WO2023185072A1 - 抗cd7纳米抗体、衍生物及其在肿瘤治疗中的应用 - Google Patents

抗cd7纳米抗体、衍生物及其在肿瘤治疗中的应用 Download PDF

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WO2023185072A1
WO2023185072A1 PCT/CN2022/136785 CN2022136785W WO2023185072A1 WO 2023185072 A1 WO2023185072 A1 WO 2023185072A1 CN 2022136785 W CN2022136785 W CN 2022136785W WO 2023185072 A1 WO2023185072 A1 WO 2023185072A1
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seq
chimeric antigen
antigen receptor
nanobody
tumor
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李建强
王琳
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河北森朗生物科技有限公司
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Definitions

  • the invention belongs to the field of biomedicine technology. Specifically, the invention relates to anti-CD7 nanobodies, derivatives and their applications in tumor treatment.
  • CD7 antigen is a single-chain glycoprotein and a signature antigen molecule during T cell development.
  • hematopoietic stem and progenitor cells such as lymphoid and myeloid precursor cells also express CD7.
  • a large number of studies have shown that CD7 molecules are expressed on most human T lymphocyte leukemias and lymphomas (Karube K, Ohshima K, Tsuchiya T, et al. Non-B, non-T neoplasms with lymphoblast morphology: further clarification and classification[J] .
  • CD7 useful as a An antigen receptor suitable for targeted delivery of various functional molecules into CD7-positive cells; related research also shows that there is a group of CD7-negative T lymphocytes in the human body. This group of cells can maintain the body's normal immune function and avoid the use of CD7 The loss of immune function caused by the elimination of all CD7-positive cells by nanobody-related immune cells shows that targeting CD7 is a highly potential anti-tumor direction.
  • Chimeric antigen receptor modified T cells Chimeric antigen receptor modification T cells, CAR-T
  • chimeric antigen receptor modified NK cells Chimeric antigen receptor modification NK cells, CAR-NK
  • the effective activation of CAR-T/CAR-NK cells depends heavily on the specificity of the antibody that recognizes the tumor-associated antigen and the affinity of the antigen binding. Therefore, under the current situation that the design of the intracellular signal transduction region of CAR-T/CAR-NK cells has become mature, the design of the antigen-binding region has become the focus and key to the development of new CAR-T technology.
  • Heavy chain antibody Heavy chain antibody, HCAb
  • This antibody only contains one heavy chain variable region and two conventional CH2 and CH3 regions. Its heavy chain variable region has the same stability and antigen-binding activity as heavy chain antibodies. Its size is only 2.4 ⁇ 4nm, which is the smallest fragment capable of binding antigens. It is called a single domain antibody (Variable domain of heavy chain of heavy- chain antibody (VHH) or Nanobodies. Compared with traditional antibodies, VHH single domain antibodies have small molecular weight, high expression levels, good chemical stability, high affinity, high homology with human antibodies, and low immunogenicity.
  • VHH single domain antibodies have small molecular weight, high expression levels, good chemical stability, high affinity, high homology with human antibodies, and low immunogenicity.
  • VHH has good tissue penetration and has the possibility of accessing relatively hidden targets that cannot be accessed by conventional antibodies during tumor treatment. Because of these advantages, the use of single-domain antibodies as the antigen-binding region of CAR for CAR modification and CAR-T/CAR-NK cell therapy can provide a new tumor treatment strategy for this field.
  • the purpose of the present invention is to provide anti-CD7 Nanobodies, derivatives and their applications in tumor treatment to the field.
  • a first aspect of the invention provides anti-CD7 Nanobodies.
  • the Nanobody is any one of VHH01, VHH03, VHH04, VHH06, VHH07, VHH08, VHH09, VHH10, VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH19 or VHH20;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the VHH01 are shown in SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7 respectively or are the same as SEQ ID NO:3 and SEQ ID NO: respectively.
  • SEQ ID NO:7 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the VHH03 are as shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15 respectively or are the same as SEQ ID NO: 11 and SEQ ID NO: 13.
  • SEQ ID NO:15 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the VHH04 are as shown in SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23 respectively or are the same as SEQ ID NO: 19 and SEQ ID NO: 21.
  • SEQ ID NO:23 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • amino acid sequences of CDR1, CDR2, and CDR3 of VHH06 are as shown in SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31 respectively or are the same as SEQ ID NO:27 and SEQ ID NO: 29.
  • SEQ ID NO:31 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the VHH07 are as shown in SEQ ID NO:35, SEQ ID NO:37 and SEQ ID NO:39 respectively or are the same as SEQ ID NO:35 and SEQ ID NO: 37.
  • SEQ ID NO:39 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the VHH08 are as shown in SEQ ID NO: 43, SEQ ID NO: 45, and SEQ ID NO: 47 respectively or are the same as SEQ ID NO: 43, SEQ ID NO: 45.
  • SEQ ID NO:47 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the VHH09 are as shown in SEQ ID NO:51, SEQ ID NO:53, and SEQ ID NO:55 respectively or are the same as SEQ ID NO:51 and SEQ ID NO: 53.
  • SEQ ID NO:55 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the VHH10 are shown in SEQ ID NO:59, SEQ ID NO:61 and SEQ ID NO:63 respectively or are the same as SEQ ID NO:59 and SEQ ID NO: 61.
  • SEQ ID NO:63 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the VHH12 are as shown in SEQ ID NO:67, SEQ ID NO:69, and SEQ ID NO:71 respectively or are the same as SEQ ID NO:67 and SEQ ID NO: 69.
  • SEQ ID NO:71 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • amino acid sequences of CDR1, CDR2, and CDR3 of VHH13 are as shown in SEQ ID NO:75, SEQ ID NO:77, and SEQ ID NO:79 respectively or are the same as SEQ ID NO:75 and SEQ ID NO: 77.
  • SEQ ID NO:79 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the VHH14 are as shown in SEQ ID NO:83, SEQ ID NO:85 and SEQ ID NO:87 respectively or are the same as SEQ ID NO:83 and SEQ ID NO: 85.
  • SEQ ID NO:87 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the VHH15 are as shown in SEQ ID NO:91, SEQ ID NO:93, and SEQ ID NO:95 respectively or are the same as SEQ ID NO:91 and SEQ ID NO: 93.
  • SEQ ID NO:95 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the VHH16 are as shown in SEQ ID NO:99, SEQ ID NO:101, and SEQ ID NO:103 respectively or are the same as SEQ ID NO:99 and SEQ ID NO: 101.
  • SEQ ID NO:103 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • the amino acid sequences of CDR1, CDR2, and CDR3 of the VHH17 are as shown in SEQ ID NO: 107, SEQ ID NO: 109, and SEQ ID NO: 111 respectively or are the same as SEQ ID NO: 107 and SEQ ID NO: 109.
  • SEQ ID NO: 111 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the VHH18 are as shown in SEQ ID NO: 115, SEQ ID NO: 117, and SEQ ID NO: 119 respectively or are the same as SEQ ID NO: 115 and SEQ ID NO: 117.
  • SEQ ID NO: 119 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • the amino acid sequences of CDR1, CDR2, and CDR3 of the VHH19 are as shown in SEQ ID NO: 123, SEQ ID NO: 125, and SEQ ID NO: 127 respectively or are the same as SEQ ID NO: 123 and SEQ ID NO: 125.
  • SEQ ID NO: 127 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • the amino acid sequences of CDR1, CDR2, and CDR3 of the VHH20 are as shown in SEQ ID NO: 131, SEQ ID NO: 133, and SEQ ID NO: 135 respectively or are the same as SEQ ID NO: 131 and SEQ ID NO: 133.
  • SEQ ID NO: 135 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • nucleotide sequences of CDR1, CDR2, and CDR3 of the VHH01 are as shown in SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8 respectively or are the same as SEQ ID NO: 4 and SEQ ID NO: 4, respectively.
  • ID NO:6, SEQ ID NO:8 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences;
  • nucleotide sequences of CDR1, CDR2, and CDR3 of VHH03 are as shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16 respectively or are the same as SEQ ID NO: 12 and SEQ ID NO: 12, respectively.
  • ID NO:14, SEQ ID NO:16 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences;
  • nucleotide sequences of CDR1, CDR2, and CDR3 of the VHH04 are as shown in SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NO:24 respectively or are the same as SEQ ID NO:20 and SEQ ID NO:20, respectively.
  • ID NO:22, SEQ ID NO:24 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences;
  • nucleotide sequences of CDR1, CDR2, and CDR3 of VHH06 are as shown in SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:32 respectively or are the same as SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences;
  • nucleotide sequences of CDR1, CDR2, and CDR3 of VHH07 are as shown in SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:40 respectively or are the same as SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences;
  • nucleotide sequences of CDR1, CDR2, and CDR3 of VHH08 are as shown in SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48 respectively or are the same as SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences;
  • nucleotide sequences of CDR1, CDR2 and CDR3 of VHH09 are as shown in SEQ ID NO:52, SEQ ID NO:54 and SEQ ID NO:56 respectively or are the same as SEQ ID NO:52 and SEQ ID NO:52 respectively.
  • ID NO:54, SEQ ID NO:56 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences;
  • nucleotide sequences of CDR1, CDR2, and CDR3 of VHH10 are as shown in SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:64 respectively or are the same as SEQ ID NO:60 and SEQ ID NO:60, respectively.
  • ID NO:62, SEQ ID NO:64 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences;
  • nucleotide sequences of CDR1, CDR2, and CDR3 of the VHH12 are as shown in SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO: 72 respectively or are the same as SEQ ID NO: 68, SEQ ID NO:70, SEQ ID NO:72 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences;
  • nucleotide sequences of CDR1, CDR2, and CDR3 of VHH13 are as shown in SEQ ID NO:76, SEQ ID NO:78, and SEQ ID NO:80 respectively or are the same as SEQ ID NO:76 and SEQ ID NO:76, respectively.
  • ID NO:78, SEQ ID NO:80 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences;
  • nucleotide sequences of CDR1, CDR2 and CDR3 of the VHH14 are as shown in SEQ ID NO:84, SEQ ID NO:86 and SEQ ID NO:88 respectively or are the same as SEQ ID NO:84 and SEQ ID NO:84 respectively.
  • ID NO:86, SEQ ID NO:88 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences;
  • nucleotide sequences of CDR1, CDR2, and CDR3 of VHH15 are as shown in SEQ ID NO:92, SEQ ID NO:94, and SEQ ID NO:96 respectively or are the same as SEQ ID NO:92 and SEQ ID NO:92, respectively.
  • ID NO:94, SEQ ID NO:96 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences;
  • nucleotide sequences of CDR1, CDR2, and CDR3 of the VHH16 are as shown in SEQ ID NO: 100, SEQ ID NO: 102, and SEQ ID NO: 104 respectively or are the same as SEQ ID NO: 100, SEQ ID NO: 100, and SEQ ID NO: 104.
  • ID NO:102, SEQ ID NO:104 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences;
  • nucleotide sequences of CDR1, CDR2, and CDR3 of VHH17 are as shown in SEQ ID NO: 108, SEQ ID NO: 110, and SEQ ID NO: 112 respectively or are the same as SEQ ID NO: 108, SEQ ID NO: 112, respectively.
  • ID NO:110, SEQ ID NO:112 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences;
  • nucleotide sequences of CDR1, CDR2, and CDR3 of the VHH18 are as shown in SEQ ID NO: 116, SEQ ID NO: 118, and SEQ ID NO: 120 respectively or are the same as SEQ ID NO: 116, SEQ ID NO: 116, and SEQ ID NO: 118, respectively.
  • ID NO:118, SEQ ID NO:120 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences;
  • nucleotide sequences of CDR1, CDR2, and CDR3 of VHH19 are as shown in SEQ ID NO: 124, SEQ ID NO: 126, and SEQ ID NO: 128 respectively or are the same as SEQ ID NO: 124 and SEQ ID NO: 124, respectively.
  • ID NO:126, SEQ ID NO:128 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences;
  • nucleotide sequences of CDR1, CDR2, and CDR3 of the VHH20 are as shown in SEQ ID NO: 132, SEQ ID NO: 134, and SEQ ID NO: 136 respectively or are the same as SEQ ID NO: 132 and SEQ ID NO: 132, respectively.
  • ID NO:134, SEQ ID NO:136 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences;
  • amino acid sequences of VHH01, VHH03, VHH04, VHH06, VHH07, VHH08, VHH09, VHH10, VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH19, and VHH20 are respectively as SEQ ID NO: 1, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:41, SEQ ID NO:49, SEQ ID NO:57, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:81, SEQ ID NO:89, SEQ ID NO:97, SEQ ID NO:105, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:129 are shown or respectively.
  • SEQ ID NO:1 SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:41, SEQ ID NO:49, SEQ ID NO:57, SEQ ID NO:65 ⁇ SEQ ID NO:73 ⁇ SEQ ID NO:81 ⁇ SEQ ID NO:89 ⁇ SEQ ID NO:97 ⁇ SEQ ID NO:105 ⁇ SEQ ID NO:113 ⁇ SEQ ID NO:121 ⁇ SEQ ID NO :129 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • nucleotide sequences of VHH01, VHH03, VHH04, VHH06, VHH07, VHH08, VHH09, VHH10, VHH12, VHH13, VHH14, VHH15, VHH16, VHH17, VHH18, VHH19, and VHH20 are respectively as SEQ ID NO: 2.
  • CD7 is a very stable marker on the surface of T cells. Both naive T cells and mature T cells express CD7, so whether it is a patient with naive T cell tumors (T-ALL/LBL/NKT cell leukemia), Patients with mature T-cell tumors (peripheral T-cell lymphoma, NKT-cell lymphoma, anaplastic large cell lymphoma) basically express CD7 highly. Therefore, CAR-T targeting this target is currently the clinical choice for the treatment of T-lineage hematological tumors. One of the most rapidly developing technologies in the world.
  • a second aspect of the invention provides humanized anti-CD7 Nanobodies.
  • the humanized anti-CD7 Nanobody is obtained by humanizing the residues at key positions of the Nanobody by comparing it with DP-47 using the universal humanization framework h-NbBcII10FGLA as a reference. of;
  • the humanized anti-CD7 Nanobody is any of hVHH01, hVHH03, hVHH04, hVHH06, hVHH07, hVHH08, hVHH09, hVHH10, hVHH12, hVHH13, hVHH14, hVHH15, hVHH16, hVHH17, hVHH18, hVHH19 or hVHH20 A sort of;
  • amino acid sequences of CDR1, CDR2, and CDR3 of hVHH06 are as shown in SEQ ID NO: 139, SEQ ID NO: 141, and SEQ ID NO: 143 respectively or are the same as SEQ ID NO: 139 and SEQ ID NO. :141, SEQ ID NO:143 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • nucleotide sequences of CDR1, CDR2, and CDR3 of hVHH06 are as shown in SEQ ID NO: 140, SEQ ID NO: 142, and SEQ ID NO: 144 respectively or are the same as SEQ ID NO: 140 and SEQ ID NO: 140, respectively.
  • ID NO:142, SEQ ID NO:144 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical nucleotide sequences;
  • the amino acid sequence of hVHH06 is as shown in SEQ ID NO: 137 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% with SEQ ID NO: 137 , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % identity of the amino acid sequence;
  • the nucleotide sequence of hVHH06 is as shown in SEQ ID NO:138 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% identical nucleotide sequence.
  • a third aspect of the invention provides single Nanobody-based chimeric antigen receptors.
