养基中培养。待菌液OD达到0.5-1.0时，加入IPTG并调整终浓度为1mM，37℃诱导表达12h。通过3600rpm离心收集菌体并加入PBS重悬沉淀，超声破碎2min，加入裂解液并以12000rpm、4℃离心，弃去沉淀，收集上清进行蛋白纯化。Mix 100ng of the pComb3xss plasmid of positive phage clones with 100μL of competent Rosetta gami (DE3) bacteria, put them in an ice bath for 5min, heat shock at 42°C for 90s, and ice bath for 3min, and spread them on the LB plate containing ampicillin resistance. Incubate overnight in a constant temperature incubator at 37°C. The monoclonal plaque formed by picking was placed in 4ml of LB medium, and after cultivating on a shaker at 37°C and 250rpm for 8h, the bacterial liquid was transferred to 200mL of LB medium for cultivation. When the OD of the bacterial solution reached 0.5-1.0, IPTG was added to adjust the final concentration to 1 mM, and the expression was induced at 37°C for 12 hours. The bacteria were collected by centrifugation at 3600rpm, and the pellet was resuspended by adding PBS, sonicated for 2min, the lysate was added and centrifuged at 12000rpm, 4°C, the pellet was discarded, and the supernatant was collected for protein purification.

菌液裂解上清通过0.22μm滤膜过滤，以等体积pH7.2 PBS稀释后，以GE Ni Sepharose excel纯化柱富集ScFv。5倍柱体积PBS流洗后，以5倍柱体积的含30mM咪唑PBS溶液流洗以去除杂质。使用含500mM咪唑的PBS溶液洗脱蛋白，收集洗胶液并将其以3KDa的超滤管浓缩。使用GE HiLoad Superdex 16/600 200pg分子排阻色谱柱上样，PBS流洗并收集紫外吸收峰，通过SDS-PAGE鉴定ScFv纯化效果后，进行流式染色特异性检测。The bacterial lysate supernatant was filtered through a 0.22 μm filter membrane, diluted with an equal volume of PBS at pH 7.2, and ScFv was enriched with a GE Ni Sepharose excel purification column. After washing with 5 column volumes of PBS, wash with 5 column volumes of PBS solution containing 30 mM imidazole to remove impurities. Use the PBS solution containing 500mM imidazole to elute the protein, collect the gel solution and concentrate it with a 3KDa ultrafiltration tube. Use GE HiLoad Superdex 16/600 200pg molecular exclusion chromatography column to load the sample, wash with PBS and collect the UV absorption peak, after identifying the ScFv purification effect by SDS-PAGE, perform flow staining specificity detection.

1.6流式染色能力鉴定1.6 Identification of Flow Cytometry Staining Ability

将K562、K562-CD70细胞各自分装至1.5mL Ep管，每管1×10 ⁶个细胞作为靶细胞，均以1000g离心5min后弃尽上清，分别用50μL 30μg/mL A-7-18(人源化改造的抗人CD70ScFv(申请人的另外一篇未公开的专利202010559366.8中的抗体)，实验中作为阳性对照)、DK-10-A01、DK-10-A02、DK-10-A03、DK-10-A04、DK-10-A05、DK-10-A06和DK-10-A07ScFv溶液重悬并4℃避光孵育。30min后加入PBS重悬细胞，并以1000g洗涤5min，弃尽上清。加入30μL检测用Anti-His-647荧光二抗并重悬细胞后，4℃避光孵育30min。以1mL PBS重悬并以1000g、5min洗涤细胞两次，弃尽上清并以200μL PBS重悬。所有细胞管上机检测流式染色阳性率。对比A-7-18染色组与淘选得到抗CD70ScFv染色组的染色结果，以评估ScFv的流式染色特异性。A-7-18 ScFv对K562的染色结果结果如图2所示。A-7-18 ScFv对K562-CD70细胞的染色结果如图3所示。淘选得到CD70特异性ScFv对K562细胞的染色结果如图4、图5、图6、图7、图8、图9及图10所示，从图中看出，CD70特异性ScFv对阴性细胞无结合。对K562-CD70的染色结果如图11、图12、图13、图14、图15、图16及图17所示，从图中看出，CD70特异性ScFv对阳性细胞有结合，证明筛选的CD70特异性ScFv可以作为检测试剂检测CD70的表达。 Separate K562 and K562-CD70 cells into 1.5mL Ep tubes, 1× ¹⁰⁶ cells in each tube as target cells, centrifuge at 1000g for 5min, discard the supernatant, and use 50μL 30μg/mL A-7-18 (humanized anti-human CD70ScFv (an antibody in another unpublished patent 202010559366.8 of the applicant), used as a positive control in the experiment), DK-10-A01, DK-10-A02, DK-10-A03 , DK-10-A04, DK-10-A05, DK-10-A06 and DK-10-A07 ScFv solutions were resuspended and incubated at 4°C in the dark. After 30 min, PBS was added to resuspend the cells, washed with 1000 g for 5 min, and the supernatant was discarded. After adding 30 μL Anti-His-647 fluorescent secondary antibody for detection and resuspending the cells, incubate at 4°C in the dark for 30 min. Resuspend in 1 mL of PBS and wash the cells twice with 1000 g for 5 min, discard the supernatant and resuspend in 200 μL of PBS. The positive rate of flow cytometry staining was tested on all cell tubes. The staining results of the A-7-18 staining group and the anti-CD70 ScFv staining group obtained by panning were compared to evaluate the flow staining specificity of ScFv. The results of staining of K562 by A-7-18 ScFv are shown in FIG. 2 . The staining results of A-7-18 ScFv on K562-CD70 cells are shown in FIG. 3 . The staining results of CD70-specific ScFv on K562 cells obtained by panning are shown in Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9 and Figure 10. It can be seen from the figure that the CD70-specific ScFv on negative cells No binding. The staining results of K562-CD70 are shown in Figure 11, Figure 12, Figure 13, Figure 14, Figure 15, Figure 16 and Figure 17. It can be seen from the figure that CD70-specific ScFv binds to positive cells, which proves that the screened CD70-specific ScFv can be used as a detection reagent to detect the expression of CD70.