  • the chimeric antigen receptor includes any one of the Nanobody described in the first aspect of the present invention or the humanized anti-CD7 Nanobody described in the second aspect of the present invention;
  • the chimeric antigen receptor further comprises a transmembrane domain
  • the chimeric antigen receptor further comprises an intracellular signaling domain
  • the chimeric antigen receptor further comprises a hinge region
  • the chimeric antigen receptor further comprises a signal peptide
  • the chimeric antigen receptor further comprises a costimulatory signaling domain
  • the chimeric antigen receptor further comprises promoter EF1 ⁇ ;
  • the chimeric antigen receptor further comprises a self-cleaving peptide T2A;
  • the chimeric antigen receptor further includes a detection tag/auxiliary functional element tEGFR;
  • the transmembrane domain includes the transmembrane domain of the following molecules: CD8 ⁇ , CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3 ⁇ , IL-2 receptor, IL-7 Receptor, IL-11 receptor;
  • the intracellular signaling domain includes the intracellular signaling domain of the following molecules: CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , TCR ⁇ , CD4, CD5, CD8, CD21, CD22, CD79a, CD79b , CD278, Fc ⁇ RI, DAP10, DAP12, CD66d;
  • the hinge region includes the hinge region of the following molecules: CD8 ⁇ , CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3 ⁇ , IL-2 receptor, IL-7 receptor, IL -11 receptor;
  • the signal peptide includes the signal peptide of the following molecules: ⁇ chain and ⁇ chain of T cell receptor, CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR, GM-CSF;
  • the costimulatory signal domain includes the costimulatory signal domain of the following molecules: 4-1BB (CD137), CD27, CD19, CD4, CD28, ICOS (CD278), CD8 ⁇ , CD8 ⁇ , BAFFR, HVEM, LIGHT , KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TIM1, CD2, CD7, CD226;
  • the promoter is not limited to the EF1 ⁇ promoter, but also includes: CMV promoter, EFS promoter, CAG promoter, CBh promoter, SFFV promoter, MSCV promoter, SV40 promoter, mPGK promoter , hPGK promoter, UBC promoter;
  • the self-cleaving peptide is not limited to T2A, but also includes: P2A, E2A, and F2A;
  • the detection label/auxiliary functional element is not limited to tEGFR, but also includes: tCD34, tCD19, tCD20, tCD22, immune checkpoint inhibitors (CTLA-4, PD-1/PD-L1, LAG-3 , TIM-3, TIGIT, CD226, CD155, CD47, B7-H3, B7-H4) Nanobodies, cytokines and their receptors (IL2, IL2 receptor, IL7, IL7 receptor, IL15, IL15 receptor);
  • the transmembrane domain is a CD8 ⁇ transmembrane domain
  • the intracellular signaling domain is a CD3 ⁇ intracellular signaling domain
  • the hinge region is a CD8 ⁇ hinge region
  • the costimulatory signal domain is a 4-1BB costimulatory signal domain
  • the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 145 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% with SEQ ID NO: 145 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Amino acid sequence with 99% identity;
  • the nucleotide sequence of the signal peptide is as shown in SEQ ID NO: 146 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81% with SEQ ID NO: 146 , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% identical nucleotide sequence;
  • the amino acid sequence of the CD8 ⁇ hinge region is as shown in SEQ ID NO: 147 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% identical amino acid sequence;
  • the nucleotide sequence of the CD8 ⁇ hinge region is as shown in SEQ ID NO: 148 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81% similarity with SEQ ID NO: 148 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Nucleotide sequences with 98% or 99% identity;
  • the amino acid sequence of the CD8 ⁇ transmembrane domain is as shown in SEQ ID NO: 149 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81% with SEQ ID NO: 149 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98% or 99% identity;
  • the nucleotide sequence of the CD8 ⁇ transmembrane domain is as shown in SEQ ID NO: 150 or has at least 75%, 76%, 77%, 78%, 79%, 80% with SEQ ID NO: 150 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 Nucleotide sequences with %, 98%, and 99% identity;
  • the amino acid sequence of the 4-1BB costimulatory signal domain is as shown in SEQ ID NO: 151 or has at least 75%, 76%, 77%, 78%, 79%, 80% with SEQ ID NO: 151 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Amino acid sequences with 97%, 98%, and 99% identity;
  • the nucleotide sequence of the 4-1BB costimulatory signal domain is as shown in SEQ ID NO: 152 or has at least 75%, 76%, 77%, 78%, 79% with SEQ ID NO: 152 , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 Nucleotide sequences with %, 97%, 98%, and 99% identity;
  • the amino acid sequence of the CD3 ⁇ intracellular signaling domain is as shown in SEQ ID NO: 153 or has at least 75%, 76%, 77%, 78%, 79%, 80% with SEQ ID NO: 153 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% identical amino acid sequences;
  • the nucleotide sequence of the CD3 ⁇ intracellular signaling domain is as shown in SEQ ID NO: 154 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% identical nucleotide sequences;
  • the amino acid sequence of T2A is as shown in SEQ ID NO: 155 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% with SEQ ID NO: 155 , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % identity of the amino acid sequence;
  • the nucleotide sequence of the T2A is as shown in SEQ ID NO: 156 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% identical nucleotide sequence;
  • nucleotide sequence of EF1 ⁇ is as shown in SEQ ID NO:157 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% identical nucleotide sequence;
  • the amino acid sequence of the tEGFR signal peptide is as shown in SEQ ID NO: 158 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% identical amino acid sequence;
  • the nucleotide sequence of the tEGFR signal peptide is as shown in SEQ ID NO: 159 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81% similarity with SEQ ID NO: 159 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Nucleotide sequences with 98% or 99% identity;
  • the amino acid sequence of tEGFR is as shown in SEQ ID NO: 160 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% with SEQ ID NO: 160 , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % identity of the amino acid sequence;
  • nucleotide sequence of tEGFR is as shown in SEQ ID NO:161 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% identical nucleotide sequence;
  • the chimeric antigen receptor is any one of EF1 ⁇ , a signal peptide, the Nanobody described in the first aspect of the present invention or the humanized anti-CD7 Nanobody described in the second aspect of the present invention,
  • the CD8 ⁇ hinge region, CD8 ⁇ transmembrane domain, 4-1BB costimulatory signaling domain, CD3 ⁇ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR are obtained in series.
  • a fourth aspect of the invention provides dual Nanobody-based chimeric antigen receptors.
  • the chimeric antigen receptor includes any two of the Nanobodies described in the first aspect of the present invention or the humanized anti-CD7 Nanobodies described in the second aspect of the present invention;
  • the chimeric antigen receptor further comprises a transmembrane domain
  • the chimeric antigen receptor further comprises an intracellular signaling domain
  • the chimeric antigen receptor further comprises a hinge region
  • the chimeric antigen receptor further comprises a signal peptide
  • the chimeric antigen receptor further comprises a costimulatory signaling domain
  • the chimeric antigen receptor further comprises promoter EF1 ⁇ ;
  • the chimeric antigen receptor further comprises a self-cleaving peptide T2A;
  • the chimeric antigen receptor further includes a detection tag/auxiliary functional element tEGFR;
  • the transmembrane domain includes the transmembrane domain of the following molecules: CD8 ⁇ , CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3 ⁇ , IL-2 receptor, IL-7 Receptor, IL-11 receptor;
  • the intracellular signaling domain includes the intracellular signaling domain of the following molecules: CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , TCR ⁇ , CD4, CD5, CD8, CD21, CD22, CD79a, CD79b , CD278, Fc ⁇ RI, DAP10, DAP12, CD66d;
  • the hinge region includes the hinge region of the following molecules: CD8 ⁇ , CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3 ⁇ , IL-2 receptor, IL-7 receptor, IL -11 receptor;
  • the signal peptide includes the signal peptide of the following molecules: ⁇ chain and ⁇ chain of T cell receptor, CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR, GM-CSF;
  • the costimulatory signal domain includes the costimulatory signal domain of the following molecules: 4-1BB (CD137), CD27, CD19, CD4, CD28, ICOS (CD278), CD8 ⁇ , CD8 ⁇ , BAFFR, HVEM, LIGHT , KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TIM1, CD2, CD7, CD226;
  • the promoter is not limited to the EF1 ⁇ promoter, but also includes: CMV promoter, EFS promoter, CAG promoter, CBh promoter, SFFV promoter, MSCV promoter, SV40 promoter, mPGK promoter , hPGK promoter, UBC promoter;
  • the self-cleaving peptide is not limited to T2A, but also includes: P2A, E2A, and F2A;
  • the detection label/auxiliary functional element is not limited to tEGFR, but also includes: tCD34, tCD19, tCD20, tCD22, immune checkpoint inhibitors (CTLA-4, PD-1/PD-L1, LAG-3 , TIM-3, TIGIT, CD226, CD155, CD47, B7-H3, B7-H4) Nanobodies, cytokines and their receptors (IL2, IL2 receptor, IL7, IL7 receptor, IL15, IL15 receptor);
  • the transmembrane domain is a CD8 ⁇ transmembrane domain
  • the intracellular signaling domain is a CD3 ⁇ intracellular signaling domain
  • the hinge region is a CD8 ⁇ hinge region
  • the costimulatory signal domain is a 4-1BB costimulatory signal domain
  • any two Nanobodies among the Nanobodies described in the first aspect of the present invention or the humanized anti-CD7 Nanobodies described in the second aspect of the present invention are connected through a linking peptide Linker;
  • the chimeric antigen receptor is any two of EF1 ⁇ , a signal peptide, the Nanobody described in the first aspect of the present invention or the humanized anti-CD7 Nanobody described in the second aspect of the present invention,
  • the CD8 ⁇ hinge region, CD8 ⁇ transmembrane domain, 4-1BB costimulatory signaling domain, CD3 ⁇ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR are sequentially connected in series;
  • the chimeric antigen receptor is any one of EF1 ⁇ , a signal peptide, and the Nanobody described in the first aspect of the present invention, Linker, or any one of the Nanobodies described in the first aspect of the present invention.
  • CD8 ⁇ hinge region, CD8 ⁇ transmembrane domain, 4-1BB costimulatory signal domain, CD3 ⁇ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR are obtained by serial connection;
  • any one of the Nanobodies described in the first aspect of the present invention is VHH03, VHH06, VHH10, or VHH12;
  • the chimeric antigen receptor is EF1 ⁇ , signal peptide, VHH06 described in the first aspect of the present invention, Linker, VHH03 described in the first aspect of the present invention, CD8 ⁇ hinge region, CD8 ⁇ transmembrane domain , 4-1BB costimulatory signaling domain, CD3 ⁇ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR are obtained by serial connection;
  • the chimeric antigen receptor is EF1 ⁇ , signal peptide, VHH06 described in the first aspect of the present invention, Linker, VHH12 described in the first aspect of the present invention, CD8 ⁇ hinge region, CD8 ⁇ transmembrane domain , 4-1BB costimulatory signaling domain, CD3 ⁇ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR are obtained by serial connection;
  • the chimeric antigen receptor is EF1 ⁇ , signal peptide, VHH10 described in the first aspect of the present invention, Linker, VHH12 described in the first aspect of the present invention, CD8 ⁇ hinge region, CD8 ⁇ transmembrane domain , 4-1BB costimulatory signaling domain, CD3 ⁇ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR are obtained by serial connection;
  • the chimeric antigen receptor is EF1 ⁇ , signal peptide, VHH10 described in the first aspect of the present invention, Linker, VHH10 described in the first aspect of the present invention, CD8 ⁇ hinge region, CD8 ⁇ transmembrane domain , 4-1BB costimulatory signaling domain, CD3 ⁇ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR are obtained by serial connection;
  • the chimeric antigen receptor is EF1 ⁇ , signal peptide, VHH12 described in the first aspect of the present invention, Linker, VHH12 described in the first aspect of the present invention, CD8 ⁇ hinge region, CD8 ⁇ transmembrane domain , 4-1BB costimulatory signaling domain, CD3 ⁇ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR are obtained by serial connection;
  • the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 145 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% with SEQ ID NO: 145 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Amino acid sequence with 99% identity;
  • the nucleotide sequence of the signal peptide is as shown in SEQ ID NO: 146 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81% with SEQ ID NO: 146 , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% identical nucleotide sequence;
  • the amino acid sequence of the CD8 ⁇ hinge region is as shown in SEQ ID NO: 147 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% identical amino acid sequence;
  • the nucleotide sequence of the CD8 ⁇ hinge region is as shown in SEQ ID NO: 148 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81% similarity with SEQ ID NO: 148 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Nucleotide sequences with 98% or 99% identity;
  • the amino acid sequence of the CD8 ⁇ transmembrane domain is as shown in SEQ ID NO: 149 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81% with SEQ ID NO: 149 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98% or 99% identity;
  • the nucleotide sequence of the CD8 ⁇ transmembrane domain is as shown in SEQ ID NO: 150 or has at least 75%, 76%, 77%, 78%, 79%, 80% with SEQ ID NO: 150 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 Nucleotide sequences with %, 98%, and 99% identity;
  • the amino acid sequence of the 4-1BB costimulatory signal domain is as shown in SEQ ID NO: 151 or has at least 75%, 76%, 77%, 78%, 79%, 80% with SEQ ID NO: 151 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Amino acid sequences with 97%, 98%, and 99% identity;
  • the nucleotide sequence of the 4-1BB costimulatory signal domain is as shown in SEQ ID NO: 152 or has at least 75%, 76%, 77%, 78%, 79% with SEQ ID NO: 152 , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 Nucleotide sequences with %, 97%, 98%, and 99% identity;
  • the amino acid sequence of the CD3 ⁇ intracellular signaling domain is as shown in SEQ ID NO: 153 or has at least 75%, 76%, 77%, 78%, 79%, 80% with SEQ ID NO: 153 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% identical amino acid sequences;
  • the nucleotide sequence of the CD3 ⁇ intracellular signaling domain is as shown in SEQ ID NO: 154 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% identical nucleotide sequences;
  • the amino acid sequence of T2A is as shown in SEQ ID NO: 155 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% with SEQ ID NO: 155 , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % identity of the amino acid sequence;
  • the nucleotide sequence of the T2A is as shown in SEQ ID NO: 156 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% identical nucleotide sequence;
  • nucleotide sequence of EF1 ⁇ is as shown in SEQ ID NO:157 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% identical nucleotide sequence;
  • the amino acid sequence of the tEGFR signal peptide is as shown in SEQ ID NO: 158 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% identical amino acid sequence;
  • the nucleotide sequence of the tEGFR signal peptide is as shown in SEQ ID NO: 159 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81% similarity with SEQ ID NO: 159 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Nucleotide sequences with 98% or 99% identity;
  • the amino acid sequence of tEGFR is as shown in SEQ ID NO: 160 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% with SEQ ID NO: 160 , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % identity of the amino acid sequence;
  • the nucleotide sequence of tEGFR is as shown in SEQ ID NO:161 or has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% identical nucleotide sequence.
  • the term "functional portion” refers to any portion or fragment of one or more chimeric antigen receptors disclosed herein that retains the chimeric antigen Biological activity of the receptor of which the portion or fragment is a part (the parent chimeric antigen receptor).
  • Functional portions encompass, for example, those portions of a chimeric antigen receptor that retain recognition of target cells or detection, treatment, or prevention to a similar extent, to the same extent, or to a greater extent than the parent chimeric antigen receptor. disease capacity.
  • the functional moiety may comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent chimeric antigen receptor.
  • the functional moiety may comprise additional amino acids at the amino or carboxy terminus of the moiety, or at both termini, that are not present in the amino acid sequence of the parent chimeric antigen receptor.
  • the additional amino acids do not interfere with the biological function of the functional moiety, such as identifying target cells, detecting cancer, treating or preventing cancer, etc. More desirably, the additional amino acids enhance the biological activity compared to the biological activity of the parent chimeric antigen receptor.
  • the term "functional variant” as used herein refers to a chimeric antigen receptor, polypeptide or protein that has clear or significant sequence identity or similarity to a parent chimeric antigen receptor that retains chimeric Biological activity of the antigen receptor of which the functional variant is a variant.
  • Functional variants encompass, for example, those variants of the chimeric antigen receptors described herein (the parent chimeric antigen receptor) that are to a similar extent, to the same extent, or to the same extent as the parent chimeric antigen receptor. The ability to recognize target cells is retained to a higher extent.
  • the functional variant may, for example, be at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical in amino acid sequence to the parent chimeric antigen receptor. .
  • Functional variants may, for example, comprise the amino acid sequence of the parent chimeric antigen receptor with at least one conservative amino acid substitution.
  • functional variants may comprise the amino acid sequence of the parent chimeric antigen receptor with at least one non-conservative amino acid substitution.
  • the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant.
  • Non-conservative amino acid substitutions can enhance the biological activity of the functional variant such that the functional variant has increased biological activity compared to the parent chimeric antigen receptor.
  • Amino acid substitutions in the chimeric antigen receptor are preferably conservative amino acid substitutions.
  • Conservative amino acid substitutions are known in the art and include substitutions in which one amino acid with specific physical and/or chemical properties is exchanged for another amino acid with the same or similar chemical or physical properties.
  • a conservative amino acid substitution may be an acidic/negatively charged polar amino acid replacing another acidic/negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a non-polar side chain replacing another with a non-polar side chain.
  • An amino acid with a polar side chain e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.
  • a basic/positively charged polar amino acid replaces another basic/charged A positively charged polar amino acid (e.g., Lys, His, Arg, etc.)
  • an uncharged amino acid with a polar side chain replaces another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.)
  • an amino acid with a ⁇ -branched side chain replaces another amino acid with a ⁇ -branched side chain (e.g., He, Thr, and Val)
  • an amino acid with an aromatic side chain replaces another with an aromatic side chain amino acids (for example, His, Phe, Trp and Tyr), etc.
  • a chimeric antigen receptor can consist essentially of one or more specific amino acid sequences described herein, such that other components (eg, other amino acids) do not materially alter the biological activity of the functional variant.
  • the chimeric antigen receptor (including functional portions and functional variants) may be of any length, i.e., contain any number of amino acids, as long as the chimeric antigen receptor (or functional portion or functional variant thereof) retains its biological Activity, such as the ability to specifically bind to an antigen, detect diseased cells in mammals, or treat or prevent diseases in mammals, is sufficient.
  • a chimeric antigen receptor can be about 50 to about 5000 amino acids long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900 in length , 1000 or more amino acids.
  • Chimeric antigen receptors may contain synthetic amino acids that replace one or more naturally occurring amino acids.
  • synthetic amino acids include, for example, aminocyclohexanecarboxylic acid, norleucine, 10-aminon-decanoic acid, homoserine, S-acetamidomethyl-cysteine, trans -3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine , ⁇ -phenylserine, ⁇ -hydroxyphenylalanine, phenylglycine, ⁇ -naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2 , 3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid, aminomalonic acid, aminomalonic acid, aminomalonic
  • the chimeric antigen receptors of the invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (via, for example, di- sulfur bridge) or converted into acid addition salts and/or optionally dimeric or multimeric, or conjugated.
  • a fifth aspect of the invention provides nucleic acid molecules.
  • the nucleic acid molecule includes a DNA encoding the Nanobody described in the first aspect of the present invention, the humanized anti-CD7 Nanobody described in the second aspect of the present invention, the chimeric antigen receptor described in the third aspect of the present invention, or The nucleotide sequence of the chimeric antigen receptor according to the fourth aspect of the present invention;
  • the nucleotide sequence is such as SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO: 50.
  • nucleic acid molecule may comprise natural, non-natural or altered nucleotides; and it may comprise natural, non-natural or altered inter-nucleotide linkages, such as phosphoramidate linkages or Phosphorothioate linkages replace the phosphodiesters present between the nucleotides of unmodified oligonucleotides.
  • nucleic acid does not contain any insertions, deletions, inversions and/or substitutions.
  • nucleic acid may contain one or more insertions, deletions, inversions and/or substitutions such that the nucleic acid formed by these insertions, deletions, inversions and/or substitutions is also within the protection scope of the present invention.
  • the nucleic acid molecules include a variety of nucleic acids that may use naturally occurring nucleotides or be designed to increase the biological stability of the molecule or to increase the physical stability of the doublet formed upon hybridization.
  • Nucleic acid molecules obtained by chemical synthesis of modified nucleotides (such as phosphorothioate derivatives and acridine-substituted nucleotides).
  • modified nucleotides that can be used to produce nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine Pyrimidine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, ⁇ -D-semi- Lactosyl queosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine , 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy methylaminomethyl-2-thiouracil,
  • a sixth aspect of the invention provides recombinant expression vectors.
  • the recombinant expression vector contains the nucleic acid molecule described in the fifth aspect of the present invention.
  • the expression vector includes a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector, or a viral vector;
  • the viral vector includes a lentiviral vector, an adenoviral vector, and a retroviral vector.
  • the recombinant expression vector can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host cell.
  • Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses.
  • Vectors can be selected from the pUC series (Fermentas Life Sciences, Glen Bumie, MD), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series ( Clontech, Palo Alto, CA).
  • Phage vectors such as ⁇ ZapII (Stratagene), EMBL4 and ⁇ NMI149 can also be used.
  • plant expression vectors include pBIO1, pBI101.2, pBHO1.3, pBI121 and pBIN19 (Clontech).
  • animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech).
  • the recombinant expression vector may be a viral vector, such as a retroviral vector or a lentiviral vector.
  • a lentiviral vector is a vector derived from at least a portion of a lentiviral genome, including in particular self-inactivating lentiviral vectors, such as those provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009).
  • Other examples of lentiviral vectors that can be used clinically include, for example, but are not limited to, LENTIVECTOR.RTM. gene delivery technology from Oxford BioMedica plc, LENTIMAX.TM. vector system from Lentigen, etc.
  • Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.
  • the recombinant expression vector may contain regulatory sequences, such as transcriptional and translational initiation and termination codons, that are specific to the type of host cell (e.g., bacterial, fungal, plant, or animal) into which the vector is to be introduced. , which depends on the situation and takes into account whether the vector is DNA- or RNA-based.
  • Recombinant expression vectors may contain restriction sites to facilitate cloning.
  • the recombinant expression vector may contain one or more marker genes that allow selection of transformed or transfected host cells.
  • Marker genes include biocide resistance (eg, resistance to antibiotics, heavy metals, etc.); complementation to provide prototrophy in auxotrophic hosts, etc.
  • Suitable marker genes for use in the expression vector of the present invention include, for example, neomycin/G418 resistance gene, hygromycin resistance gene, histidinol resistance gene, tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene toxin resistance genes, puromycin resistance genes, etc.
  • the recombinant expression vector may comprise a nucleotide sequence encoding the chimeric antigen receptor (including functional portions and functional variants thereof) or complementary to a nucleotide sequence encoding the chimeric antigen receptor or
  • the hybridizing nucleotide sequence may be operably linked to a natural or non-natural promoter. Selection of promoters (eg, strong, weak, inducible, tissue-specific, and development-specific) is within the ordinary skill of the skilled artisan. Similarly, the combination of nucleotide sequences and promoters is within the skill of the skilled artisan.
  • the promoter can be a non-viral promoter or a viral promoter, such as type III promoter (U6 promoter, H1 promoter), mammalian constitutive promoter (generalized promoter): CMV (cytomegalovirus) promoter; EF1A (elongation factor-1 ⁇ ) promoter; EFS promoter; CAG (composed of cytomegalovirus enhancer and chicken ⁇ -actin promoter) promoter; CBh promoter; SFFV promoter; MSCV promoter; SV40 (simian virus Source) promoter; mPGK promoter; hPGK (phosphoglycerate kinase) promoter; UBC (ubiquitin C) promoter, RSV promoter, or promoter found in the long terminal repeat of murine stem cell virus.
  • type III promoter U6 promoter, H1 promoter
  • mammalian constitutive promoter generally known promoter
  • CMV cytomegalovirus
  • EF1A elongation factor-1 ⁇
  • the recombinant expression vector can be designed for transient expression, for stable expression, or for both. Additionally, recombinant expression vectors can be made for constitutive expression or for inducible expression.
  • the recombinant expression vector also contains a suicide gene.
  • suicide gene refers to a gene that causes the death of cells expressing the suicide gene.
  • a suicide gene may be a gene that confers sensitivity to an agent (eg, a drug) in a cell in which it is expressed, and causes the cell to die when the cell is brought into contact with or exposed to the agent.
  • agent e.g, a drug
  • Suicide genes are known in the art (see, e.g., Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J.
  • HSV herpes simplex virus
  • TK thymidine kinase
  • a seventh aspect of the invention provides engineered host cells.
  • the engineered host cells express the Nanobody described in the first aspect of the present invention, the humanized anti-CD7 Nanobody described in the second aspect of the present invention, and the chimeric antigen described in the third aspect of the present invention.
  • Receptor or chimeric antigen receptor according to the fourth aspect of the present invention are also included in the engineered host cells.
  • the engineered host cell contains the recombinant expression vector described in the sixth aspect of the present invention.
  • the engineered host cells comprise engineered immune cells
  • the engineered immune cells comprise T cells, NK cells, iNKT cells, CTL cells, monocytes, macrophages, dendritic cells, NKT cells, or any combination thereof.
  • the term "host cell” as used herein refers to any type of cell.
  • the host cell may be a eukaryotic cell, such as a plant, animal, fungus, or algae, or a prokaryotic cell, such as a bacterium or protozoa.
  • Host cells may be cultured cells or primary cells, ie, isolated directly from an organism (eg, human).
  • Host cells can be adherent cells or suspension cells, that is, cells that grow in suspension. Suitable host cells are known in the art and include, for example, DH5a E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like.
  • the host cell may be a prokaryotic cell, such as a DH5 ⁇ cell.
  • the host cell may be a mammalian cell.
  • the host cell can be a human cell. While the host cell can be any cell type, derived from any type of tissue, and at any stage of development, the host cell can be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBL). cell, PBMC).
  • the host cells are T cells.
  • the engineered host cells include an engineered host cell population.
  • the engineered host cell population includes non-expressing Nanobodies described in the first aspect of the present invention, humanized anti-CD7 Nanobodies described in the second aspect of the present invention, and Nanobodies described in the third aspect of the present invention.
  • the host cells are immune cells.
  • the immune cells include T cells, NK cells, iNKT cells, CTL cells, monocytes, macrophages, dendritic cells, NKT cells or any combination thereof.
  • An eighth aspect of the invention provides a conjugate.
  • the conjugate includes the Nanobody according to the first aspect of the present invention or the humanized anti-CD7 Nanobody according to the second aspect of the present invention, and a modified part connected to the Nanobody, and the modification Some include detectable markers, therapeutic agents;
  • the detectable label includes an enzyme, a radionuclide, a fluorescent dye, a luminescent substance, and biotin,
  • the therapeutic agent includes a drug with anti-tumor activity or a cytotoxic agent.
  • a ninth aspect of the invention provides a pharmaceutical composition.
  • the pharmaceutical composition includes the Nanobody described in the first aspect of the present invention, the humanized anti-CD7 Nanobody described in the second aspect of the present invention, the chimeric antigen receptor described in the third aspect of the present invention, The chimeric antigen receptor described in the fourth aspect of the present invention, the nucleic acid molecule described in the fifth aspect of the present invention, the recombinant expression vector described in the sixth aspect of the present invention, and the engineered host described in the seventh aspect of the present invention cells or the conjugate according to the eighth aspect of the present invention.
  • composition also contains additional pharmaceutically active agents
  • the additional pharmaceutically active agent includes an additional antibody, fusion protein or drug (such as an anti-tumor drug, such as a drug for radiotherapy or a chemotherapeutic drug).
  • an additional antibody, fusion protein or drug such as an anti-tumor drug, such as a drug for radiotherapy or a chemotherapeutic drug.
  • the pharmaceutical composition also contains pharmaceutically acceptable carriers and/or excipients.
  • the pharmaceutically acceptable carriers and/or excipients are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). These substances are used to help the stability of the drug or help improve the effectiveness as needed.
  • the activity of the ingredients, the preparations that can be used in this pharmaceutical composition can be in the form of the original compound itself, or optionally in the form of a pharmaceutically acceptable salt thereof, the pharmaceutical composition so formulated can be selected as needed.
  • the drug is administered in any suitable manner known to those skilled in the art.
  • Pharmaceutically acceptable carriers and/or excipients may additionally contain liquids such as water, physiological saline, glycerin and ethanol.
  • auxiliary substances such as wetting or emulsifying agents or pH buffering substances may be present in the compositions.
  • Suitable forms of administration include those suitable for parenteral administration, eg by injection or infusion, eg by bolus injection or continuous infusion, intravenous, inhalable or subcutaneous forms.
  • the product may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents such as suspending agents, preservatives, stabilizers and/or Dispersant.
  • Nanobodies provided according to the invention may be in dry form for reconstitution with a suitable sterile liquid prior to use. Solid forms suitable for solution in or suspension in liquid vehicles prior to injection may also be prepared.
  • a tenth aspect of the present invention provides a kit.
  • the kit includes the Nanobody according to the first aspect of the present invention, the humanized anti-CD7 Nanobody according to the second aspect of the present invention, the chimeric antigen receptor according to the third aspect of the present invention, the The chimeric antigen receptor according to the fourth aspect of the invention, the nucleic acid molecule according to the fifth aspect of the invention, the recombinant expression vector according to the sixth aspect of the invention, and the engineered host cell according to the seventh aspect of the invention Or the conjugate described in the eighth aspect of the present invention.
  • An eleventh aspect of the present invention provides a reagent for detecting CD7 protein or antigen fragment thereof.
  • the reagent for detecting CD7 protein or its antigen fragment includes the Nanobody described in the first aspect of the present invention, the humanized anti-CD7 Nanobody described in the second aspect of the present invention, or the Nanobody described in the eighth aspect of the present invention. conjugate.
  • a twelfth aspect of the invention provides a biological preparation.
  • the biological preparation contains the engineered host cell described in the seventh aspect of the present invention and/or the pharmaceutical composition described in the ninth aspect of the present invention.
  • biological preparations described in the present invention are any pharmaceutical products manufactured, extracted or semi-synthesized from biological sources. They include, but are not limited to, vaccines, blood or blood components, and recombinant therapeutic proteins (such as antibodies, cytokines, chemokines, and fusion proteins). Biologics can consist of sugars, proteins, or nucleic acids or complex combinations of these substances.
  • compositions or biologics for gene therapy, immunotherapy and/or cell therapy comprising in a carrier (eg, a pharmaceutically acceptable carrier) a combination of one of the or One or more of the disclosed chimeric antigen receptors, immune cells expressing chimeric antigen receptors, Nanobodies, conjugates that specifically bind to a wider variety of antigens.
  • a carrier eg, a pharmaceutically acceptable carrier
  • the pharmaceutical composition or biologic or composition thereof may be prepared in unit dosage form for administration to a subject. The amount and timing of administration are determined by the treating clinician to achieve the desired outcome.
  • Compositions may be formulated for systemic (eg, intravenous) or local (eg, intratumoral) administration.
  • the disclosed chimeric antigen receptors, immune cells expressing the chimeric antigen receptors (in particular, T cells expressing the chimeric antigen receptors), Nanobodies, and conjugates are formulated For parenteral administration, such as intravenous administration.
  • Treatment and detection of tumors including, but not limited to, CD7-expressing tumors.
  • the above-mentioned pharmaceutical compositions or biological agents or combinations thereof can be used in the treatment and/or detection of tumors.
  • a chimeric antigen receptor as disclosed herein, an immune cell expressing the chimeric antigen receptor (in particular, a T cell expressing the chimeric antigen receptor), a Nanobody, a conjugate, or a combination thereof Materials are also used, for example, in the detection of CD7 antigen.
  • compositions or biologics for administration, or compositions thereof may comprise chimeric antigen receptors, immune cells expressing said chimeric antigen receptors (in particular, T cells expressing the chimeric antigen receptor), conjugates, and solutions of Nanobodies.
  • aqueous vehicles can be used, such as buffered saline and the like. These solutions are sterile and generally free of undesirable substances.
  • These pharmaceutical compositions or biologics or compositions thereof may be sterilized by conventional, well-known sterilization techniques.
  • compositions or biological preparations or compositions thereof may contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjusters and buffers, toxicity adjusters, auxiliary agents, etc., such as sodium acetate, sodium chloride, Potassium chloride, calcium chloride, sodium lactate, etc.
  • concentration of chimeric antigen receptors, immune cells expressing said chimeric antigen receptors (in particular, T cells expressing said chimeric antigen receptors), conjugates, Nanobodies in these preparations can vary widely, And depending on the specific mode of administration selected and the needs of the subject, selection will be based primarily on fluid volume, viscosity, body weight, etc. Practical methods of preparing such dosage forms for gene therapy, immunotherapy and/or cell therapy are known or will be apparent to those skilled in the art.
  • compositions for intravenous administration comprise from about 0.01 mg/kg to about 30 mg/kg of the Nanobody of the first aspect of the invention or of the second aspect of the invention.
  • Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in greater detail in publications such as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995) .
  • the chimeric antigen receptors, immune cells expressing the chimeric antigen receptors (in particular, T cells expressing the chimeric antigen receptors), Nanobodies or conjugates described herein can be in lyophilized form Provided and rehydrated with sterile water prior to administration, but it is also provided as a sterile solution of known concentration.
  • the chimeric antigen receptor, immune cells expressing the chimeric antigen receptor (in particular, T cells expressing the chimeric antigen receptor), Nanobody or conjugate solution are added to a solution containing 0.9% chloride Sodium (USP) in an infusion bag and in some cases administered at a dose of 0.5 mg/kg body weight to 15 mg/kg body weight.
  • USP chloride Sodium
  • Chimeric antigen receptors, immune cells expressing chimeric antigen receptors, antibodies, antigen-binding fragments and conjugates thereof, and the like may be administered by slow infusion rather than by intravenous push or bolus. In one embodiment, a higher loading dose is administered, followed by a maintenance dose at a lower level.
  • an initial loading dose of 4 mg/kg of antibody or antigen-binding fragment may be infused over approximately 90 minutes, followed by, if the previous dose is well tolerated, A weekly maintenance dose of 2 mg/kg is administered over 30 minutes for 4 to 8 weeks.
  • Controlled release parenteral formulations may be formulated as implants, oily injections, or as particulate systems.
  • Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres and nanoparticles.
  • Microcapsules contain therapeutic proteins (eg, cytotoxins or drugs) as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres and microcapsules less than about 1 ⁇ m are generally referred to as nanoparticles, nanospheres and nanocapsules, respectively.
  • the diameter of the capillaries is approximately 5 ⁇ m allowing only nanoparticles to be administered intravenously.
  • Microparticles are typically about 100 ⁇ m in diameter and are administered subcutaneously or intramuscularly. See, e.g., Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).
  • Polymers may be used in the chimeric antigen receptors disclosed herein, immune cells expressing the chimeric antigen receptor (in particular, T cells expressing the chimeric antigen receptor), Nanobodies, conjugates, or Ion controlled release of its composition.
  • immune cells expressing the chimeric antigen receptor in particular, T cells expressing the chimeric antigen receptor
  • Nanobodies conjugates
  • Ion controlled release of its composition A variety of degradable and nondegradable polymer matrices for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993).
  • the block copolymer Poloxamer 407 exists as a viscous but mobile liquid at low temperatures but forms a semi-solid gel at body temperature.
  • Existing studies have shown that it is an effective carrier for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm.
  • hydroxyapatite has been used as microcarriers for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994).
  • liposomes are used for controlled release of lipid-encapsulated drugs as well as drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993) ).
  • Many additional systems for controlled delivery of therapeutic proteins are known (see U.S. Patent No. 5,055,303; U.S. Patent No.
  • a thirteenth aspect of the invention provides a method of stimulating an immune response to a target cell population or tissue in a mammal.
  • the method includes the following steps: administering to the mammal an effective amount of the engineered host cell described in the seventh aspect of the present invention, the conjugate described in the eighth aspect of the present invention, or the ninth aspect of the present invention.
  • the pharmaceutical composition or the biological preparation according to the twelfth aspect of the present invention is administered to the mammal an effective amount of the engineered host cell described in the seventh aspect of the present invention, the conjugate described in the eighth aspect of the present invention, or the ninth aspect of the present invention.
  • a fourteenth aspect of the invention provides a method of modulating an immune response in a subject.
  • the method includes the following steps: administering to the subject an effective amount of the engineered host cell according to the seventh aspect of the present invention, the conjugate according to the eighth aspect of the present invention, and the ninth aspect of the present invention.
  • the pharmaceutical composition described above or the biological preparation described in the twelfth aspect of the present invention is administered to the subject an effective amount of the engineered host cell according to the seventh aspect of the present invention, the conjugate according to the eighth aspect of the present invention, and the ninth aspect of the present invention.
  • the pharmaceutical composition described above or the biological preparation described in the twelfth aspect of the present invention is administered to the subject an effective amount of the engineered host cell according to the seventh aspect of the present invention, the conjugate according to the eighth aspect of the present invention, and the ninth aspect of the present invention.
  • the fifteenth aspect of the present invention provides a method for producing the Nanobody described in the first aspect of the present invention or the humanized anti-CD7 Nanobody described in the fifth aspect of the present invention.
  • the method includes the following steps: culturing the engineered host cells described in the seventh aspect of the present invention, and isolating the Nanobody described in the first aspect of the present invention or the Nanobody described in the second aspect of the present invention from the culture. Humanized anti-CD7 Nanobody.
  • a sixteenth aspect of the present invention provides a method for specifically inhibiting CD7 activity.
  • the method includes the following steps: introducing the nucleic acid molecule described in the fifth aspect of the present invention into biological cells, by expressing the Nanobody described in the first aspect of the present invention or the human source described in the second aspect of the present invention.
  • Anti-CD7 Nanobodies inhibit CD7 activity.
  • a seventeenth aspect of the present invention provides a method of preventing and/or treating a CD7-associated disease or disorder.
  • the method includes the following steps: adding an effective amount of the Nanobody according to the first aspect of the present invention, the humanized anti-CD7 Nanobody according to the second aspect of the present invention, and the nucleic acid according to the fifth aspect of the present invention.
  • Molecule, the recombinant expression vector according to the sixth aspect of the present invention, the engineered host cell according to the seventh aspect of the present invention, the conjugate according to the eighth aspect of the present invention, and the drug according to the ninth aspect of the present invention The composition or biologic of the twelfth aspect of the invention is administered to a subject having a disease or disorder associated with CD7.