1.7抗原抗体结合的动力学参数检测1.7 Detection of Kinetic Parameters of Antigen-Antibody Binding

将ProA生物传感器在分析缓冲液中“baseline1”60s，在30ug/mL CD70-hFc溶液中“loading”120s以进行配体蛋白固化，转入分析缓冲液中“baseline2”120s，转入梯度稀释的分析物(CD70ScFv)溶液中“association”120s，最后在分析缓冲液中“dissociation”300s。通过对响应值R的变化，判断是否存在特异性结合。一般而言，扣平Reference后的最大响应值Rmax大于0.05nm且存在浓度依赖与解离现象时，即可以判断为存在特异性结合。通过Fortebio Octet K2仪器自带的Data Analysis分析软件可以给出结合-解离曲线代表的动力学参数，如结合常数(Kon)、解离常数(Kdis)以及平衡解离常数(KD)。其中Kon的单位是1/Ms，用以表述抗原与抗体结合的速率，Kon越高，抗体与抗原结合形成复合物的速度越快。Kdis的单位是1/s，用以表述抗原与抗体解离速率，Kdis越高，抗原-抗体复合物解离的速度越快。KD为Kdis与Kon的比值，用以综合描述抗原抗体结合的难易程度，KD越小，通常认为抗体亲和力越高。A-7-18 ScFv与CD70抗原的结合动力学数据如表1所示，作图如图18所示。淘选得到CD70特异性ScFv与CD70抗原的结合动力学数据如表2-表7所示，作图分别如图19、图20、图21、图22、图23及图24所示。Put ProA biosensor in "baseline1" in analysis buffer for 60s, "loading" in 30ug/mL CD70-hFc solution for 120s to immobilize ligand protein, transfer to "baseline2" in analysis buffer for 120s, transfer to serially diluted "Association" for 120s in analyte (CD70ScFv) solution, and finally "dissociation" for 300s in assay buffer. Through the change of the response value R, it is judged whether there is specific binding. Generally speaking, when the maximum response value Rmax after deducting the reference is greater than 0.05nm and there are concentration dependence and dissociation phenomena, it can be judged that there is specific binding. The data analysis analysis software that comes with the Fortebio Octet K2 instrument can give the kinetic parameters represented by the binding-dissociation curve, such as binding constant (Kon), dissociation constant (Kdis) and equilibrium dissociation constant (KD). The unit of Kon is 1/Ms, which is used to express the binding rate of antigen and antibody. The higher the Kon, the faster the binding speed of antibody and antigen to form a complex. The unit of Kdis is 1/s, which is used to express the dissociation rate of antigen and antibody. The higher the Kdis, the faster the dissociation speed of the antigen-antibody complex. KD is the ratio of Kdis to Kon, which is used to comprehensively describe the difficulty of antigen-antibody binding. The smaller the KD, the higher the antibody affinity is generally considered. The binding kinetic data of A-7-18 ScFv to CD70 antigen is shown in Table 1, and the plot is shown in Figure 18. The binding kinetics data of CD70-specific ScFv and CD70 antigen obtained by panning are shown in Table 2-Table 7, and the plots are shown in Figure 19, Figure 20, Figure 21, Figure 22, Figure 23 and Figure 24, respectively.

表1人源化ScFv A-7-18与CD70抗原的结合动力学参数Table 1 The binding kinetic parameters of humanized ScFv A-7-18 and CD70 antigen

表2 DK-10-A01与CD70抗原的结合动力学参数Table 2 The binding kinetic parameters of DK-10-A01 and CD70 antigen

表3 DK-10-A02与CD70抗原的结合动力学参数Table 3 The binding kinetic parameters of DK-10-A02 and CD70 antigen

表4 DK-10-A03与CD70抗原的结合动力学参数Table 4 The binding kinetic parameters of DK-10-A03 and CD70 antigen

表5 DK-10-A04与CD70抗原的结合动力学参数Table 5 The binding kinetic parameters of DK-10-A04 and CD70 antigen

表6 DK-10-A05与CD70抗原的结合动力学参数Table 6 The binding kinetic parameters of DK-10-A05 and CD70 antigen

表7 DK-10-A07与CD70抗原的结合动力学参数Table 7 The binding kinetic parameters of DK-10-A07 and CD70 antigen

实施例二：靶向全人源化CD70的嵌合抗原受体及其应用Example 2: Chimeric antigen receptor targeting fully humanized CD70 and its application

本实施例中的人源化CD70的制备方法同实施例一中的1.1、1.2和1.3。The preparation method of humanized CD70 in this example is the same as 1.1, 1.2 and 1.3 in Example 1.

淘选结束后，挑取最终出库的单克隆菌斑进行ELISA检测筛选，得到与CD70抗原有结合的噬菌体克隆，淘选得到靶向CD70的ScFv，其具备人CD70抗原的特异性结合能力。检测结果如图25所示，淘选了多条靶向CD70的ScFv，其中基于SEQ ID NO:23所示的氨基酸序列的这一条靶向CD70的ScFv，不仅能够成功地构建CAR，而且表现出优异的体内体外疗效。After panning, the final monoclonal plaques were selected for ELISA detection and screening to obtain phage clones that bind to CD70 antigen, and scFv targeting CD70 was obtained by panning, which has the specific binding ability of human CD70 antigen. The test results are shown in Figure 25. Multiple CD70-targeting ScFvs were panned, and the CD70-targeting ScFv based on the amino acid sequence shown in SEQ ID NO: 23 was not only able to successfully construct a CAR, but also showed Excellent in vivo and in vitro efficacy.

本实施例中的抗原淘选和ELISA检测的具体实施过程同实施例一。The specific implementation process of antigen panning and ELISA detection in this example is the same as that in Example 1.

在本实施例中，载体结构元件的序列如下表8所示；其中8H、7H、8hdc、G4H和G4HH2H3mt为不同的铰链结构；8TM和28TM为不同的跨膜结构；28z、BBz、28z(YMFM)为不同的胞内信号结构。In this embodiment, the sequence of the carrier structural elements is shown in Table 8 below; where 8H, 7H, 8hdc, G4H and G4HH2H3mt are different hinge structures; 8TM and 28TM are different transmembrane structures; 28z, BBz, 28z (YMFM ) are different intracellular signaling structures.