  • CD7-related diseases or conditions include tumors expressing CD7.
  • the tumor is a hematological tumor of T lymphocyte lineage.
  • the tumors include acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), lymphoblastic lymphoma (LBL), NKT cell leukemia, peripheral T cell lymphoma (NHL), NKT cell lymphoma, mesenchymal lymphoma, Anaplastic large cell lymphoma (ALCL).
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • LBL lymphoblastic lymphoma
  • NKT cell leukemia peripheral T cell lymphoma
  • NHL peripheral T cell lymphoma
  • NKT cell lymphoma mesenchymal lymphoma
  • ACL Anaplastic large cell lymphoma
  • CD7-related diseases or disorders described in the present invention refer to any disease or disorder related to the expression of CD7, including but not limited to the disease types listed above. As long as the disease or disorder is related to the expression of CD7, all diseases or disorders are related to the expression of CD7. within the protection scope of the present invention.
  • treatment and “prevention” and the words from which they are derived do not necessarily mean 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art would consider potentially beneficial or therapeutically effective.
  • the methods can provide any amount or level of cancer treatment or prevention in a mammal.
  • Treatment or prevention provided by the methods may include treating or preventing one or more conditions or symptoms of the disease (eg, cancer) being treated or prevented. Additionally, for purposes herein, "prevention" may encompass delaying the onset of a disease or a symptom or condition thereof.
  • the mammal referred to herein may be any mammal, including but not limited to mammals of the order Rodentia, such as mice and hamsters; and mammals of the order Lagomorpha, such as rabbits.
  • Mammals may be from the order Carnivora, including the families Felidae (cats) and Canidae (dogs).
  • Mammals may be from the order Artiodactyla, which includes the family Bovidae (cows) and Suidae (pig), or the order Perissodactyla, which includes the family Equidae (horses).
  • a mammal may be of the order Primates, Ceboid, or Simoid (monkeys); or of the order Anthropoid (humans and apes).
  • the mammal is a human.
  • engineered host cells comprise the single Nanobody-based chimeric antigen receptor (CAR) described in the third aspect of the present invention or the dual Nanobody-based CAR described in the fourth aspect of the present invention that expresses anti-CD7.
  • CAR Nanobody-based chimeric antigen receptor
  • the engineered host cells comprise the single Nanobody-based chimeric antigen receptor (CAR) described in the third aspect of the present invention or the dual Nanobody-based CAR described in the fourth aspect of the present invention that expresses anti-CD7.
  • CAR Nanobody-based chimeric antigen receptor
  • the immune cells include T cells, NK cells, iNKT cells, CTL cells, monocytes, macrophages, dendritic cells, and NKT cells;
  • the engineered host cells are CAR-T cells.
  • the engineered host cells are administered to a subject suffering from a CD7-associated disease or disorder in an amount selected from:
  • the subject's weight is less than 100 kilograms (kg) and the age is less than 18 years old, then from about or about 0.05 ⁇ 10 6 CAR-T cells/kg of the subject's weight to about or about 5.0 ⁇ 10 7 CAR-T cells/kg body weight of the subject;
  • the subject's weight is or is greater than 100 kilograms (kg) or the age is greater than 18 years old, then from or about 0.05 ⁇ 10 6 CAR-T cells/kg of the subject's body weight to or approximately 5.0 ⁇ 10 8 CAR-T cells/kg of the subject's body weight.
  • the CAR-T cell therapy is performed by autologous transfer, wherein the CAR-T cell therapy is isolated from a subject receiving cell therapy or from a sample derived from such a subject and/or otherwise way to prepare cells.
  • cells are derived from a subject (eg, a patient) in need of treatment, and after isolation and processing, the cells are administered to the same subject.
  • the CAR-T cell therapy is performed by allogeneic transfer, in which a subject other than the subject who will receive or ultimately receives the cell therapy (e.g., the first subject or) isolate and/or otherwise prepare cells.
  • the cells are then administered to a different subject of the same species, such as a second subject.
  • the first and second subjects are genetically identical.
  • the first and second subjects are genetically similar.
  • the second subject expresses the same HLA class or supertype as the first subject.
  • the CAR-T cells may be administered by any suitable means, such as by bolus infusion, by injection such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection Injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, anterior chamber injection, subconjunctival (Subconjunctival) injection, subtenon injection (Sub-Tenon) injection, retrobulbar injection, peribulbar injection, or Posteriorjuxtascleral delivery. In some embodiments, they are administered parenterally, intrapulmonary and intranasally, and if necessary for local treatment, intralesional administration.
  • injection such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection Injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, anterior chamber injection, subconjunctival (Subconjunctival) injection, subtenon injection (Sub-Tenon) injection
  • Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • a given dose is administered by a single bolus administration of the cells.
  • a given dose is administered, for example, by multiple bolus injections of cells over a period of no more than 3 days, or by continuous infusion of cells.
  • administration of the cell dose or any other therapy is by outpatient delivery.
  • the appropriate dosage may depend on the type of disease to be treated, the type of cells, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, prior treatments, the subject clinical history and response to cells and the discretion of the attending physician.
  • the pharmaceutical composition and/or CAR-T cells are suitably administered to the subject at one time or in a series of treatments.
  • the CAR-T cells are administered as part of a combination therapy, such as with another or additional therapeutic intervention (such as an antibody or engineered cell or receptor or agent, such as a cytotoxic agent or Therapeutic agents) are administered simultaneously or sequentially in any order.
  • the CAR-T cells are co-administered with one or more additional therapeutic agents or in conjunction with another therapeutic intervention (simultaneously or sequentially in any order).
  • the additional therapeutic agent is any intervention agent or agent that is known in the art to be useful in the interventional treatment of tumors.
  • the one or more additional therapeutic agents are The therapeutic agent is administered to the cells prior to administration.
  • the cells are administered after the one or more additional therapeutic agents.
  • the one or more additional agents include cytokines such as IL-2, for example, to enhance persistence.
  • the method includes administering a chemotherapeutic agent.
  • the method includes administering a chemotherapeutic agent (eg, a conditioning chemotherapeutic agent) prior to the administering, eg, to reduce tumor burden.
  • a chemotherapeutic agent eg, a conditioning chemotherapeutic agent
  • preconditioning a subject with immune depletion (eg, lymphodepletion) therapy can improve the effectiveness of cell therapy.
  • the provided methods and applications involve the administration of all or part of a CAR-T cell or pharmaceutical composition, the cells expressing the chimeric antigen receptor CAR of the second aspect of the invention or the present invention.
  • the engineered immune cells of the chimeric antigen receptor according to the third aspect of the invention in some embodiments, a specific amount or number of cells, or a specific amount containing the specific amount or number of cells, is administered to the subject.
  • Pharmaceutical compositions is administered one or more cellular doses containing a specific amount or number of cells or a specific amount of a pharmaceutical composition containing the specific amount or number of cells.
  • a dose of cells is administered to a subject according to the provided methods and/or with the provided preparations or compositions.
  • the size, amount, or timing of the dose is determined based on the age of the subject. In some embodiments, the size, amount, or timing of the dose is determined based on the subject's body weight. In some embodiments, the size, amount, or timing of the dose is determined based on the particular type of tumor in the subject.
  • the eighteenth aspect of the present invention provides a method for detecting CD7 protein or antigen fragment thereof.
  • the method includes the following steps:
  • Nanobody is a Nanobody labeled with a label that can be used for detection.
  • markers that can be used for detection include fluorescent dyes, avidin, paramagnetic atoms, and radioactive isotopes.
  • the fluorescent pigment is fluorescein, rhodamine, Texas red, phycoerythrin, phycocyanin, allophycocyanin, polydinophycoxanthin-chlorophyll protein.
  • the avidin is biotin, avidin, streptavidin, vitellavidin, or avidin.
  • radioactive isotope is radioactive iodine, radioactive cesium, radioactive iridium, and radioactive cobalt.
  • a nineteenth aspect of the present invention provides a method for preparing the engineered host cell described in the seventh aspect of the present invention.
  • the method includes the following steps: introducing the recombinant expression vector described in the sixth aspect of the present invention into the host cell.
  • the introduced methods include lipofection, microinjection, electroporation, DNA vectors, RNA vectors, retroviral vectors, lentiviral vectors, poxvirus vectors, herpes simplex virus vectors, adenovirus vectors, adeno-associated Viral vectors.
  • a twentieth aspect of the invention provides a method for diagnosing whether a subject has a CD7-expressing tumor.
  • the method includes the following steps:
  • the sample in step (1) includes urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue associated cells, tissue or histological preparations.
  • the CD7-expressing tumor is a hematological tumor of T lymphocyte lineage.
  • the tumors include acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), lymphoblastic lymphoma (LBL), NKT cell leukemia, peripheral T cell lymphoma (NHL), NKT cell lymphoma, mesenchymal lymphoma, Anaplastic large cell lymphoma (ALCL).
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • LBL lymphoblastic lymphoma
  • NKT cell leukemia peripheral T cell lymphoma
  • NHL peripheral T cell lymphoma
  • NKT cell lymphoma mesenchymal lymphoma
  • ACL Anaplastic large cell lymphoma
  • a twenty-first aspect of the present invention provides the use of the Nanobody described in the first aspect of the present invention or the humanized anti-CD7 Nanobody described in the second aspect of the present invention in detecting CD7 protein or antigen fragments thereof.
  • the twenty-second aspect of the present invention provides the use of the Nanobody described in the first aspect of the present invention or the humanized anti-CD7 Nanobody described in the second aspect of the present invention in the preparation of reagents for detecting CD7 protein or antigen fragments thereof. or application in a kit.
  • a twenty-third aspect of the present invention provides the use of the nucleic acid molecule described in the fifth aspect of the present invention or the recombinant expression vector described in the sixth aspect of the present invention in preparing engineered host cells.
  • the engineered host cell is the engineered host cell described in the seventh aspect of the present invention.
  • the twenty-fourth aspect of the present invention provides the Nanobody according to the first aspect of the present invention, the humanized anti-CD7 Nanobody according to the second aspect of the present invention, and the chimeric antigen according to the third aspect of the present invention.
  • the engineered host cells, the conjugate according to the eighth aspect of the present invention, the pharmaceutical composition according to the ninth aspect of the present invention or the biological preparation according to the twelfth aspect of the present invention are used in the preparation of anti-tumor Applications in medicine.
  • anti-tumor drugs include anti-tumor immune cell therapy agents and anti-tumor gene therapy drugs.
  • the tumor is a tumor expressing CD7.
  • the tumor is a hematological tumor of T lymphocyte lineage.
  • the tumors include acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), lymphoblastic lymphoma (LBL), NKT cell leukemia, peripheral T cell lymphoma (NHL), NKT cell lymphoma, mesenchymal lymphoma, Anaplastic large cell lymphoma (ALCL).
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • LBL lymphoblastic lymphoma
  • NKT cell leukemia peripheral T cell lymphoma
  • NHL peripheral T cell lymphoma
  • NKT cell lymphoma mesenchymal lymphoma
  • ACL Anaplastic large cell lymphoma
  • the twenty-fifth aspect of the present invention provides the Nanobody according to the first aspect of the present invention, the humanized anti-CD7 Nanobody according to the second aspect of the present invention, and the chimeric nanobody according to the third aspect of the present invention.
  • the engineered host cell, the conjugate according to the eighth aspect of the present invention, the pharmaceutical composition according to the ninth aspect of the present invention or the biological preparation according to the twelfth aspect of the present invention are prepared in Application in kits for preparing immune cells for preventing and/or treating tumors.
  • the tumor is a tumor expressing CD7.
  • the tumor is a hematological tumor of T lymphocyte lineage.
  • the tumors include acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), lymphoblastic lymphoma (LBL), NKT cell leukemia, peripheral T cell lymphoma (NHL), NKT cell lymphoma, mesenchymal lymphoma, Anaplastic large cell lymphoma (ALCL).
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • LBL lymphoblastic lymphoma
  • NKT cell leukemia peripheral T cell lymphoma
  • NHL peripheral T cell lymphoma
  • NKT cell lymphoma mesenchymal lymphoma
  • ACL Anaplastic large cell lymphoma
  • the twenty-sixth aspect of the present invention provides the Nanobody according to the first aspect of the present invention, the humanized anti-CD7 Nanobody according to the second aspect of the present invention, and the chimeric nanobody according to the third aspect of the present invention.
  • the engineered host cell, the conjugate according to the eighth aspect of the present invention or the pharmaceutical composition according to the ninth aspect of the present invention are used in the preparation of biological preparations for preventing and/or treating tumors. application.
  • the tumor is a tumor expressing CD7.
  • the tumor is a hematological tumor of T lymphocyte lineage.
  • the tumors include acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), lymphoblastic lymphoma (LBL), NKT cell leukemia, peripheral T cell lymphoma (NHL), NKT cell lymphoma, mesenchymal lymphoma, Anaplastic large cell lymphoma (ALCL).
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • LBL lymphoblastic lymphoma
  • NKT cell leukemia peripheral T cell lymphoma
  • NHL peripheral T cell lymphoma
  • NKT cell lymphoma mesenchymal lymphoma
  • ACL Anaplastic large cell lymphoma
  • a twenty-seventh aspect of the present invention provides the use of the engineered host cells described in the seventh aspect of the present invention in preventing and/or treating tumors.
  • the tumor is a tumor expressing CD7.
  • the tumor is a hematological tumor of T lymphocyte lineage.
  • the tumors include acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), lymphoblastic lymphoma (LBL), NKT cell leukemia, peripheral T cell lymphoma (NHL), NKT cell lymphoma, mesenchymal lymphoma, Anaplastic large cell lymphoma (ALCL).
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • LBL lymphoblastic lymphoma
  • NKT cell leukemia peripheral T cell lymphoma
  • NHL peripheral T cell lymphoma
  • NKT cell lymphoma mesenchymal lymphoma
  • ACL Anaplastic large cell lymphoma
  • the twenty-eighth aspect of the present invention provides the use of the conjugate according to the eighth aspect of the present invention in preventing and/or treating tumors.
  • the tumor is a tumor expressing CD7.
  • the tumor is a hematological tumor of T lymphocyte lineage.
  • the tumors include acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), lymphoblastic lymphoma (LBL), NKT cell leukemia, peripheral T cell lymphoma (NHL), NKT cell lymphoma, mesenchymal lymphoma, Anaplastic large cell lymphoma (ALCL).
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • LBL lymphoblastic lymphoma
  • NKT cell leukemia peripheral T cell lymphoma
  • NHL peripheral T cell lymphoma
  • NKT cell lymphoma mesenchymal lymphoma
  • ACL Anaplastic large cell lymphoma
  • the twenty-ninth aspect of the present invention provides the use of the pharmaceutical composition described in the ninth aspect of the present invention in preventing and/or treating tumors.
  • the tumor is a tumor expressing CD7.
  • the tumor is a hematological tumor of T lymphocyte lineage.
  • the tumors include acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), lymphoblastic lymphoma (LBL), NKT cell leukemia, peripheral T cell lymphoma (NHL), NKT cell lymphoma, mesenchymal lymphoma, Anaplastic large cell lymphoma (ALCL).
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • LBL lymphoblastic lymphoma
  • NKT cell leukemia peripheral T cell lymphoma
  • NHL peripheral T cell lymphoma
  • NKT cell lymphoma mesenchymal lymphoma
  • ACL Anaplastic large cell lymphoma
  • a thirtieth aspect of the present invention provides the use of the kit described in the tenth aspect of the present invention in preparing immune cells for preventing and/or treating tumors.
  • the immune cells are the engineered host cells described in the seventh aspect of the present invention.
  • a thirty-first aspect of the present invention provides the use of the reagent for detecting CD7 protein or antigen fragments thereof described in the eleventh aspect of the present invention in detecting CD7 protein or antigen fragments thereof.
  • a thirty-second aspect of the present invention provides the use of the biological agent described in the twelfth aspect of the present invention in preventing and/or treating tumors.
  • the tumor is a tumor expressing CD7.
  • the tumor is a hematological tumor of T lymphocyte lineage.
  • the tumors include acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), lymphoblastic lymphoma (LBL), NKT cell leukemia, peripheral T cell lymphoma (NHL), NKT cell lymphoma, mesenchymal lymphoma, Anaplastic large cell lymphoma (ALCL).
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • LBL lymphoblastic lymphoma
  • NKT cell leukemia peripheral T cell lymphoma
  • NHL peripheral T cell lymphoma
  • NKT cell lymphoma mesenchymal lymphoma
  • ACL Anaplastic large cell lymphoma
  • the present invention provides anti-CD7 Nanobodies, which have good affinity with CD7.
  • the Nanobodies are used as the antigen-binding region of a chimeric antigen receptor and are modified with chimeric antigen receptors and used in CAR-T.
  • the CAR-T cells constructed based on single nanobodies or double nanobodies of the present invention have multiple advantages. They not only effectively avoid It overcomes the common shortcomings of traditional CAR-T designed based on scFv, such as difficulty in expression and poor stability, and significantly enhances the ability of immune cells to target and recognize tumor antigens, strengthens the killing activity against tumor cells, and has broad application prospects. .