在本实施例中，CD70(1)、CD70(2)、CD70(3)和CD70(4)是不同的靶向CD70的ScFv。不同的ScFv包括CD70 ScFv、CD70 ScFv2、CD70 ScFv3和CD70 ScFv4。In this example, CD70(1), CD70(2), CD70(3) and CD70(4) are different ScFvs targeting CD70. Different ScFvs include CD70 ScFv, CD70 ScFv2, CD70 ScFv3 and CD70 ScFv4.

表8载体结构元件的序列Table 8 Sequences of Vector Structural Elements

2.1质粒构建2.1 Plasmid construction

通过PCR扩增，获得CD70抗体的ScFv序列。再通过限制性内酶酶切的方式，将序列连接于含不同启动子、不同铰链、不同跨膜、不同共刺激信号的慢病毒载体中，即获得不同结构的靶向CD70的CAR T质粒载体。通过测序比对验证后，CAR结构构建成功，具体简称及结构对应见表9。ScFv sequence of CD70 antibody was obtained by PCR amplification. Then, by means of restriction endonuclease digestion, the sequences were connected to lentiviral vectors containing different promoters, different hinges, different transmembrane, and different costimulatory signals, so as to obtain CAR T plasmid vectors targeting CD70 with different structures . After verification by sequencing comparison, the CAR structure was successfully constructed, and the specific abbreviation and corresponding structure are shown in Table 9.

表9 CAR结构及简称Table 9 CAR structure and abbreviation

CAR结构CAR structure	简称Abbreviation
启动子1-CD70(2)-8H-8TM-BBzPromoter 1-CD70(2)-8H-8TM-BBz	CAR-2CAR-2
启动子1-CD70(3)-8H-8TM-BBzPromoter 1-CD70(3)-8H-8TM-BBz	CAR-3CAR-3
启动子1-CD70(4)-8H-8TM-BBzPromoter 1-CD70(4)-8H-8TM-BBz	CAR-4CAR-4
启动子1-CD70(1)-8H-8TM-BBzPromoter 1-CD70(1)-8H-8TM-BBz	CAR-5CAR-5
启动子2-CD70(1)-8H-8TM-BBzPromoter 2-CD70(1)-8H-8TM-BBz	CAR-7CAR-7
启动子2-CD70(1)-7H-8TM-BBzPromoter 2-CD70(1)-7H-8TM-BBz	CAR-8CAR-8
启动子2-CD70(1)-8hdc-8TM-BBzPromoter2-CD70(1)-8hdc-8TM-BBz	CAR-9CAR-9
启动子2-CD70(1)-G4HH2H3mt-8TM-BBzPromoter 2-CD70(1)-G4HH2H3mt-8TM-BBz	CAR-11CAR-11
启动子2-CD70(1)-8H-28TM-28z(YMFM)Promoter 2-CD70(1)-8H-28TM-28z(YMFM)	CAR-12CAR-12
启动子1-CD70(1)-G4HH2H3mt-28TM-28zPromoter 1-CD70(1)-G4HH2H3mt-28TM-28z	CAR-13CAR-13
启动子2-CD70(1)-G4HH2H3mt-28TM-28zPromoter 2-CD70(1)-G4HH2H3mt-28TM-28z	CAR-14CAR-14
启动子1-CD70(1)-7h-28TM-28zPromoter 1-CD70(1)-7h-28TM-28z	CAR-16CAR-16
启动子2-CD70(1)-7h-28TM-28zPromoter 2-CD70(1)-7h-28TM-28z	CAR-17CAR-17
启动子2-CD70(1)-7h-28TM-28z-shCD70Promoter2-CD70(1)-7h-28TM-28z-shCD70	CAR-19CAR-19

CD70是活化的T细胞和NK细胞会表达的分子，如果肿瘤微环境中存在上述这种表达CD70的免疫细胞；或者CAR-T细胞自身表达的CD70由于CAR-T细胞的活化而表达升高，就可能会造成“本底较高”。为了验证T细胞内源CD70的表达与CAR-T功能的影响，也为了阻止这种CAR与机体自身的免疫细胞上的CD70结合并杀死机体自身的免疫细胞，本发明实施例还设计了包含CD70干扰RNA的CAR-T序列，如表2中的CAR-19所示，以避免“本底较高”的情况。CAR-17和CAR-19的区别是，CAR-19加了CD70干扰RNA序列。本实施例是在本底较低的情况下做的实验，因此CAR-17和CAR-19做出来的体内与体外结果相差不大。但可以大胆猜测，如果本底较高，含有CD70干扰RNA序列的CAR的效果可能会明显优于没有CD70干扰RNA序列的CAR的效果。应当理解，除了本实施例的设计外，还可以采用其他手段以敲除或者破坏CAR-T细胞自身内源性表达的CD70而降低CAR-T可能的耗竭。在某些实施例中，还可以将携带CD70干扰RNA的载体与CAR载体共转染T细胞，以达到干扰内源性CD70的目的。在某些实施例中，还可以使用基因编辑的方式(例如锌指技术、TELN、Cas9、Cas12等基因编辑方式)导致内源性CD70表达降低或破坏从而失活，例如敲除内源性CD70表达。在某些实施例中，还可以使用专利CN111826352A和专利CN113088495A的技术导致内源性CD70表达降低或破坏从而失活。CD70 is a molecule expressed by activated T cells and NK cells. If the above-mentioned CD70-expressing immune cells exist in the tumor microenvironment; or the expression of CD70 expressed by CAR-T cells is increased due to the activation of CAR-T cells, It may cause "high background". In order to verify the expression of endogenous CD70 in T cells and the influence of CAR-T function, and to prevent this CAR from binding to CD70 on the body's own immune cells and killing the body's own immune cells, the embodiment of the present invention also designed a The CAR-T sequence of CD70 interfering RNA, as shown in CAR-19 in Table 2, to avoid the situation of "higher background". The difference between CAR-17 and CAR-19 is that CAR-19 adds a CD70 interference RNA sequence. This example is an experiment conducted under low background conditions, so the in vivo and in vitro results of CAR-17 and CAR-19 are not much different. However, it can be boldly guessed that if the background is higher, the effect of CAR containing CD70 interfering RNA sequence may be significantly better than that of CAR without CD70 interfering RNA sequence. It should be understood that, in addition to the design of this example, other means can also be used to knock out or destroy CD70 endogenously expressed by CAR-T cells themselves to reduce the possible exhaustion of CAR-T. In some embodiments, T cells can also be co-transfected with a vector carrying CD70 interfering RNA and a CAR vector to achieve the purpose of interfering with endogenous CD70. In some embodiments, gene editing methods (such as zinc finger technology, TELN, Cas9, Cas12 and other gene editing methods) can also be used to reduce or destroy the expression of endogenous CD70 and inactivate it, such as knocking out endogenous CD70 Express. In some embodiments, the technology of patent CN111826352A and patent CN113088495A can also be used to reduce or destroy the expression of endogenous CD70 to inactivate it.