  • FIG 1 shows the SDS-PAGE results of CD7 antigen purification
  • Figure 2 is an alpaca immunization flow chart
  • Figure 3 shows the results of the first round of PCR amplification during the construction of the Nanobody library
  • Figure 4 shows the results of the second round of PCR amplification during the construction of the Nanobody library
  • Figure 5 shows the PCR results of nanobody library diversity detection
  • Figure 6 is a graph showing the ratio results of the positive group and the negative group after phage panning
  • Figure 7 is a graph showing the statistical results of monoclonal screening OD values
  • Figure 8 shows the statistical results of single clone screening and identification
  • Figure 9 is a schematic structural diagram of a single VHH CAR-T
  • Figure 10 is a flow chart of lentivirus packaging
  • Figure 11 is a flow chart of CAR-T cell culture
  • Figure 12 shows the representative results of flow cytometry detection of single VHH CAR-T cells
  • Figure 13 is a statistical chart of the average MFI results of single VHH CAR-T cell CD7+;
  • Figure 14 shows the statistical results of the killing ratio of single VHH CAR-T cells
  • Figure 15 shows the statistical results of the average MFI value of a single VHH K562 cell line
  • FIG. 16 shows the results of the single VHH specificity test
  • Figure 17 shows the representative results of flow cytometry detection of single VHH CAR-T cells
  • Figure 18 shows the average MFI results of single VHH CAR-T cell CD7+
  • Figure 19 shows the comparison results between the sourced sequence and DP-47, the template h-NbBcII10PGLA and the original sequence, in which the highlighted sites are mutation sites;
  • Figure 20 is a schematic structural diagram of dual VHH CAR-T
  • Figure 21 shows the representative results of dual VHH CAR-T cell flow cytometry detection
  • Figure 22 is a statistical chart of the CD7+ average MFI results of single VHH CAR-T cells and double VHH CAR-T cells;
  • Figure 23 shows the dVHH-D CAR-T cell expansion curve results
  • Figure 24 shows the killing results of dVHH-D CAR-T cells
  • Figure 25 shows the dVHH-E CAR-T cell expansion curve results
  • Figure 26 shows the killing results of dVHH-E CAR-T cells.
  • RNA extraction kit Refer to the instruction manual of SuperScript TM II Reverse Transcriptase and use random primers to perform reverse transcription to obtain cDNA.
  • cDNA as a template, the extracellular region gene sequence of the antigen CD7 was obtained through PCR.
  • the CD7 extracellular region gene sequence was connected into a protein expression vector for expression, and Ni column purification was performed to obtain purified CD7-His protein.
  • Example 1 Use the self-purified CD7-His protein of the present invention in Example 1 to immunize alpaca.
  • the specific alpaca immunization flow chart is shown in Figure 2. Immunization was performed once a week, with a total of 6 consecutive immunizations; (3) 100 mL of peripheral blood was collected 7 days after the last immunization, and peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and RNA was extracted and reversed.
  • the first round of PCR amplification results are shown in Figure 3.
  • the results show that after the first round of PCR, a DNA fragment of approximately 700 bp was recovered.
  • the second round of PCR amplification results are shown in Figure 4.
  • the results show that the second round of PCR After the completion, DNA fragments with a size of about 400 bp were recovered.
  • 25 clones were randomly selected for colony PCR using primers MP57 and GIII, and the PCR products were subjected to Sanger sequencing. The results showed that the insertion efficiency The rate is close to 95% (see Figure 5).
  • SEQ ID NO:41, SEQ ID NO:49, SEQ ID NO:57, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:81, SEQ ID NO:89, SEQ ID NO:97, SEQ ID NO:105, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:129 are shown, and their corresponding variable region nucleic acid sequences are as follows: SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO :82, SEQ ID NO:90, SEQ ID NO:98, SEQ ID NO:106, SEQ ID NO:114, SEQ ID NO:122, SEQ ID NO:130.
  • NCAR-F1 5’-CTGCAGGAGTCTGGRGGAGG-3’
  • NCAR-R1 5’-TGAGGAGACGGTGACCTGGG-3’
  • the primers for the second round of PCR are:
  • NCAR-F2 5’-TTTCTGCTGATCCCCCAGGTGCAGCTGCAGGAGTCTGGRGGAGG-3’
  • NCAR-R2 5’-TAGGAGCCGGGGTGGGCGGCCGCGTCTGGGGTAGTTGAGGAGACGGTGACCTGGG-3’
  • leader is the coding sequence of the signal peptide
  • VHH is the coding sequence of the anti-CD7 nanobody
  • CD8H+TM is the hinge region and transmembrane region of CD8
  • the intracellular signal region of 4-1BB and CD3 ⁇ is the intracellular costimulatory domain
  • T2A Peptide linkage expresses the extracellular region of tEGFR to detect CAR expression after viral transduction.
  • lentivirus Before preparing CAR-T cells, lentivirus is first packaged: (1) The target plasmid and three helper plasmids (pMD2.G, pRSV-REV, pMDLg) are co-transfected into 293FT cells under the action of PEI-Pro; (2) ) Pack for 6 hours and change the medium; (3) Harvest the lentivirus after 48 hours of packaging; (4) Concentrate the harvested lentivirus stock solution by ultracentrifugation, resuspend the lentiviral particles in DMEM high-glucose medium, and aliquot for use ; The lentivirus packaging process is shown in Figure 10;
  • CAR-T cells After lentivirus packaging is completed, prepare CAR-T cells: (1) collect peripheral blood mononuclear cells (PBMC) from patients or healthy donors; (2) sort ⁇ T cells using CD3 magnetic beads; (3) sorting The completed ⁇ T cells are cultured in TexMACS GMP medium (MACS); (4) lentivirus transduction is performed 2 days later; (5) CAR-T cells are harvested after continuing to be cultured for 12-14 days to obtain CD7-targeted cells.
  • PBMC peripheral blood mononuclear cells
  • CD3 magnetic beads sorting The completed ⁇ T cells are cultured in TexMACS GMP medium (MACS); (4) lentivirus transduction is performed 2 days later; (5) CAR-T cells are harvested after continuing to be cultured for 12-14 days to obtain CD7-targeted cells.
  • VHH NS CAR-T cells (named VHH01-VHH20, excluding VHH02, VHH05 and VHH11), the CAR-T cell culture process is shown in Figure 11; (6) Flow cytometry is performed during the culture process to determine the concentration of CAR+ cells Ratio and average MFI value of CD7+ cells.
  • the results of in vitro functional verification are shown in Figure 14.
  • the results show that the killing ratio of blank T cells is 13.65%, and the killing ratios of VHH01 to VHH20 (VHH02 and, VHH05 and VHH11) CAR-T cells are 4.79%, 75.7%, and 57% respectively. , 62.19%, 61.63%, 68.52%, 71.47%, 91.36%, 78.18%, 1.18%, 60.44%, 56.77%, 72.87%, 8.58%, 74.52%, 61.71%, 9.38%.
  • the human myeloid leukemia cell line K562 was transduced with 17 VHH CAR structures corresponding to single VHHs identified through screening in Example 4. On the 4th day after transduction, flow cytometry was performed using CD7-His protein to count positive cells. the average MFI value. According to the difference in affinity between each VHH and CD7, the average MFI value of positive cells will be different. The higher the MFI value, the higher the affinity of the corresponding nanobody to the antigen CD7. A total of three parallel experiments were conducted.
  • amino acid sequences of CDR1, CDR2 and CDR3 of hVHH06 are as SEQ ID NO: 139 and SEQ ID respectively.
  • NO:141, SEQ ID NO:143, the nucleotide sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO:140, SEQ ID NO:142, and SEQ ID NO:144, respectively.
  • the results of the flow cytometry test are shown in Figure 17.
  • the results show that the transduction rates of VHH06 and hVHH06 are 12.4% and 21% respectively, and the CD7 positive rates are 0.249% and 0.157% respectively.
  • the results of the killing experiment are shown in Figure 18.
  • VHHs VHH03, VHH06, VHH10, VHH12
  • the structural schematic diagram is shown in Figure 20.
  • the first round of PCR primers are:
  • the primers for the second round of PCR are:
  • dNCAR-F2 5’-CCAGGTCACCGTCTCCTCAGGAGGAGGAGGATCCGGAGGAGGAGGATCTGGCGGCGGCGGCAGTGGCGGCGGCGGCTCCGGCGGCGGCGGCTCTCAGGTGCAGCTGCAGGAG-3’
  • dNCAR-R2 5’-TAGGAGCCGGGGTGGGCGGCCGCGGTGCTGGGGTAGTTGAGGAGACGGTGACCTGG-3’
  • the second-round PCR product was ligated into the vector VHH-XX (XX represents the sequence number of the CAR structure targeting CD7 single VHH) through homologous recombination, and the vector was digested with Not I single enzyme.
  • XX represents the sequence number of the CAR structure targeting CD7 single VHH
  • the vector was digested with Not I single enzyme.
  • the double VHH structures constructed with VHH-06 as the carrier and VHH03 and VHH12 were named dVHH-B and dVHH-C respectively.
  • VHH-10 as a carrier
  • the double VHH structures constructed together with VHH12 and VHH10 were named dVHH-D and dVHH-E respectively.
  • the double VHH structure constructed with VHH12 as a vector was named dVHH-F.
  • the preparation process of dual VHH CAR-T cells is consistent with the preparation process of single VHH CAR-T cells in Example 5.
  • the lentivirus packaging process is shown in Figure 10, and the culture process is shown in Figure 11.
  • CAR-T cells, VHH10 CAR-T cells, and VHH12 CAR-T cells prepared from 5 double VHH structures and transduced T cells were cultured for 6 days and then analyzed by flow cytometry.
  • dVHH-B to dVHH-F a total of 5 double VHH structures were constructed, named dVHH-B to dVHH-F (VHH-06+VHH-03, VHH-06+VHH-12, VHH-10+VHH-12, VHH-10+ VHH-10, VHH-12+VHH-12), the structure is shown in Figure 20, EF1 ⁇ is the promoter of elongation factor 1 ⁇ , leader is the coding sequence of the signal peptide, VHH is the coding sequence of anti-CD7 nanobody, CD8H+TM It is the CD8 hinge region and transmembrane region, the 4-1BB and CD3 ⁇ intracellular signal regions are intracellular costimulatory domains, and the tEGFR extracellular region is expressed through T2A peptide connection, so that the expression of CAR can be detected after viral transduction;
  • the results corresponding to the proportion of CAR+ cells and CD7MFI values are shown in Figure 21 and Figure 22 respectively.
  • the results show that the CAR positive rates of dVHH-B to dVHH-F are: 22.59%, 53.6%, 68.68%, 55.34%, respectively, and the CAR positive rates of VHH10 are: The positive rate was 72.2%, and the positive rate of VHH12 was 86.7%; the average MFI value of CD7-positive cells in blank T cells was 10109, and the average MFI value of CD7-positive cells in CAR-T cells from dVHH-B to dVHH-F were: 708, 797 respectively. , 648, 577, 1057, the average MFI value of CD7-positive cells of VHH10 CAR-T cells was 2302, and the average MFI value of CD7-positive cells of VHH12 CAR-T cells was 1238.
  • the CAR-T cell preparation method described in Example 8 was used to culture the dVHH-D, VHH10, and VHH12 structures in vitro, and the cell expansion folds during the culture process were counted.
  • CAR-T cells and blank T cells were compared with the CD7-expressing positive cell line CCRF-CEM ( Leukemia T lymphoma cells)
  • E:T 0.5:1, 1:1, 2:1
  • the dVHH-D amplification curve is shown in Figure 23.
  • the results show that on the 14th day of amplification, the average amplification fold of dVHH-D was 46.35, the average amplification fold of VHH-12 was 26.3, and the average amplification fold of VHH-10 is 22.75, it can be seen that the amplification fold of dVHH-D is significantly better than that of single VHH CAR-T;
  • the results of the in vitro functional verification of dVHH-D are shown in Figure 24.
  • the results show that when the killing ratios are 0.5:1, 1:1, and 2:1, the average killing values of dVHH-D against CCRF-CEM are 86.925%, 92.115%, and 94.465%.
  • the average killing values of VHH10 against CCRF-CEM are 45.55%, 69.95% and 85.85% respectively.
  • the average killing values of VHH12 against CCRF-CEM are 58.65%, 80.9% and 82.95% respectively. It can be seen that dVHH-D has the ability to kill CCRF-CEM. Higher lethality.
  • the CAR-T cell preparation method described in Example 8 was used to culture the dVHH-E, VHH10, and VHH12 structures in vitro, and the cell expansion folds during the culture process were counted.
  • Example 9 Use the method described in Example 9 to perform in vitro functional experimental verification of dVHH-E dual VHH CAR-T.
  • the dVHH-E amplification curve is shown in Figure 25.
  • the results show that on the 13th day of amplification, the average amplification fold of dVHH-E was 45.875, the average amplification fold of VHH-12 was 19.775, and the average amplification fold of VHH-10 is 21.36, it can be seen that the amplification fold of dVHH-E is significantly better than that of single VHH CAR-T;
  • the results of the in vitro functional verification of dVHH-E are shown in Figure 26.
  • the results show that when the killing ratios are 0.5:1, 1:1, and 2:1, the average killing values of dVHH-E against CCRF-CEM are 58.15%, 84.385%, and 84.385%, respectively. 91.775%, the average killing values of VHH10 to CCRF-CEM are 45.55%, 69.95%, and 85.85% respectively, and the average killing values of VHH12 to CCRF-CEM are 58.65%, 80.9%, and 82.95% respectively. It can be seen that dVHH-E shows a strong resistance to CCRF-CEM. CEM has relatively high lethality.
  • the SPR method surface plasmon resonance method
  • the CD7-His protein prepared in Example 1 is immobilized on the CM5 chip through the amino coupling method.
  • the antibody (dVHH-D, dVHH- E, VHH10, VHH12) were used as analytes in experiments to detect the affinity between each antibody and the CD7 antigen.
  • the results show that the affinity constant between dVHH-D and CD7-His protein is 3.35E-09M, the affinity constant between dVHH-E and CD7-His protein is 4.51E-09M, and the affinity constant between VHH10 and CD7-His protein is 3.35E-09M.
  • the affinity constant is 9.99E-08M, and the affinity constant between VHH12 and CD7-His protein is 1.34E-09M (see Table 2).
  • the above results further show that dVHH-D, dVHH-E, VHH10, and VHH12 can all bind to CD7 The antigen binds specifically with strong affinity.