2.2慢病毒制备2.2 Lentivirus preparation

本实施例采用磷酸钙法来包装慢病毒，具体为：用含10％FBS(w/v)的DMEM培养基培养293T细胞至较佳状态。将包装质粒(RRE:REV:2G)和表达质粒按一定比列加入到1.5mL的离心管中，再加入CaCl ₂和2×HBS，混匀后室温静置后，加入到处理好的293T细胞培养液(将前面培养至较佳状态的293T细胞，预先更换无血清培养基，以预饥饿处理几个小时)中，3-5h后再次换液至10mL含10％FBS的DMEM培养基，48h或72h后收集细胞上清，纯化病毒。获得2.1中15个病毒颗粒。 In this embodiment, the calcium phosphate method is used to package the lentivirus, specifically: 293T cells are cultured to a better state with DMEM medium containing 10% FBS (w/v). Add the packaging plasmid (RRE:REV:2G) and the expression plasmid into a 1.5mL centrifuge tube according to a certain ratio, then add CaCl ₂ and 2×HBS, mix well, let it stand at room temperature, and then add it to the treated 293T cells In the culture medium (the 293T cells that have been cultivated to a better state before, replace the serum-free medium in advance to pre-starve for several hours), change the medium again to 10mL DMEM medium containing 10% FBS after 3-5h, 48h Or collect the cell supernatant after 72 hours, and purify the virus. Obtain 15 virus particles in 2.1.

2.3 CAR-T细胞制备2.3 Preparation of CAR-T cells

利用梯度离心法进行淋巴细胞分离；离心后，取第二层白色淋巴细胞层，生理盐水洗涤，加入含有10％FBS的RPMI 1640完全培养基培养，获得人PBMC细胞。获得的PBMC细胞经抗CD3、CD28单克隆抗体活化24h后，按一定的感染复数(MOI)感染已活化的PBMC。在病毒感染的第8天检测CAR-T的阳性率，检测方法为流式检测，抗体为：Protein-L-PE。Protein-L可识别抗体轻链，即CAR抗原识别区的ScFv序列的轻链可被Protein-L识别。因此利用Protein-L可检测CAR阳性率和CAR表达强度。Lymphocytes were separated by gradient centrifugation; after centrifugation, the second white lymphocyte layer was taken, washed with saline, and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human PBMC cells. After the obtained PBMC cells were activated by anti-CD3 and CD28 monoclonal antibodies for 24 hours, the activated PBMCs were infected according to a certain multiplicity of infection (MOI). On the 8th day of virus infection, the positive rate of CAR-T was detected by flow cytometry, and the antibody was Protein-L-PE. Protein-L can recognize the light chain of the antibody, that is, the light chain of the ScFv sequence of the CAR antigen recognition region can be recognized by Protein-L. Therefore, the positive rate of CAR and the expression intensity of CAR can be detected by using Protein-L.

2.4不同人源化ScFv体内外药效学评价2.4 In vivo and in vitro pharmacodynamic evaluation of different humanized ScFv

使用通过2.3制备包含CAR-2、CAR-3、CAR-4、CAR-5的CAR-T细胞制剂(这4种CAR-T只有ScFv不同，其余均相同)验证不同ScFv制备CAR-T的可行性。通过Protein-L检测CAR阳性率，具体结果见表10。Use the CAR-T cell preparations prepared in 2.3 containing CAR-2, CAR-3, CAR-4, and CAR-5 (these 4 CAR-Ts are only different in ScFv, and the rest are the same) to verify the feasibility of preparing CAR-T with different ScFv sex. The positive rate of CAR was detected by Protein-L, and the specific results are shown in Table 10.

表10不同CAR-T细胞制剂的阳性率Table 10 Positive rate of different CAR-T cell preparations

CAR-T细胞制剂名称Name of CAR-T cell preparation	CAR阳性率CAR positive rate
CAR-2CAR-2	92.86％92.86%
CAR-3CAR-3	74.81％74.81%
CAR-4CAR-4	3.75％3.75%
CAR-5CAR-5	89.79％89.79%

以Control T为对照组，实验组设置为CAR-2组、CAR-3组、CAR-4组、CAR-5组，以786-O-Luc-GFP(CD70阳性)、A549-Luc-GFP(CD70阴性)为靶细胞，通过体外杀伤和体外细胞因子分泌验证体外有效性。结果显示，相比于对照组，CAR-2、CAR-3和CAR-4有一定体外杀伤及细胞因子分泌，但效果欠佳。CAR-5组体外杀伤及细胞因子分泌均较高，详细结果见图25、图26和图27。Control T was used as the control group, and the experimental group was set as CAR-2 group, CAR-3 group, CAR-4 group, and CAR-5 group. 786-O-Luc-GFP (CD70 positive), A549-Luc-GFP ( CD70 negative) as the target cells, and the in vitro effectiveness was verified by in vitro killing and in vitro cytokine secretion. The results showed that, compared with the control group, CAR-2, CAR-3 and CAR-4 had certain in vitro killing and cytokine secretion, but the effect was not good. The in vitro killing and cytokine secretion of the CAR-5 group were higher, and the detailed results are shown in Figure 25, Figure 26 and Figure 27.