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Abstract

提供了一种抗CD7纳米抗体及其衍生物，该衍生物包括人源化的抗CD7纳米抗体、基于单纳米抗体的嵌合抗原受体、基于双纳米抗体的嵌合抗原受体、重组表达载体、经工程改造的宿主细胞、缀合物、药物组合物、试剂盒、检测细胞表面CD7的试剂。该纳米抗体与CD7具有较好的亲和力，制备的CAR-T细胞靶向识别肿瘤抗原且对肿瘤细胞的杀伤活性高。

Description

抗CD7纳米抗体、衍生物及其在肿瘤治疗中的应用
相关申请的交叉引用
本申请要求享有以下申请文件的优先权：2022年3月30日提交的申请号为2022103314787名称为“抗CD7纳米抗体、衍生物及其在肿瘤治疗中的应用”的发明专利申请，其内容以全文引用的方式并入。
技术领域
本发明属于生物医药技术领域，具体地，本发明涉及抗CD7纳米抗体、衍生物及其在肿瘤治疗中的应用。
背景技术
CD7抗原是一种单链糖蛋白，是T细胞发育过程中的标志性抗原分子。除健康人胸腺细胞、T细胞和自然杀伤细胞外，淋系、髓系前体细胞等造血干祖细胞也表达CD7。大量研究显示在多数的人T淋巴细胞白血病和淋巴瘤上表达CD7分子(Karube K,Ohshima K,Tsuchiya T,et al.Non-B,non-T neoplasms with lymphoblast morphology:further clarification and classification[J].The American journal of surgical pathology,2003,27(10):1366-1374.；Shiyong Li,Jonathan Juco,Karen P.Mann,et al.Flow Cytometry in the Differential Diagnosis of Lymphocyte-Rich Thymoma From Precursor T-Cell Acute Lymphoblastic Leukemia/Lymphoblastic Lymphoma[J].,American Journal of Clinical Pathology,2004,121:268-274.)和约10％的急性髓系白血病(Acute myeloidleukemia，AML)上表达CD7抗原(Foon K A,Todd R F.Immunologic classification of leukemia and lymphoma[J].Blood.1986,68:1-31)；此外，当CD7分子与其对应的抗体结合后会快速地发生内吞作用，这一特性使得CD7可作为一个适合靶向递送各类功能分子到CD7阳性细胞内的抗原受体；相关研究还表明人体内存在一群CD7阴性的T淋巴细胞群，这一群细胞可以维持人体正常的免疫功能，避免了使用CD7纳米抗体相关的免疫细胞清除全部CD7阳性细胞而造成的免疫功能丢失，可见靶向CD7是一个极具潜力的抗肿瘤方向。
嵌合抗原受体修饰T细胞(Chimeric antigen receptor modification T cells，CAR-T)和嵌合抗原受体修饰NK细胞(Chimeric antigen receptor modification NK cells，CAR-NK)免疫疗法是目前研究进展最为迅速的两种肿瘤细胞免疫疗法，而CAR-T/CAR-NK细胞的有效激活均严重依赖识别肿瘤相关抗原的抗体的特异性以及抗原结合的亲和力高低等性质。所以在目前CAR-T/CAR-NK细胞胞内信号转导区的设计已经趋于成熟的现状下，抗原结合区的设计成为新型CAR-T技术开发的重点和关键。骆驼科(骆驼、羊驼)或鲨鱼体内存在一种天然缺乏轻链的重链抗体(Heavy chain antibody，HCAb)，该抗体只包含一个重链可变区和两个常规的CH2与CH3区。其重链可变区具有与重链抗体相当的稳定性和抗原结合活性，其大小仅为2.4×4nm，是能够结合抗原的最小片段，称为单域抗体(Variable domain of heavy chain of heavy-chain antibody，VHH)或纳米抗体。与传统抗体相比，VHH单域抗体分子量小且表达量高，化学稳定性好，亲和力高并且与人源抗体同源性高，免疫原性低。分子量小易于进行基因工程改造，构建双重或多重特异性的单域抗体组合，达到一个分子多靶点或多种功能的效果。VHH组织渗透性好，在进行肿瘤治疗时，具有接触到不能被常规抗体接触的较为隐蔽的靶点的可能性。正由于这些优点，利用单域抗体作为CAR的抗原结合区进行CAR修饰及CAR-T/CAR-NK细胞疗法能够为本领域提供全新的肿瘤治疗策略。
发明内容
鉴于此，本发明的目的在于为本领域提供抗CD7纳米抗体、衍生物及其在肿瘤治疗中的应用。
本发明的上述目的通过以下技术方案得以实现：
本发明的第一方面提供了抗CD7纳米抗体。
进一步，所述纳米抗体为VHH01、VHH03、VHH04、VHH06、VHH07、VHH08、VHH09、VHH10、VHH12、VHH13、VHH14、VHH15、VHH16、VHH17、VHH18、VHH19或VHH20中的任意一种；
优选地，所述VHH01的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7所示或分别为与SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
优选地，所述VHH03的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15所示或分别为与SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
优选地，所述VHH04的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23所示或分别为与SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
优选地，所述VHH06的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31所示或分别为与SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
优选地，所述VHH07的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39所示或分别为与SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
优选地，所述VHH08的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47所示或分别为与SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
优选地，所述VHH09的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:55所示或分别为与SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:55具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
优选地，所述VHH10的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63所示或分别为与SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
优选地，所述VHH12的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71所示或分别为与SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
优选地，所述VHH13的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79所示或分别为与SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
优选地，所述VHH14的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87所示或分别为与SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
优选地，所述VHH15的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95所示或分别为与SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
优选地，所述VHH16的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103所示或分别为与SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
优选地，所述VHH17的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111所示或分别为与SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
优选地，所述VHH18的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:115、SEQ ID NO:117、SEQ ID NO:119所示或分别为与SEQ ID NO:115、SEQ ID NO:117、SEQ ID NO:119具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
优选地，所述VHH19的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:123、SEQ ID NO:125、SEQ ID NO:127所示或分别为与SEQ ID NO:123、SEQ ID NO:125、SEQ ID NO:127具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
优选地，所述VHH20的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135所示或分别为与SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
更优选地，所述VHH01的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8所示或分别为与SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
更优选地，所述VHH03的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16所示或分别为与SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
更优选地，所述VHH04的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24所示或分别为与SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
更优选地，所述VHH06的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32所示或分别为与SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
更优选地，所述VHH07的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40所示或分别为与SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
更优选地，所述VHH08的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48所示或分别为与SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
更优选地，所述VHH09的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56所示或分别为与SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
更优选地，所述VHH10的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64所示或分别为与SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
更优选地，所述VHH12的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72所示或分别为与SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
更优选地，所述VHH13的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80所示或分别为与SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
更优选地，所述VHH14的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:88所示或分别为与SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:88具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
更优选地，所述VHH15的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96所示或分别为与SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
更优选地，所述VHH16的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104所示或分别为与SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
更优选地，所述VHH17的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:112所示或分别为与SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:112具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
更优选地，所述VHH18的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:116、SEQ ID NO:118、SEQ ID NO:120所示或分别为与SEQ ID NO:116、SEQ ID NO:118、SEQ ID NO:120具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
更优选地，所述VHH19的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:124、SEQ ID NO:126、SEQ ID NO:128所示或分别为与SEQ ID NO:124、SEQ ID NO:126、SEQ ID NO:128具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
更优选地，所述VHH20的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:132、SEQ ID NO:134、SEQ ID NO:136所示或分别为与SEQ ID NO:132、SEQ ID NO:134、SEQ ID NO:136具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述VHH01、VHH03、VHH04、VHH06、VHH07、VHH08、VHH09、VHH10、VHH12、VHH13、VHH14、VHH15、VHH16、VHH17、VHH18、VHH19、VHH20的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:9、SEQ ID NO:17、SEQ ID NO:25、SEQ ID NO:33、SEQ ID NO:41、SEQ ID NO:49、SEQ ID NO:57、SEQ ID NO:65、SEQ ID NO:73、SEQ ID NO:81、SEQ ID NO:89、SEQ ID NO:97、SEQ ID NO:105、SEQ ID NO:113、SEQ ID NO:121、SEQ ID NO:129所示或分别为与SEQ ID NO:1、SEQ ID NO:9、SEQ ID NO:17、SEQ ID NO:25、SEQ ID NO:33、SEQ ID NO:41、SEQ ID NO:49、SEQ ID NO:57、SEQ ID NO:65、SEQ ID NO:73、SEQ ID NO:81、SEQ ID NO:89、SEQ ID NO:97、SEQ ID NO:105、SEQ ID NO:113、SEQ ID NO:121、SEQ ID NO:129具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述VHH01、VHH03、VHH04、VHH06、VHH07、VHH08、VHH09、VHH10、VHH12、VHH13、VHH14、VHH15、VHH16、VHH17、VHH18、VHH19、VHH20的核苷酸序列分别如SEQ ID NO:2、SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:26、SEQ ID NO:34、SEQ ID NO:42、SEQ ID NO:50、SEQ ID  NO:58、SEQ ID NO:66、SEQ ID NO:74、SEQ ID NO:82、SEQ ID NO:90、SEQ ID NO:98、SEQ ID NO:106、SEQ ID NO:114、SEQ ID NO:122、SEQ ID NO:130所示或分别为与SEQ ID NO:2、SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:26、SEQ ID NO:34、SEQ ID NO:42、SEQ ID NO:50、SEQ ID NO:58、SEQ ID NO:66、SEQ ID NO:74、SEQ ID NO:82、SEQ ID NO:90、SEQ ID NO:98、SEQ ID NO:106、SEQ ID NO:114、SEQ ID NO:122、SEQ ID NO:130具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列。
进一步，上述VHH01、VHH03、VHH04、VHH06、VHH07、VHH08、VHH09、VHH10、VHH12、VHH13、VHH14、VHH15、VHH16、VHH17、VHH18、VHH19或VHH20中的一种或多种的CDR1、CDR2、CDR3的氨基酸序列或核苷酸序列任意组合得到的纳米抗体同样在本发明的保护范围内。
进一步，CD7是T细胞表面一个非常稳定的标志物，无论是幼稚的T细胞还是成熟的T细胞均会表达CD7，所以不管是幼稚T细胞肿瘤(T-ALL/LBL/NKT细胞白血病)患者，还是成熟T细胞肿瘤(外周T细胞淋巴瘤、NKT细胞淋巴瘤、间变大细胞淋巴瘤)患者，基本都高表达CD7，因此，针对该靶点的CAR-T治疗T系血液肿瘤是目前临床上发展最为迅速的技术之一。
本发明的第二方面提供了人源化的抗CD7纳米抗体。
进一步，所述人源化的抗CD7纳米抗体为以通用人源化框架h-NbBcII10FGLA为参考，并通过与DP-47进行比对，对纳米抗体关键位置的残基进行人源化改造后得到的；
优选地，所述人源化的抗CD7纳米抗体为hVHH01、hVHH03、hVHH04、hVHH06、hVHH07、hVHH08、hVHH09、hVHH10、hVHH12、hVHH13、hVHH14、hVHH15、hVHH16、hVHH17、hVHH18、hVHH19或hVHH20中的任意一种；
更优选地，所述hVHH06的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:139、SEQ ID NO:141、SEQ ID NO:143所示或分别为与SEQ ID NO:139、SEQ ID NO:141、SEQ ID NO:143具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述hVHH06的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:140、SEQ ID NO:142、SEQ ID NO:144所示或分别为与SEQ ID NO:140、SEQ ID NO:142、SEQ ID NO:144具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述hVHH06的氨基酸序列如SEQ ID NO:137所示或与SEQ ID NO:137具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述hVHH06的核苷酸序列如SEQ ID NO:138所示或与SEQ ID NO:138具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列。
本发明的第三方面提供了基于单纳米抗体的嵌合抗原受体。
进一步，所述嵌合抗原受体包含本发明第一方面所述的纳米抗体或本发明第二方面所述的人源化的抗CD7纳米抗体中的任意一种；
优选地，所述嵌合抗原受体还包含跨膜结构域；
优选地，所述嵌合抗原受体还包含胞内信号传导结构域；
优选地，所述嵌合抗原受体还包含铰链区；
优选地，所述嵌合抗原受体还包含信号肽；
优选地，所述嵌合抗原受体还包含共刺激信号结构域；
优选地，所述嵌合抗原受体还包含启动子EF1α；
优选地，所述嵌合抗原受体还包含自断裂肽T2A；
优选地，所述嵌合抗原受体还包含检测标签/辅助功能元件tEGFR；
更优选地，所述跨膜结构域包括下列分子的跨膜结构域：CD8α、CD28、IgG1、IgG4、4-1BB、PD-1、 CD34、OX40、CD3ε、IL-2受体、IL-7受体、IL-11受体；
更优选地，所述胞内信号传导结构域包括下列分子的胞内信号传导结构域：CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、TCRζ、CD4、CD5、CD8、CD21、CD22、CD79a、CD79b、CD278、FcεRI、DAP10、DAP12、CD66d；
更优选地，所述铰链区包括下列分子的铰链区：CD8α、CD28、IgG1、IgG4、4-1BB、PD-1、CD34、OX40、CD3ε、IL-2受体、IL-7受体、IL-11受体；
更优选地，所述信号肽包括下列分子的信号肽：T细胞受体的α链及β链、CD3ζ、CD3ε、CD4、CD5、CD8、CD9、CD28、CD16、CD22、CD33、CD37、CD45、CD64、CD80、CD86、CD134、CD137、CD154、GITR、GM-CSF；
更优选地，所述共刺激信号结构域包括下列分子的共刺激信号结构域：4-1BB(CD137)、CD27、CD19、CD4、CD28、ICOS(CD278)、CD8α、CD8β、BAFFR、HVEM、LIGHT、KIRDS2、SLAMF7、NKp30、NKp46、CD40、CDS、ICAM-1、B7-H3、OX40、DR3、GITR、CD30、TIM1、CD2、CD7、CD226；
更优选地，所述启动子并不局限于EF1α启动子，还包括：CMV启动子、EFS启动子、CAG启动子、CBh启动子、SFFV启动子、MSCV启动子、SV40启动子、mPGK启动子、hPGK启动子、UBC启动子；
更优选地，所述自断裂肽并不局限于T2A，还包括：P2A、E2A、F2A；
更优选地，所述检测标签/辅助功能元件并不局限于tEGFR，还包括：tCD34、tCD19、tCD20、tCD22、免疫检查点抑制剂(CTLA-4、PD-1/PD-L1、LAG-3、TIM-3、TIGIT、CD226、CD155、CD47、B7-H3、B7-H4)纳米抗体、细胞因子及其受体(IL2、IL2受体、IL7、IL7受体、IL15、IL15受体)；
最优选地，所述跨膜结构域为CD8α跨膜结构域；
最优选地，所述胞内信号传导结构域为CD3ζ胞内信号传导结构域；
最优选地，所述铰链区为CD8α铰链区；
最优选地，所述共刺激信号结构域为4-1BB共刺激信号结构域；
最优选地，所述信号肽的氨基酸序列如SEQ ID NO:145所示或与SEQ ID NO:145具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述信号肽的核苷酸序列如SEQ ID NO:146所示或与SEQ ID NO:146具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述CD8α铰链区的氨基酸序列如SEQ ID NO:147所示或与SEQ ID NO:147具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述CD8α铰链区的核苷酸序列如SEQ ID NO:148所示或与SEQ ID NO:148具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述CD8α跨膜结构域的氨基酸序列如SEQ ID NO:149所示或与SEQ ID NO:149具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述CD8α跨膜结构域的核苷酸序列如SEQ ID NO:150所示或与SEQ ID NO:150具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述4-1BB共刺激信号结构域的氨基酸序列如SEQ ID NO:151所示或与SEQ ID NO:151具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述4-1BB共刺激信号结构域的核苷酸序列如SEQ ID NO:152所示或与SEQ ID NO:152具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述CD3ζ胞内信号传导结构域的氨基酸序列如SEQ ID NO:153所示或与SEQ ID NO:153具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述CD3ζ胞内信号传导结构域的核苷酸序列如SEQ ID NO:154所示或与SEQ ID NO:154具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述T2A的氨基酸序列如SEQ ID NO:155所示或与SEQ ID NO:155具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述T2A的核苷酸序列如SEQ ID NO:156所示或与SEQ ID NO:156具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述EF1α的核苷酸序列如SEQ ID NO:157所示或与SEQ ID NO:157具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述tEGFR信号肽的氨基酸序列如SEQ ID NO:158所示或与SEQ ID NO:158具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述tEGFR信号肽的核苷酸序列如SEQ ID NO:159所示或与SEQ ID NO:159具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述tEGFR的氨基酸序列如SEQ ID NO:160所示或与SEQ ID NO:160具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述tEGFR的核苷酸序列如SEQ ID NO:161所示或与SEQ ID NO:161具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述嵌合抗原受体为EF1α、信号肽、本发明第一方面所述的纳米抗体或本发明第二方面所述的人源化的抗CD7纳米抗体中的任意一种、CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次串联得到。
本发明的第四方面提供了基于双纳米抗体的嵌合抗原受体。
进一步，所述嵌合抗原受体包含本发明第一方面所述的纳米抗体或本发明第二方面所述的人源化的抗CD7纳米抗体中的任意两种；
优选地，所述嵌合抗原受体还包含跨膜结构域；
优选地，所述嵌合抗原受体还包含胞内信号传导结构域；
优选地，所述嵌合抗原受体还包含铰链区；
优选地，所述嵌合抗原受体还包含信号肽；
优选地，所述嵌合抗原受体还包含共刺激信号结构域；
优选地，所述嵌合抗原受体还包含启动子EF1α；
优选地，所述嵌合抗原受体还包含自断裂肽T2A；
优选地，所述嵌合抗原受体还包含检测标签/辅助功能元件tEGFR；
更优选地，所述跨膜结构域包括下列分子的跨膜结构域：CD8α、CD28、IgG1、IgG4、4-1BB、PD-1、CD34、OX40、CD3ε、IL-2受体、IL-7受体、IL-11受体；
更优选地，所述胞内信号传导结构域包括下列分子的胞内信号传导结构域：CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、TCRζ、CD4、CD5、CD8、CD21、CD22、CD79a、CD79b、CD278、FcεRI、DAP10、DAP12、CD66d；
更优选地，所述铰链区包括下列分子的铰链区：CD8α、CD28、IgG1、IgG4、4-1BB、PD-1、CD34、OX40、CD3ε、IL-2受体、IL-7受体、IL-11受体；
更优选地，所述信号肽包括下列分子的信号肽：T细胞受体的α链及β链、CD3ζ、CD3ε、CD4、CD5、CD8、CD9、CD28、CD16、CD22、CD33、CD37、CD45、CD64、CD80、CD86、CD134、CD137、CD154、GITR、GM-CSF；
更优选地，所述共刺激信号结构域包括下列分子的共刺激信号结构域：4-1BB(CD137)、CD27、CD19、CD4、CD28、ICOS(CD278)、CD8α、CD8β、BAFFR、HVEM、LIGHT、KIRDS2、SLAMF7、NKp30、NKp46、CD40、CDS、ICAM-1、B7-H3、OX40、DR3、GITR、CD30、TIM1、CD2、CD7、CD226；
更优选地，所述启动子并不局限于EF1α启动子，还包括：CMV启动子、EFS启动子、CAG启动子、CBh启动子、SFFV启动子、MSCV启动子、SV40启动子、mPGK启动子、hPGK启动子、UBC启动子；
更优选地，所述自断裂肽并不局限于T2A，还包括：P2A、E2A、F2A；
更优选地，所述检测标签/辅助功能元件并不局限于tEGFR，还包括：tCD34、tCD19、tCD20、tCD22、免疫检查点抑制剂(CTLA-4、PD-1/PD-L1、LAG-3、TIM-3、TIGIT、CD226、CD155、CD47、B7-H3、B7-H4)纳米抗体、细胞因子及其受体(IL2、IL2受体、IL7、IL7受体、IL15、IL15受体)；
最优选地，所述跨膜结构域为CD8α跨膜结构域；
最优选地，所述胞内信号传导结构域为CD3ζ胞内信号传导结构域；
最优选地，所述铰链区为CD8α铰链区；
最优选地，所述共刺激信号结构域为4-1BB共刺激信号结构域；
最优选地，所述本发明第一方面所述的纳米抗体或本发明第二方面所述的人源化的抗CD7纳米抗体中的任意两种纳米抗体通过连接肽Linker进行连接；
最优选地，所述嵌合抗原受体为EF1α、信号肽、本发明第一方面所述的纳米抗体或本发明第二方面所述的人源化的抗CD7纳米抗体中的任意两种、CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次串联得到；
最优选地，所述嵌合抗原受体为EF1α、信号肽、本发明第一方面所述的纳米抗体中的任意一种、Linker、本发明第一方面所述的纳米抗体中的任意一种、CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次串联得到；
最优选地，所述本发明第一方面所述的纳米抗体中的任意一种为VHH03、VHH06、VHH10、VHH12；
最优选地，所述嵌合抗原受体为EF1α、信号肽、本发明第一方面中所述的VHH06、Linker、本发明第一方面中所述的VHH03、CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次串联得到；
最优选地，所述嵌合抗原受体为EF1α、信号肽、本发明第一方面中所述的VHH06、Linker、本发明第一方面中所述的VHH12、CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次串联得到；
最优选地，所述嵌合抗原受体为EF1α、信号肽、本发明第一方面中所述的VHH10、Linker、本发明第一方面中所述的VHH12、CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次串联得到；
最优选地，所述嵌合抗原受体为EF1α、信号肽、本发明第一方面中所述的VHH10、Linker、本发明第一方面中所述的VHH10、CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次串联得到；
最优选地，所述嵌合抗原受体为EF1α、信号肽、本发明第一方面中所述的VHH12、Linker、本发明第一方面中所述的VHH12、CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次串联得到；
最优选地，所述信号肽的氨基酸序列如SEQ ID NO:145所示或与SEQ ID NO:145具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述信号肽的核苷酸序列如SEQ ID NO:146所示或与SEQ ID NO:146具有至少75％、76％、 77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述CD8α铰链区的氨基酸序列如SEQ ID NO:147所示或与SEQ ID NO:147具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述CD8α铰链区的核苷酸序列如SEQ ID NO:148所示或与SEQ ID NO:148具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述CD8α跨膜结构域的氨基酸序列如SEQ ID NO:149所示或与SEQ ID NO:149具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述CD8α跨膜结构域的核苷酸序列如SEQ ID NO:150所示或与SEQ ID NO:150具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述4-1BB共刺激信号结构域的氨基酸序列如SEQ ID NO:151所示或与SEQ ID NO:151具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述4-1BB共刺激信号结构域的核苷酸序列如SEQ ID NO:152所示或与SEQ ID NO:152具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述CD3ζ胞内信号传导结构域的氨基酸序列如SEQ ID NO:153所示或与SEQ ID NO:153具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述CD3ζ胞内信号传导结构域的核苷酸序列如SEQ ID NO:154所示或与SEQ ID NO:154具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述T2A的氨基酸序列如SEQ ID NO:155所示或与SEQ ID NO:155具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述T2A的核苷酸序列如SEQ ID NO:156所示或与SEQ ID NO:156具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述EF1α的核苷酸序列如SEQ ID NO:157所示或与SEQ ID NO:157具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述tEGFR信号肽的氨基酸序列如SEQ ID NO:158所示或与SEQ ID NO:158具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述tEGFR信号肽的核苷酸序列如SEQ ID NO:159所示或与SEQ ID NO:159具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
最优选地，所述tEGFR的氨基酸序列如SEQ ID NO:160所示或与SEQ ID NO:160具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
最优选地，所述tEGFR的核苷酸序列如SEQ ID NO:161所示或与SEQ ID NO:161具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列。
还明确地包括在本发明的范围内的是本文中所公开的嵌合抗原受体的功能性部分。当参考嵌合抗原受体使用时，术语“功能性部分”是指本文中所公开的一种或更多种嵌合抗原受体的以下任何部分或片段，所述部分或片段保留嵌合抗原受体(所述部分或片段为其一部分)(亲本嵌合抗原受体)的生物活性。功能性部分涵盖例如嵌合抗原受体的以下那些部分，其与亲本嵌合抗原受体相比在类似程度上、在相同程度上或在更高程度上保留识别靶细胞或者检测、治疗或预防疾病的能力。参考亲本嵌合抗原受体，功能性部分可包含例如亲本嵌合抗原受体的约10％、25％、30％、50％、68％、80％、90％、95％或更多。
功能性部分可在该部分的氨基或羧基端或在这两端包含另外的氨基酸，所述另外的氨基酸在亲本嵌合抗原受体的氨基酸序列中不存在。期望地，另外的氨基酸不干扰功能性部分的生物功能，例如识别靶细胞、检测癌症、治疗或预防癌症等。更期望地，与亲本嵌合抗原受体的生物活性相比，另外的氨基酸增强生物活性。
包括在本公开内容的范围内的是本文中所公开嵌合抗原受体的功能性变体。本文中使用的术语“功能性变体”是指与亲本嵌合抗原受体具有明显或显著序列同一性或相似性的嵌合抗原受体、多肽或蛋白质，所述功能性变体保留嵌合抗原受体(所述功能性变体为其变体)的生物活性。功能性变体涵盖例如本文中所述嵌合抗原受体(亲本嵌合抗原受体)的以下那些变体，其与亲本嵌合抗原受体相比在类似程度上、在相同程度上或在更高程度上保留识别靶细胞的能力。参考亲本嵌合抗原受体，功能性变体可例如与亲本嵌合抗原受体在氨基酸序列方面具有至少约30％、50％、75％、80％、90％、98％或更高同一性。
功能性变体可例如包含具有至少一个保守性氨基酸替换的亲本嵌合抗原受体的氨基酸序列。作为替选或补充，功能性变体可包含具有至少一个非保守性氨基酸替换的亲本嵌合抗原受体的氨基酸序列。在这种情况下，非保守性氨基酸替换优选不干扰或抑制功能性变体的生物活性。非保守性氨基酸替换可增强功能性变体的生物活性，使得与亲本嵌合抗原受体相比，功能性变体的生物活性提高。
嵌合抗原受体的氨基酸替换优选是保守性氨基酸替换。保守性氨基酸替换是本领域已知的，并且包括其中将一个具有特定物理和/或化学特性的氨基酸交换为另一个具有相同或相似化学或物理特性的氨基酸替换。例如，保守性氨基酸替换可以是酸性/带负电荷的极性氨基酸替换另一个酸性/带负电荷的极性氨基酸(例如，Asp或Glu)、具有非极性侧链的氨基酸替换另一个具有非极性侧链的氨基酸(例如，Ala、Gly、Val、He、Leu、Met、Phe、Pro、Trp、Cys、Val等)、碱性/带正电荷的极性氨基酸替换另一个碱性/带正电荷的极性氨基酸(例如，Lys、His、Arg等)、具有极性侧链的不带电荷氨基酸替换另一个具有极性侧链的不带电荷的氨基酸(例如，Asn、Gin、Ser、Thr、Tyr等)、具有β支化侧链的氨基酸替换另一个具有β支化侧链的氨基酸(例如，He、Thr和Val)、具有芳香族侧链的氨基酸替换另一个具有芳香族侧链的氨基酸(例如，His、Phe、Trp和Tyr)等。
嵌合抗原受体可基本上由本文中所述的一个或更多个特定氨基酸序列组成，使得其他组分(例如，其他氨基酸)不会实质上改变功能性变体的生物活性。嵌合抗原受体(包括功能性部分和功能性变体)可具有任意长度，即可包含任意数目的氨基酸，只要嵌合抗原受体(或其功能性部分或功能性变体)保留其生物活性，例如与抗原特异性结合、在哺乳动物中检测患病细胞或在哺乳动物中治疗或预防疾病等的能力即可。例如，嵌合抗原受体可为约50至约5000个氨基酸长，例如长度为50、70、75、100、125、150、175、200、300、400、500、600、700、800、900、1000或更多个氨基酸。
嵌合抗原受体(包括本发明的功能性部分和功能性变体)可包含替换一个或更多个天然存在氨基酸的合成氨基酸。这样的合成氨基酸是本领域已知的，并且包括例如氨基环己烷羧酸、正亮氨酸、10-氨基正癸酸、高丝氨酸、S-乙酰氨基甲基-半胱氨酸、反式-3-羟脯氨酸和反式-4-羟脯氨酸、4-氨基苯丙氨酸、4-硝基苯丙氨酸、4-氯苯丙氨酸、4-羧基苯丙氨酸、β-苯基丝氨酸、β-羟基苯丙氨酸、苯基甘氨酸、α-萘基丙氨酸、环己基丙氨酸、环己基甘氨酸、二氢吲哚-2-羧酸、1，2，3，4-四氢异喹啉-3-羧酸、氨基丙二酸、氨基丙二酸一酰胺、N’-苄基-N’-甲基-赖氨酸、N’，N’-二苄基-赖氨酸、6-羟赖氨酸、鸟氨酸、-氨基环戊烷羧酸、α-氨基环己烷羧酸、α-氨基环庚烷羧酸、α-(2-氨基-2-降冰片烷)-羧酸、γ-二氨基丁酸、β-二氨基丙酸、高苯丙氨酸和α-叔丁基甘氨酸。
本发明所述的嵌合抗原受体(包括功能性部分和功能性变体)可被糖基化、酰胺化、羧化、磷酸化、酯化、N-酰化、环化(通过例如二硫桥)或转化成酸加成盐和/或任选地二聚或多聚的，或缀合的。
本发明的第五方面提供了核酸分子。
进一步，所述核酸分子包含编码本发明第一方面所述的纳米抗体、本发明第二方面所述的人源化的抗CD7纳米抗体、本发明第三方面所述的嵌合抗原受体或本发明第四方面所述的嵌合抗原受体的核苷酸序列；
优选地，所述核苷酸序列如SEQ ID NO:2、SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:26、SEQ ID NO:34、SEQ ID NO:42、SEQ ID NO:50、SEQ ID NO:58、SEQ ID NO:66、SEQ ID NO:74、SEQ ID NO:82、SEQ ID NO:90、SEQ ID NO:98、SEQ ID NO:106、SEQ ID NO:114、SEQ ID NO:122、SEQ ID NO:130所示或与SEQ ID NO:2、SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:26、SEQ ID NO:34、SEQ ID NO:42、SEQ ID NO:50、SEQ ID NO:58、SEQ ID NO:66、SEQ ID NO:74、SEQ ID NO:82、SEQ ID NO:90、SEQ ID NO:98、SEQ ID NO:106、SEQ ID NO:114、SEQ ID NO:122、SEQ ID NO:130具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列。
本文中使用的“核酸分子”可包含天然的、非天然的或被改变的核苷酸；并且其可包含天然的、非天然的或被改变的核苷酸间连接，例如氨基磷酸酯连接或硫代磷酸酯连接，代替在未经修饰寡核苷酸的核苷酸之间存在的磷酸二酯。在一些实施方案中，核酸不包含任何插入、缺失、倒位和/或替换。然而，在一些情况下对核酸而言，包含一个或更多个插入、缺失、倒位和/或替换可以是合适的，因此，这些插入、缺失、倒位和/或替换形成的核酸同样在本发明的保护范围内。
在本发明的具体实施方案中，所述核酸分子包括可使用天然存在的核苷酸或被设计成提高分子的生物稳定性或提高在杂交后形成的二联体的物理稳定性的经多种修饰的核苷酸(例如硫代磷酸酯衍生物和吖啶取代的核苷酸)化学合成得到的核酸分子。可用于产生核酸的经修饰核苷酸的一些实例包括但不限于5-氟尿嘧啶、5-溴尿嘧啶、5-氯尿嘧啶、5-碘尿嘧啶、次黄嘌呤、黄嘌呤、4-乙酰胞嘧啶、5-(羧基羟甲基)尿嘧啶、5-羧甲基氨基甲基-2-硫代尿苷、5-羧甲基氨基甲基尿嘧啶、二氢尿嘧啶、β-D-半乳糖基辫苷(queosine)、肌苷、N6-异戊烯基腺嘌呤、1-甲基鸟嘌呤、1-甲基肌苷、2，2-二甲基鸟嘌呤、2-甲基腺嘌呤、2-甲基鸟嘌呤、3-甲基胞嘧啶、5-甲基胞嘧啶、N6-取代的腺嘌呤、7-甲基鸟嘌呤、5-甲基氨基甲基尿嘧啶、5-甲氧基氨基甲基-2-硫尿嘧啶、β-D-甘露糖基辫苷、5’-甲氧基羧甲基尿嘧啶、5-甲氧基尿嘧啶、2-甲硫基-N6-异戊烯基腺嘌呤、尿嘧啶-5-羟乙酸(v)、怀丁氧苷(wybutoxosine)、假尿嘧啶、辫苷、2-硫胞嘧啶、5-甲基-2-硫尿嘧啶、2-硫尿嘧啶、4-硫尿嘧啶、5-甲基尿嘧啶、尿嘧啶-5-氧基乙酸甲酯、3-(3-氨基-3-N-2-羧丙基)尿嘧啶和2，6-二氨基嘌呤。