选用NOG小鼠(雌性，6周龄)，以5×10 ⁶Cells/只的剂量皮下注射(i.v.)786-O-Luc-GFP细胞，以建立体内荷瘤模型。荷瘤后42d，以3×10 ⁶CAR-T Cells/只的剂量，通过尾静脉注射(i.v.)给予不同组别(Saline、Control、CAR-2、CAR-3、CAR-4、CAR-5)。结果显示，CAR-5组的体内抗肿瘤效果最好，具体结果见图28、图29和图30；仅有CD70(1)ScFv构建的CAR-T表现出优异的体内体外疗效。 NOG mice (female, 6 weeks old) were selected and subcutaneously injected (iv) with 786-O-Luc-GFP cells at a dose of 5×10 ⁶ Cells/mouse to establish a tumor-bearing model in vivo. 42 days after tumor bearing, different groups (Saline, Control, CAR- ² , CAR-3, CAR-4, CAR-5 ). The results showed that the CAR-5 group had the best anti-tumor effect in vivo, and the specific results are shown in Figure 28, Figure 29 and Figure 30; only CAR-T constructed with CD70(1) ScFv showed excellent in vivo and in vitro efficacy.

2.5含CD70(1)ScFv的不同CAR-T制剂体外药效评价2.5 In vitro efficacy evaluation of different CAR-T preparations containing CD70(1) ScFv

为进一步确定基于CD70(1)ScFv构建的不同CAR-T制剂的抗肿瘤效果，参考2.3制备含CD70(1)ScFv的不同CAR-T制剂：CAR-5、CAR-11、CAR-12、CAR-13、CAR-14、CAR-16。通过Protein-L检测CAR阳性率，具体结果见图31。以Control T为对照组，上述CAR-T制剂为实验组，以786-O-Luc-GFP(CD70阳性)、A549-Luc-GFP(CD70阴性)为靶细胞，通过体外杀伤和体外因子分泌验证体外有效性。结果显示，相比于对照组(Control T)，实验组(CAR-5、CAR-11、CAR-12、CAR-13、CAR-14和CAR-16)均有明显的体外杀伤及细胞因子分泌，详细结果见图32、图33。In order to further determine the anti-tumor effect of different CAR-T preparations based on CD70(1) ScFv, refer to 2.3 to prepare different CAR-T preparations containing CD70(1) ScFv: CAR-5, CAR-11, CAR-12, CAR -13, CAR-14, CAR-16. The positive rate of CAR was detected by Protein-L, and the specific results are shown in Figure 31. Control T was used as the control group, the above-mentioned CAR-T preparation was used as the experimental group, and 786-O-Luc-GFP (CD70-positive) and A549-Luc-GFP (CD70-negative) were used as target cells, which were verified by in vitro killing and in vitro factor secretion In vitro effectiveness. The results showed that compared with the control group (Control T), the experimental groups (CAR-5, CAR-11, CAR-12, CAR-13, CAR-14 and CAR-16) all had significant in vitro killing and cytokine secretion. , see Figure 32 and Figure 33 for detailed results.

2.6含CD70(1)ScFv的不同CAR-T制剂体内以及体外药效评价2.6 In vivo and in vitro efficacy evaluation of different CAR-T preparations containing CD70(1) ScFv

为进一步评估基于CD70(1)ScFv的不同CAR-T制剂的体内抗肿瘤效果，参考2.3制备含CD70(1)ScFv的不同CAR-T制剂：CAR-5、CAR-12、CAR-13、CAR-14、CAR-16、CAR-17、CAR-19。通过Protein-L检测CAR阳性率，具体结果见图34。选用NOG小鼠(雌性，6周龄)，以5×10 ⁶Cells/只的剂量皮下注射(i.v.)786-O-Luc-GFP细胞，以建立体内荷瘤模型。荷瘤后30d，以1.5×10 ⁶CAR-T Cells/只的剂量，通过尾静脉注射(i.v.)给予不同组别的CAR-T细胞制剂。通过活体成像和肿瘤测量观察肿瘤体内生长情况，以评估基于CD70(1)ScFv的不同CAR-T制剂的体内抗肿瘤效果。结果显示，各组体内抗肿瘤效果明显，详细结果见图35、图36和图37。 In order to further evaluate the in vivo anti-tumor effect of different CAR-T preparations based on CD70(1) ScFv, refer to 2.3 to prepare different CAR-T preparations containing CD70(1) ScFv: CAR-5, CAR-12, CAR-13, CAR -14, CAR-16, CAR-17, CAR-19. The positive rate of CAR was detected by Protein-L, and the specific results are shown in Figure 34. NOG mice (female, 6 weeks old) were selected and subcutaneously injected (iv) with 786-O-Luc-GFP cells at a dose of 5×10 ⁶ Cells/mouse to establish a tumor-bearing model in vivo. 30 days after tumor bearing, different groups of CAR-T cell preparations were given through tail vein injection (iv) at a dose of 1.5×10 ⁶ CAR-T Cells/mouse. In vivo tumor growth was observed by in vivo imaging and tumor measurement to evaluate the in vivo anti-tumor effects of different CD70(1) ScFv-based CAR-T formulations. The results showed that the anti-tumor effect in each group was obvious, and the detailed results are shown in Figure 35, Figure 36 and Figure 37.

2.72.7

接着，通过验证体外的药效评价，再次验证其他的CAR结构是否有效。结果如图38-图40所示，经验证，CAR-8和CAR-9也是可行的，以此类推出本专利中的ScFv可以适用于所有的CAR结构。Next, verify the effectiveness of other CAR structures by verifying the efficacy evaluation in vitro. The results are shown in Figure 38-Figure 40. It has been verified that CAR-8 and CAR-9 are also feasible. Based on this, it is deduced that the ScFv in this patent can be applied to all CAR structures.

最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be carried out Modifications or equivalent replacements without departing from the spirit and scope of the technical solution of the present invention shall be covered by the claims of the present invention.

Claims

靶向CD70的抗原结合片段，其特征在于，所述抗原结合片段包含重链可变区和轻链可变区；所述轻链可变区包含L-CDR1、L-CDR2和L-CDR3，所述重链可变区包含H-CDR1、H-CDR2和H-CDR3；The antigen-binding fragment targeting CD70 is characterized in that the antigen-binding fragment comprises a heavy chain variable region and a light chain variable region; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, The heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3;

所述L-CDR1、所述L-CDR2和所述L-CDR3选自以下氨基酸序列组合中一种：The L-CDR1, the L-CDR2 and the L-CDR3 are selected from one of the following amino acid sequence combinations:

1)QSISSY、AAS、QQSYSTPWT；1) QSISSY, AAS, QQSYSTPWT;

2)QSVSSN、GAS、QQYNNWPLT；2) QSVSSN, GAS, QQYNNWPLT;

3)QSISSS、AAS、QQSYNTPRT；3) QSISSS, AAS, QQSYNTPRT;

4)QSLLSSADDLNY、WAS、QQYYGTPT；4) QSLLSSADDLNY, WAS, QQYYGTPT;

5)QSVSSY、GAS、QQSYTLPLT；5) QSVSSY, GAS, QQSYTLPLT;

6)QTINTY、AAS、QQSYSTLIT；6) QTINTY, AAS, QQSYSTLIT;

7)QSVSSY、GAS、QQSYTLPRT；7) QSVSSY, GAS, QQSYTLPRT;

所述H-CDR1、所述H-CDR2和所述H-CDR3选自以下氨基酸序列组合中一种：The H-CDR1, the H-CDR2 and the H-CDR3 are selected from one of the following amino acid sequence combinations:

1)GYTFTDYY、INPYNGGT、ARSVYDYPFDY；1) GYTFTDYY, INPYNGGT, ARSVYDYPFDY;

2)GDSVSSNSAA、TYYRSKWYN、ARWGPAADGGFDP。2) GDSVSSNSAA, TYYRSKWYN, ARWGPAADGGFDP.
靶向CD70的全长抗体，其特征在于，所述全长抗体包含权利要求1所述的抗原结合片段。A full-length antibody targeting CD70, wherein the full-length antibody comprises the antigen-binding fragment of claim 1.
靶向CD70的单链抗体，其特征在于，所述单链抗体包含重链可变区和轻链可变区；所述轻链可变区包含L-CDR1、L-CDR2和L-CDR3，所述重链可变区包含H-CDR1、H-CDR2和H-CDR3；A single-chain antibody targeting CD70, characterized in that the single-chain antibody comprises a heavy chain variable region and a light chain variable region; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, The heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3;

所述L-CDR1、所述L-CDR2和所述L-CDR3选自以下氨基酸序列组合中一种：The L-CDR1, the L-CDR2 and the L-CDR3 are selected from one of the following amino acid sequence combinations:

1)QSISSY、AAS、QQSYSTPWT；1) QSISSY, AAS, QQSYSTPWT;

2)QSVSSN、GAS、QQYNNWPLT；2) QSVSSN, GAS, QQYNNWPLT;

3)QSISSS、AAS、QQSYNTPRT；3) QSISSS, AAS, QQSYNTPRT;

4)QSLLSSADDLNY、WAS、QQYYGTPT；4) QSLLSSADDLNY, WAS, QQYYGTPT;

5)QSVSSY、GAS、QQSYTLPLT；5) QSVSSY, GAS, QQSYTLPLT;

6)QTINTY、AAS、QQSYSTLIT；6) QTINTY, AAS, QQSYSTLIT;

7)QSVSSY、GAS、QQSYTLPRT；7) QSVSSY, GAS, QQSYTLPRT;

所述H-CDR1、所述H-CDR2和所述H-CDR3选自以下氨基酸序列组合中一种：The H-CDR1, the H-CDR2 and the H-CDR3 are selected from one of the following amino acid sequence combinations:

1)GYTFTDYY、INPYNGGT、ARSVYDYPFDY；1) GYTFTDYY, INPYNGGT, ARSVYDYPFDY;

2)GDSVSSNSAA、TYYRSKWYN、ARWGPAADGGFDP。2) GDSVSSNSAA, TYYRSKWYN, ARWGPAADGGFDP.
根据权利要求3所述的单链抗体，其特征在于，所述轻链可变区包含SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6或SEQ ID NO:7所示的氨基酸序列或其功能性变体；所述重链可变区包含SEQ ID NO:8、SEQ ID NO:9或SEQ ID NO:10所示的氨基酸序列或其功能性变体。The single-chain antibody according to claim 3, wherein the light chain variable region comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO :5, the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7 or a functional variant thereof; the heavy chain variable region comprises SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: The amino acid sequence shown in 10 or a functional variant thereof.
根据权利要求3或4所述的单链抗体，所述轻链可变区和所述重链可变区通过linker连接，所述linker包含SEQ ID NO:11所示的氨基酸序列或其功能性变体。The single-chain antibody according to claim 3 or 4, the light chain variable region and the heavy chain variable region are connected by a linker, and the linker comprises the amino acid sequence shown in SEQ ID NO: 11 or its functional Variants.
CAR结构，其特征在于，包含权利要求3-5任一所述的单链抗体。The CAR structure is characterized in that it comprises the single-chain antibody according to any one of claims 3-5.
核酸序列，其特征在于，包含编码权利要求3-5任一所述的单链抗体的核苷酸序列，编码所述轻链可变区的核苷酸序列如SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17或SEQ ID NO:18所示；编码所述重链可变区的核苷酸序列如SEQ ID NO:19、SEQ ID NO:20或SEQ ID NO:21所示。The nucleic acid sequence is characterized in that it comprises the nucleotide sequence encoding the single-chain antibody described in any one of claims 3-5, and the nucleotide sequence encoding the light chain variable region is such as SEQ ID NO: 12, SEQ ID Shown in NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18; the nucleotide sequence encoding the heavy chain variable region is as follows Shown in SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21.
重组质粒，其特征在于，包含权利要求7所述的核酸序列和表达载体。The recombinant plasmid is characterized in that it comprises the nucleic acid sequence and expression vector according to claim 7.
免疫工程细胞，其特征在于，所述免疫工程细胞是通过权利要求8所述的重组质粒转染免疫细胞得到，优选地，所述免疫细胞为T细胞、T细胞前体或NK细胞。Immunoengineering cells, characterized in that the immune engineering cells are obtained by transfecting immune cells with the recombinant plasmid according to claim 8, preferably, the immune cells are T cells, T cell precursors or NK cells.
权利要求1所述的靶向CD70的抗原结合片段或权利要求2所述的靶向CD70的全长抗体或权利要求3-5任一所述的靶向CD70的单链抗体或权利要求6所述的CAR结构或权利要求7所述的核酸序列或权利要求8所述的重组质粒或权利要求9所述的免疫工程细胞在制备抗肿瘤药物中的应用；优选地，所述肿瘤包含肾癌、血源性恶性肿瘤、胸腺肿瘤、卵巢癌、成神经胶质细胞瘤、鼻咽癌肿瘤、胰腺癌和胃肠道癌中的一种或多种；所述胃肠道癌可选地包括食道癌、结肠癌和胃癌中的一种或多种；更优选地，所述肿瘤为急性髓系白血病或肾细胞癌。The antigen-binding fragment targeting CD70 of claim 1 or the full-length antibody targeting CD70 of claim 2 or the single-chain antibody targeting CD70 of any one of claims 3-5 or the antibody of claim 6 The CAR structure described above or the nucleotide sequence described in claim 7 or the recombinant plasmid described in claim 8 or the application of the immunoengineering cell described in claim 9 in the preparation of antitumor drugs; preferably, the tumor comprises renal cancer one or more of , hematogenous malignancy, thymus tumor, ovarian cancer, glioblastoma, nasopharyngeal carcinoma tumor, pancreatic cancer, and gastrointestinal cancer; the gastrointestinal cancer optionally includes One or more of esophageal cancer, colon cancer and gastric cancer; more preferably, the tumor is acute myeloid leukemia or renal cell carcinoma.
权利要求1所述的靶向CD70的抗原结合片段或权利要求2所述的靶向CD70的全长抗体或权利要求3-5任一所述的靶向CD70的单链抗体在制备检测试剂/检测试剂盒中的用途。The antigen-binding fragment targeting CD70 according to claim 1 or the full-length antibody targeting CD70 according to claim 2 or the single-chain antibody targeting CD70 described in any one of claims 3-5 is used in the preparation of detection reagents/ Use in detection kits.
靶向CD70的嵌合抗原受体，其特征在于，包括胞外结构域、铰链区和跨膜区及胞内信号域，所述胞外结构域包括如SEQ ID NO:23所示的氨基酸序列或其功能性变体。The chimeric antigen receptor targeting CD70 is characterized in that it includes an extracellular domain, a hinge region, a transmembrane region and an intracellular signal domain, and the extracellular domain includes an amino acid sequence as shown in SEQ ID NO:23 or functional variants thereof.
根据权利要求12所述的嵌合抗原受体，其特征在于，所述铰链区包含以下氨基酸序列中的任意一种或其功能性变体：SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28。The chimeric antigen receptor according to claim 12, wherein the hinge region comprises any one of the following amino acid sequences or a functional variant thereof: SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 26, SEQ ID NO: ID NO:27, SEQ ID NO:28.
根据权利要求13所述的嵌合抗原受体，其特征在于：所述跨膜区包含以下氨基酸序列中的任意一种或其功能性变体：SEQ ID NO:29、SEQ ID NO:30。The chimeric antigen receptor according to claim 13, wherein the transmembrane region comprises any one of the following amino acid sequences or a functional variant thereof: SEQ ID NO:29, SEQ ID NO:30.
根据权利要求14所述的嵌合抗原受体，其特征在于，所述胞内信号域包含信号传导区和共刺激结构域，所述信号传导区包含SEQ ID NO:31或SEQ ID NO:56所示的氨基酸序列或其功能性变体；所述共刺激结构域包含以下氨基酸序列或其功能性变体的中的一种或多种：SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34。The chimeric antigen receptor according to claim 14, wherein the intracellular signaling domain comprises a signaling domain and a co-stimulatory domain, and the signaling domain comprises SEQ ID NO: 31 or SEQ ID NO: 56 The shown amino acid sequence or its functional variant; The co-stimulatory domain comprises one or more of the following amino acid sequences or its functional variant: SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 33, SEQ ID NO: ID NO: 34.
根据权利要求12所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体包括以下组合中的任意一种：The chimeric antigen receptor according to claim 12, wherein the chimeric antigen receptor comprises any one of the following combinations:

组合1)：所述铰链区包含如SEQ ID NO:25所示的氨基酸序列或其功能性变体，所述跨膜区包含如SEQ ID NO:29所示的氨基酸序列或其功能性变体，所述胞内信号域包含SEQ ID NO:56所示的氨基酸序列或其功能性变体和SEQ ID NO:32的所示的氨基酸序列或其功能性变体；Combination 1): the hinge region comprises an amino acid sequence as shown in SEQ ID NO:25 or a functional variant thereof, and the transmembrane region comprises an amino acid sequence as shown in SEQ ID NO:29 or a functional variant thereof , the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:56 or a functional variant thereof and the amino acid sequence shown in SEQ ID NO:32 or a functional variant thereof;

组合2)：所述铰链区包含如SEQ ID NO:26所示的氨基酸序列或其功能性变体，所述跨膜区包含如SEQ ID NO:30所示的氨基酸序列或其功能性变体，所述胞内信号域包含SEQ ID NO:56所示的氨基酸序列或其功能性变体和SEQ ID NO:33所示的氨基酸序列或其功能性变体；Combination 2): the hinge region comprises an amino acid sequence as shown in SEQ ID NO:26 or a functional variant thereof, and the transmembrane region comprises an amino acid sequence as shown in SEQ ID NO:30 or a functional variant thereof , the intracellular signaling domain comprises an amino acid sequence shown in SEQ ID NO:56 or a functional variant thereof and an amino acid sequence shown in SEQ ID NO:33 or a functional variant thereof;

组合3)：所述铰链区包含如SEQ ID NO:28所示的氨基酸序列或其功能性变体，所述跨膜区包含如SEQ ID NO:30所示的氨基酸序列或其功能性变体，所述所述胞内信号域包含如SEQ ID NO:31所示的氨基酸序列或其功能性变体和SEQ ID NO:34所示的氨基酸序列或其功能性变体；Combination 3): the hinge region comprises the amino acid sequence shown in SEQ ID NO: 28 or a functional variant thereof, and the transmembrane region comprises the amino acid sequence shown in SEQ ID NO: 30 or a functional variant thereof , the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO: 31 or a functional variant thereof and the amino acid sequence shown in SEQ ID NO: 34 or a functional variant thereof;

组合4)：所述铰链区包含如SEQ ID NO:27所示的氨基酸序列或其功能性变体，所述跨膜区包含如SEQ ID NO:30所示的氨基酸序列或其功能性变体，所述胞内信号域包含SEQ ID NO:31所示的氨基酸序列或其功能性变体和SEQ ID NO:34所示的氨基酸序列或其功能性变体；Combination 4): the hinge region comprises an amino acid sequence as shown in SEQ ID NO:27 or a functional variant thereof, and the transmembrane region comprises an amino acid sequence as shown in SEQ ID NO:30 or a functional variant thereof , the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO: 31 or a functional variant thereof and the amino acid sequence shown in SEQ ID NO: 34 or a functional variant thereof;

组合5)：所述铰链区包含如SEQ ID NO:28或SEQ ID NO:27或SEQ ID NO:43所示的氨基酸序列或其功能性变体，所述跨膜区包含如SEQ ID NO:29所示的氨基酸序列或其功能性变体，所述胞内信号域包含SEQ ID NO:56所示的氨基酸序列或其功能性变体和SEQ ID NO:32所示的氨基酸序列或其功能性变体。Combination 5): the hinge region comprises the amino acid sequence shown in SEQ ID NO:28 or SEQ ID NO:27 or SEQ ID NO:43 or a functional variant thereof, and the transmembrane region comprises such as SEQ ID NO: The amino acid sequence shown in 29 or its functional variant, the intracellular signal domain comprises the amino acid sequence shown in SEQ ID NO:56 or its functional variant and the amino acid sequence shown in SEQ ID NO:32 or its function sex variant.
编码权利要求16所述的嵌合抗原受体的核酸序列，其特征在于，所述核酸序列包括：The nucleic acid sequence encoding the chimeric antigen receptor of claim 16, wherein the nucleic acid sequence comprises:

编码所述组合1)的核酸序列，包含如SEQ ID NO:35所示的序列；或编码所述组合2)的核酸序列，包含如SEQ ID NO:36所示的序列；或编码所述组合3)的核酸序列，包含如SEQ ID NO:37所示的序列；或编码所述组合4)的核酸序列，包含如SEQ ID NO:38所示的序列；或编码所述组合5)的核酸序列，包含如SEQ ID NO:58所示的序列。The nucleic acid sequence of encoding described combination 1) comprises the sequence shown in SEQ ID NO:35; Or the nucleic acid sequence of encoding described combination 2) comprises the sequence shown in SEQ ID NO:36; Or encoding described combination 3) the nucleic acid sequence, comprising the sequence shown in SEQ ID NO:37; or encoding the combination 4) nucleic acid sequence comprising the sequence shown in SEQ ID NO:38; or encoding the combination 5) nucleic acid Sequence, comprises the sequence shown in SEQ ID NO:58.
根据权利要求17所述的嵌合抗原受体的核酸序列，其特征在于，所述嵌合抗原受体的核酸序列还包括启动子，所述启动子的核酸序列如SEQ ID NO:40或SEQ ID NO:41所示。The nucleic acid sequence of the chimeric antigen receptor according to claim 17, wherein the nucleic acid sequence of the chimeric antigen receptor also includes a promoter, and the nucleic acid sequence of the promoter is such as SEQ ID NO: 40 or SEQ ID NO: ID NO:41.
根据权利要求17或18所述的嵌合抗原受体的核酸序列，其特征在于，所述嵌合抗原受体的核酸序列还包括CD70干扰RNA，所述CD70干扰RNA序列如SEQ ID NO:42所示。The nucleic acid sequence of the chimeric antigen receptor according to claim 17 or 18, wherein the nucleic acid sequence of the chimeric antigen receptor also includes CD70 interfering RNA, and the CD70 interfering RNA sequence is such as SEQ ID NO:42 shown.
包含权利要求17-19任一所述的核酸序列的表达载体。An expression vector comprising the nucleic acid sequence of any one of claims 17-19.
根据权利要求20所述的表达载体，其特征在于：所述表达载体选自慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、腺相关病毒表达载体、DNA载体、RNA载体和质粒中的任一种。The expression vector according to claim 20, characterized in that: the expression vector is selected from lentivirus expression vectors, retrovirus expression vectors, adenovirus expression vectors, adeno-associated virus expression vectors, DNA vectors, RNA vectors and plasmids any kind.
一种工程化的细胞，其特征在于，所述细胞中转导有权利要求17-19任一所述的嵌合抗原受体的核酸序列或权利要求20或21所述的表达载体；An engineered cell, characterized in that the nucleic acid sequence of the chimeric antigen receptor according to any one of claims 17-19 or the expression vector according to claim 20 or 21 is transduced in the cell;

优选地，所述细胞为T细胞、T细胞前体或NK细胞。Preferably, the cells are T cells, T cell precursors or NK cells.
一种细胞制品，其特征在于，所述细胞制品包括权利要求22所述的工程化的细胞。A cell product, characterized in that the cell product comprises the engineered cell of claim 22.
一种药物组合物，其特征在于，所述药物组合物包括权利要求12-16任一所述的嵌合抗原受体或权利要求17-19任一所述的嵌合抗原受体的核酸序列或权利要求20或21所述的表达载体或权利要求22所述的工程化的细胞。A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the chimeric antigen receptor according to any one of claims 12-16 or the nucleic acid sequence of the chimeric antigen receptor according to any one of claims 17-19 Or the expression vector described in claim 20 or 21 or the engineered cell described in claim 22.
权利要求12-16任一所述的嵌合抗原受体或权利要求17-19任一所述的嵌合抗原受体的核酸序列或权利要求20或21所述的表达载体或权利要求22所述的工程化的细胞或权利要求24所述的药物组合物在制备抗肿瘤药物中的应用。The chimeric antigen receptor described in any one of claims 12-16 or the nucleic acid sequence of the chimeric antigen receptor described in any one of claims 17-19 or the expression vector described in claim 20 or 21 or the nucleic acid sequence described in claim 22 The application of the engineered cell described in claim 24 or the pharmaceutical composition described in claim 24 in the preparation of antitumor drugs.
如权利要求25所述的应用，其特征在于，所述肿瘤包括肾癌、血源性恶性肿瘤、胸腺肿瘤、卵巢癌、成神经胶质细胞瘤、鼻咽癌、胰腺癌和胃肠道癌中的一种或多种，所述胃肠道癌可选地包括食道癌、结肠癌和胃癌中的一种或多种。The use according to claim 25, wherein the tumors include renal cancer, hematogenous malignancies, thymic tumors, ovarian cancer, glioblastoma, nasopharyngeal cancer, pancreatic cancer, and gastrointestinal tract cancer One or more of the gastrointestinal cancers optionally include one or more of esophageal cancer, colon cancer and gastric cancer.