本发明的第六方面提供了重组表达载体。
进一步，所述重组表达载体包含本发明第五方面所述的核酸分子；
优选地，所述表达载体包括DNA载体、RNA载体、质粒、转座子载体、CRISPR/Cas9载体、或病毒载体；
更优选地，所述病毒载体包含慢病毒载体、腺病毒载体、逆转录病毒载体。
在一个实施方案中，重组表达载体可以是任何合适的重组表达载体，并且可用于转化或转染任何合适的宿主细胞。合适的载体包括针对增殖和扩增或针对表达或这二者而设计的那些，例如质粒和病毒。载体可选自pUC系列(Fermentas Life Sciences，GlenBumie，MD)、pBluescript系列(Stratagene，LaJolla，CA)、pET系列(Novagen，Madison，WI)、pGEX系列(Pharmacia Biotech，Uppsala，Sweden)和pEX系列(Clontech，Palo Alto，CA)。
还可使用噬菌体载体例如λZapII(Stratagene)、EMBL4和λNMI149。植物表达载体的一些实例包括pBIO1、pBI101.2、pBHO1.3、pBI121和pBIN19(Clontech)。动物表达载体的一些实例包括pEUK-Cl、pMAM和pMAMneo(Clontech)。重组表达载体可以是病毒载体，例如逆转录病毒载体或慢病毒载体。慢病毒载体是来自于慢病毒基因组的至少一部分的载体，尤其包括自我失活慢病毒载体，如Milone et al.，Mol.Ther.17(8)：1453-1464(2009)中提供的。可用于临床的慢病毒载体的另一些实例包括例如但不限于来自Oxford BioMedica plc的LENTIVECTOR.RTM.基因递送技术、来自Lentigen的LENTIMAX.TM.载体系统等。慢病毒载体的非临床类型也是可用的并且是本领域技术人员已知的。
所述重组表达载体可包含调节序列，例如转录和翻译起始和终止密码子，所述调节序列对需向其中引入载体的宿主细胞的类型(例如，细菌、真菌、植物或动物)具有特异性，这视情况而定并且考虑载体是基于DNA还是基于RNA。重组表达载体可包含限制性位点以有利于克隆。
所述重组表达载体可包含一个或更多个允许选择转化或转染的宿主细胞的标记基因。标记基因包括杀生物剂抗性(例如针对抗生素、重金属等的抗性)；营养缺陷型宿主中提供原养型的互补，等等。用于本发明表达载体的合适的标记基因包括例如新霉素/G418抗性基因、潮霉素抗性基因、组氨醇抗性基因、四环素抗性基因、氨苄青霉素抗性基因、卡那霉素抗性基因、嘌呤霉素抗性基因等。
所述重组表达载体可包含与编码所述嵌合抗原受体(包括其功能性部分和功能性变体)的核苷酸序列或与编码所述嵌合抗原受体的核苷酸序列互补或杂交的核苷酸序列可操作连接的天然或非天然启动子。启动子的选择(例如，强的、弱的、诱导型的、组织特异性的和发育特异性的)在技术人员的普通技术之内。类似地，核苷酸序列与启动子的组合也在技术人员的技术之内。启动子可以是非病毒启动子或病毒启动子，例如Ⅲ型启动子(U6启动子、H1启动子)、哺乳动物组成型启动子(广泛性启动子)：CMV(巨细胞病毒)启动子；EF1A(延伸因子-1α)启动子；EFS启动子；CAG(巨细胞病毒增强子和鸡β-肌动蛋白启动子组成)启动子；CBh启动子；SFFV启动子；MSCV启动子；SV40(猿猴病毒来源)启动子；mPGK启动子；hPGK(磷酸甘油酸激酶)启动子；UBC(泛素C)启动子、RSV启动子，或在鼠干细胞病毒的长末端重复中发现的启动子。
所述重组表达载体可被设计用于瞬时表达、用于稳定表达或用于二者。另外，重组表达载体可被制成用于组成型表达或用于诱导型表达。
此外，所述重组表达载体还包含自杀基因。本文中使用的术语“自杀基因”是指引起表达该自杀基因的细胞死亡的基因。自杀基因可以是以下基因：其赋予其中表达该基因的细胞以针对试剂(例如药物)的敏感性，并且当使该细胞与该试剂接触或暴露于该试剂时引起该细胞死亡。自杀基因是本领域已知的(参见例如，Suicide Gene Therapy：Methods and Reviews，Springer，Caroline J.(Cancer Research UK Centre for CancerTherapeutics at the Institute of Cancer Research，Sutton，Surrey，UK)，HumanaPress，2004)并且包括例如单纯疱疹病毒(HSV)胸苷激酶(TK)基因、胞嘧啶脱氨酶、嘌呤核苷磷酸化酶和硝基还原酶。
本发明的第七方面提供了经工程改造的宿主细胞。
进一步，所述经工程改造的宿主细胞表达本发明第一方面所述的纳米抗体、本发明第二方面所述的人源化的抗CD7纳米抗体、本发明第三方面所述的嵌合抗原受体或本发明第四方面所述的嵌合抗原受体；
优选地，所述经工程改造的宿主细胞包含本发明第六方面所述的重组表达载体；
优选地，所述经工程改造的宿主细胞包含经工程改造的免疫细胞；
更优选地，所述经工程改造的免疫细胞包含T细胞、NK细胞、iNKT细胞、CTL细胞、单核细胞、巨噬细胞、树突状细胞、NKT细胞或其任意组合。
本文中使用的术语“宿主细胞”是指任何类型的细胞。宿主细胞可以是真核细胞，例如植物、动物、真菌或藻类，或者可以是原核细胞，例如细菌或原生动物。宿主细胞可以是培养的细胞或原代细胞，即直接从生物体(例如人)分离。宿主细胞可以是黏附细胞或悬浮细胞，即悬浮生长的细胞。合适的宿主细胞是本领域已知的并且包括例如DH5a大肠杆菌(E.coli)细胞、中国仓鼠卵巢细胞、猴VERO细胞、COS细胞、HEK293细胞等。出于扩增或复制重组表达载体的目的，宿主细胞可以是原核细胞，例如DH5α细胞。出于产生重组嵌合抗原受体的目的，宿主细胞可以是哺乳动物细胞。宿主细胞可以是人细胞。当宿主细胞可以是任何细胞类型，可以来源于任何类型的组织，并且可以在任何发育阶段时，宿主细胞可以是外周血淋巴细胞(peripheral blood lymphocyte，PBL)或外周血单个核细胞(peripheral blood mononuclear cell，PBMC)。在本发明的具体实施方案中，所述宿主细胞为T细胞。
进一步，所述经工程改造的宿主细胞包括经工程改造的宿主细胞群体。
进一步，所述经工程改造的宿主细胞群体包含不表达本发明第一方面所述的纳米抗体、本发明第二方面所述的人源化的抗CD7纳米抗体、本发明第三方面所述的嵌合抗原受体或本发明第四方面所述的嵌合抗原受体的宿主细胞。
进一步，所述宿主细胞为免疫细胞。
进一步，所述免疫细胞包含T细胞、NK细胞、iNKT细胞、CTL细胞、单核细胞、巨噬细胞、树突状细胞、NKT细胞或其任意组合。
本发明的第八方面提供了一种缀合物。
进一步，所述缀合物包含本发明第一方面所述的纳米抗体或本发明第二方面所述的人源化的抗CD7纳米抗体、以及连接于所述纳米抗体的修饰部分，所述修饰部分包括可检测的标记、治疗剂；
优选地，所述可检测的标记包括酶、放射性核素、荧光染料、发光物质、生物素，
优选地，所述治疗剂包括具有抗肿瘤活性的药物或细胞毒剂。
本发明的第九方面提供了一种药物组合物。
进一步，所述药物组合物包含本发明第一方面所述的纳米抗体、本发明第二方面所述的人源化的抗CD7纳米抗体、本发明第三方面所述的嵌合抗原受体、本发明第四方面所述的嵌合抗原受体、本发明第五方面所述的核酸分子、本发明第六方面所述的重组表达载体、本发明第七方面所述的经工程改造的宿主细胞或本发明第八方面所述的缀合物。
进一步，所述药物组合物中还包含另外的药学活性剂；
优选地，所述另外的药学活性剂包括另外的抗体、融合蛋白或药物(如抗肿瘤药物，如用于放疗的药物或化疗药物)。
进一步，所述药物组合物中还包含药学上可接受的载体和/或赋形剂。
进一步，所述药学上可接受的载体和/或赋形剂在Remington's Pharmaceutical Sciences(19th ed.,1995)中有详细的记载，这些物质根据需要用于帮助药物的稳定性或有助于提高有效成分的活性，在这种药物组合物中可以使用的制剂可以是其原始化合物本身的形式，或任选地使用其药物学可接受的盐的形式，如此配制的药物组合物根据需要可选择本领域技术人员已知的任何适当的方式将药物进行给药。
药学上可接受的载体和/或辅料可另外含有液体，诸如水、生理盐水、甘油和乙醇。另外，诸如湿润剂或乳化剂或pH缓冲物质的辅助物质可存在于所述组合物中。这些载剂使得药物组合物能够配制成片剂、丸剂、糖衣丸、胶囊、液体、凝胶、糖浆、浆液和悬浮液，以便患者摄入。
合适的施用形式包括适用于肠胃外施用的形式，例如通过注射或输注，例如通过快速注射或连续输液、静脉内、可吸入或皮下形式。在产品用于注射或输注的情况下，其可采用在油性或水性媒介物中的悬浮液、溶液或乳液的形式并且其可含有配制试剂，诸如悬浮剂、防腐剂、稳定剂和/或分散剂。可替代地，根据本发明提供的纳米抗体可呈干燥形式，用于在使用之前用合适的无菌液体重构。还可制备适于在注射前溶解或悬浮于液体媒介物中的固体形式。
本发明的第十方面提供了一种试剂盒。
进一步，所述试剂盒包含本发明第一方面所述的纳米抗体、本发明第二方面所述的人源化的抗CD7纳米抗体、本发明第三方面所述的嵌合抗原受体、本发明第四方面所述的嵌合抗原受体、本发明第五方面所述的核酸分子、本发明第六方面所述的重组表达载体、本发明第七方面所述的经工程改造的宿主细胞或本发明第八方面所述的缀合物。
本发明的第十一方面提供了一种检测CD7蛋白或其抗原片段的试剂。
进一步，所述检测CD7蛋白或其抗原片段的试剂包含本发明第一方面所述的纳米抗体、本发明第二方面所述的人源化的抗CD7纳米抗体或本发明第八方面所述的缀合物。
本发明的第十二方面提供了一种生物制剂。
进一步，所述生物制剂含有本发明第七方面所述的经工程改造的宿主细胞和/或本发明第九方面所述的药物组合物。
进一步，本发明中所述的生物制剂，又称之为生物药物、生物(医学)产品或生物制品，是从生物来源制造、提取或半合成的任何药物产品。它们包括但不限于疫苗，血液或血液成分，以及重组治疗性蛋白质(例如抗体、细胞因子、趋化因子和融合蛋白)，生物制剂可以由糖、蛋白质或核酸或这些物质的复杂组合组成。
本文中提供了用于基因治疗、免疫治疗和/或细胞治疗的药物组合物或生物制剂或其组合物，其包含在载体(例如可药用载体)中的与本文中所公开的一种或更多种抗原特异性结合的一种或更多种所公开嵌合抗原受体、表达嵌合抗原受体的免疫细胞、纳米抗体、缀合物。所述药物组合物或生物制剂或其组合物可以以单位剂量形式制备用于向受试者施用。施用的量和时机由治疗临床医师决定以达到期望结局。组合物可被配制成用于系统(例如静脉内)或局部(例如瘤内)施用。在一个实例中，所公开的嵌合抗原受体、表达所述嵌合抗原受体的免疫细胞(特别地，表达所述嵌合抗原受体的T细胞)、纳米抗体、缀合物被配制用于肠胃外施用，例如静脉内施用。包含本文中所公开的嵌合抗原受体、表达嵌合抗原受体的免疫细胞(特别地，表达所述嵌合抗原受体的T细胞)、缀合物或其组合物用于例如肿瘤的治疗和检测，所述肿瘤包括但不限于表达CD7的肿瘤。在一些实施例中，上述药物组合物或生物制剂或其组合物可用于肿瘤的治疗和/或检测中。包含如本文中所公开的嵌合抗原受体、表达所述嵌合抗原受体的免疫细胞(特别地，表达所述嵌合抗原受体的T细胞)、纳米抗体、缀合物或其组合物还用于例如CD7抗原的检测中。
用于施用的药物组合物或生物制剂或其组合物可包含溶解在可药用载体(例如水性载体)中的嵌合抗 原受体、表达所述嵌合抗原受体的免疫细胞(特别地，表达所述嵌合抗原受体的T细胞)、缀合物、纳米抗体的溶液。可使用多种水性载体，例如缓冲盐水等。这些溶液是无菌的并且通常不含不期望的物质。这些药物组合物或生物制剂或其组合物可通过常规的公知灭菌技术来灭菌。这些药物组合物或生物制剂或其组合物可根据需要包含可药用的辅助物质以接近生理条件，例如pH调节剂和缓冲剂、毒性调节剂、辅助剂等，例如乙酸钠、氯化钠、氯化钾、氯化钙、乳酸钠等。这些制剂中嵌合抗原受体、表达所述嵌合抗原受体的免疫细胞(特别地，表达所述嵌合抗原受体的T细胞)、缀合物、纳米抗体的浓度可广泛地变化，并且根据所选的具体施用模式和对象的需求将主要基于流体体积、黏度、体重等来选择。制备这样的剂型用于基因治疗、免疫治疗和/或细胞治疗的实际方法对于本领域技术人员是已知的或将是明显的。
用于静脉内施用的典型组合物(药物组合物或生物制剂或其组合物)包含约0.01mg/kg至约30mg/kg的本发明第一方面所述的纳米抗体或本发明第二方面所述的人源化的抗CD7纳米抗体或本发明第七方面所述的经工程改造的宿主细胞或本发明第八方面所述的缀合物/受试者/天。用于制备可施用组合物的实际方法对本领域技术人员将是已知或明显的并且在例如Remington’s Pharmaceutical Science，19th ed.，Mack Publishing Company，Easton，PA(1995)的出版物中更详细地描述。
本文中所述的嵌合抗原受体、表达所述嵌合抗原受体的免疫细胞(特别地，表达所述嵌合抗原受体的T细胞)、纳米抗体或缀合物可以以冻干形式提供并且在施用之前用无菌水再水化，但是其也以已知浓度的无菌溶液提供。然后将嵌合抗原受体、表达所述嵌合抗原受体的免疫细胞(特别地，表达所述嵌合抗原受体的T细胞)、纳米抗体或缀合物溶液添加到包含0.9％氯化钠(USP)的输注袋中，并且在一些情况下以0.5mg/kg体重至15mg/kg体重的剂量施用。
本领域在纳米抗体和缀合物相关药物的施用方面有相当多的经验可供使用；例如自1997年利妥昔单抗(RITUXAN)获批以来，抗体药物一直在美国销售。嵌合抗原受体、表达嵌合抗原受体的免疫细胞、抗体、抗原结合片段及其缀合物等可通过缓慢输注而不是以静脉内推送(push)或推注(bolus)来施用。在一个实施例中，施用较高的负荷剂量，随后以较低水平施用维持剂量。例如，可在大约90分钟的时间内输注4mg/kg抗体或抗原结合片段的初始负荷剂量(或包含抗体或抗原结合片段的缀合物的对应剂量)，随后如果在前剂量耐受良好，则在30分钟时间内输注2mg/kg的每周维持剂量进行4至8周。
受控释放肠胃外制剂可被制备成植入物、油性注射剂，或制备成颗粒系统。对于蛋白质递送系统的概述，参见Banga，A.J.，Therapeutic Peptides and Proteins：Formulation，Processing，and Delivery Systems，Technomic Publishing Company，Inc.，Lancaster，PA，(1995)。颗粒系统包括微球体、微粒、微胶囊剂、纳米胶囊剂、纳米球体和纳米粒。微胶囊剂包含治疗性蛋白质(例如细胞毒素或药物)作为中心核。在微球体中，治疗剂分散在整个颗粒中。小于约1μm的颗粒、微球体和微胶囊剂通常分别是指纳米粒、纳米球体和纳米胶囊剂。毛细管的直径为约5μm使得只有纳米粒静脉内施用。微粒的直径通常为约100μm并且皮下或肌内施用。参见例如，Kreuter，J.，Colloidal Drug DeliverySystems，J.Kreuter，ed.，Marcel Dekker，Inc.，New York，NY，pp.219-342(1994)；和Tice&Tabibi，Treatise on Controlled Drug Delivery，A.Kydonieus，ed.，Marcel Dekker，Inc.New York，NY，pp.315-339，(1992)。
聚合物可用于本文中所公开的嵌合抗原受体、表达所述嵌合抗原受体的免疫细胞(特别地，表达所述嵌合抗原受体的T细胞)、纳米抗体、缀合物或其组合物的离子控制释放。用于受控药物递送的多种可降解和不可降解的聚合物基质是本领域已知的(Langer，Accounts Chem.Res.26：537-542，1993)。例如，嵌段共聚物泊洛沙姆407在低温下作为黏性但是可活动的液体存在，但在体温下形成半固体凝胶。目前现有的研究已经表明，其是用于重组白介素-2和脲酶的配制和持续递送的有效载剂(Johnston et al.，Pharm.Res.9：425-434，1992；和Pec et al.，J.Parent.Sci.Tech.44(2)：58-65，1990)。或者，羟基磷灰石已经作为微载体用于蛋白质的受控释放(Ijntema et al.，Int.J.Pharm.112：215-224，1994)。在另一方面中，脂质体被用于脂质包封的药物的受控释放以及药物靶向(Betageri et al.，Liposome Drug Delivery Systems，TechnomicPublishing Co.，Inc.，Lancaster，PA(1993))。用于治疗性蛋白质的受控递送的许多另外的系统是已知的(参见美国专利No.5,055,303；美国专利No.5,188,837；美国专利No.4,235,871；美国专利No.4,501,728；美国专利No.4,837,028；美国专利No.4,957,735；美国专利No.5,019,369；美国专利No.5,055,303；美国专利No.5,514,670；美国专利No.5,413,797；美国专利No.5,268,164；美国专利No.5,004,697；美国专利No.4,902,505；美国专利No.5,506,206；美国专利No.5,271,961；美国专利No.5,254,342和美国专利No.5,534,496)。
本发明的第十三方面提供了一种刺激对哺乳动物中靶细胞群或组织的免疫应答的方法。
进一步，所述方法包括如下步骤：给哺乳动物施用有效量的本发明第七方面所述的经工程改造的宿主 细胞、本发明第八方面所述的缀合物、本发明第九方面所述的药物组合物或本发明第十二方面所述的生物制剂。
本发明的第十四方面提供了一种调节受试者体内的免疫应答的方法。
进一步，所述方法包括如下步骤：向受试者施用有效量的本发明第七方面所述的经工程改造的宿主细胞、本发明第八方面所述的缀合物、本发明第九方面所述的药物组合物或本发明第十二方面所述的生物制剂。
本发明的第十五方面提供了一种生产本发明第一方面所述的纳米抗体或本发明第五方面所述的人源化的抗CD7纳米抗体的方法。
进一步，所述方法包括如下步骤：培养本发明第七方面所述的经工程改造的宿主细胞，从培养物中分离出本发明第一方面所述的纳米抗体或本发明第二方面所述的人源化的抗CD7纳米抗体。
本发明的第十六方面提供了一种特异性地抑制CD7活性的方法。
进一步，所述方法包括如下步骤：将本发明第五方面所述的核酸分子导入到生物体细胞中，通过表达本发明第一方面所述的纳米抗体或本发明第二方面所述的人源化的抗CD7纳米抗体抑制CD7的活性。
本发明的第十七方面提供了一种预防和/或治疗CD7相关联的疾病或病症的方法。
进一步，所述方法包括如下步骤：将有效量的本发明第一方面所述的纳米抗体、本发明第二方面所述的人源化的抗CD7纳米抗体、本发明第五方面所述的核酸分子、本发明第六方面所述的重组表达载体、本发明第七方面所述的经工程改造的宿主细胞、本发明第八方面所述的缀合物、本发明第九方面所述的药物组合物或本发明第十二方面所述的生物制剂给予具有与CD7相关联的疾病或病症的受试者。
进一步，所述CD7相关联的疾病或病症包括表达CD7的肿瘤。
进一步，所述肿瘤为T淋巴细胞系血液肿瘤。
进一步，所述肿瘤包括急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、NKT细胞白血病、外周T细胞淋巴瘤(NHL)、NKT细胞淋巴瘤、间变性大细胞淋巴瘤(ALCL)。
进一步，本发明所述的CD7相关联的疾病或病症是指与CD7表达相关的任何疾病或病症，包括但不限于上述列举的疾病类型，只要所述疾病或病症与CD7的表达相关，就都在本发明的保护范围内。
进一步，所述“治疗”和“预防”以及从其来源的词语并不一定意指100％或完全的治疗或预防。相反，存在本领域普通技术人员视为具有潜在益处或治疗效果的不同程度的治疗或预防。在这一方面，所述方法可在哺乳动物中提供任意量或任意水平的癌症治疗或预防。
由所述方法提供的治疗或预防可包括治疗或预防被治疗或预防的疾病(例如，癌症)的一种或更多种状况或症状。另外，出于本文中的目的，“预防”可涵盖延迟疾病或其症状或状况的发作。
本文中提及的哺乳动物可以是任何哺乳动物，包括但不限于啮齿目的哺乳动物，例如小鼠和仓鼠；和兔形目的哺乳动物，例如兔。哺乳动物可来自食肉目，包括猫科(猫)和犬科(犬)。哺乳动物可来自偶蹄目，包括牛科(牛)和猪科(猪)；或者是奇蹄目，包括马科(马)。哺乳动物可以是灵长目、Ceboid目或Simoid目(猴)；或者是类人猿目(人和猿)。优选地，在本发明的具体实施方案中，所述哺乳动物是人。
进一步，所述经工程改造的宿主细胞包含表达抗CD7的本发明第三方面所述的基于单纳米抗体的嵌合抗原受体(CAR)或本发明第四方面所述的基于双纳米抗体的嵌合抗原受体的免疫细胞；
优选地，所述免疫细胞包括T细胞、NK细胞、iNKT细胞、CTL细胞、单核细胞、巨噬细胞、树突状细胞、NKT细胞；
更优选地，所述经工程改造的宿主细胞为CAR-T细胞。
进一步，选自以下的量给患有CD7相关联的疾病或病症的受试者施用所述的经工程改造的宿主细胞：
(1)若所述受试者的体重小于100公斤(kg)并且年龄小于18岁，则从为或约0.05×10 6个CAR-T细胞/kg所述受试者的体重至为或约5.0×10 7个CAR-T细胞/kg所述受试者的体重；
(2)如果所述受试者的体重为或大于100公斤(kg)或年龄大于18岁，则从为或约0.05×10 6个CAR-T细胞/kg所述受试者的体重至为或约5.0×10 8个CAR-T细胞/kg所述受试者的体重。
在本发明的一些实施方案中，所述CAR-T细胞疗法是通过自体转移进行的，其中从接受细胞疗法的受试者或从源自这种受试者的样品中分离和/或以其他方式制备细胞。因此，在一些方面，细胞源自需要治疗的受试者(例如，患者)，并且在分离和处理后将细胞施用于同一受试者。
在本发明的另一些实施方案中，所述CAR-T细胞疗法是通过同种异体转移进行，其中从将要接受或最终接受细胞疗法的受试者以外的受试者(例如，第一受试者)分离和/或以其他方式制备细胞。在此类实施方案中，然后将细胞施用于相同物种的不同受试者，例如第二受试者。在一些实施方案中，所述第一和第二受试者在遗传上是相同的。在一些实施方案中，所述第一和第二受试者在遗传上是相似的。在一些实施方案中，所述第二受试者与所述第一受试者表达相同的HLA类别或超类型。
在本发明的具体实施方案中，可以将所述CAR-T细胞通过任何合适的方式施用，例如通过推注输注，通过注射例如静脉内或皮下注射、眼内注射、眼周注射、视网膜下注射、玻璃体内注射、经中隔注射、巩膜下注射、脉络膜内注射、前房注射、结膜下(Subconjectval)注射、眼球筋膜囊下(Sub-Tenon)注射、眼球后注射、眼球周注射或后近巩膜(Posteriorjuxtascleral)递送。在一些实施方案中，将它们通过肠胃外、肺内和鼻内以及(如果需要用于局部治疗的话)病灶内施用来施用。肠胃外输注包括肌肉内、静脉内、动脉内、腹膜内或皮下施用。在一些实施方案中，给定剂量是通过所述细胞的单次推注施用来施用。在一些实施方案中，给定剂量通过例如在不超过3天的时间段内细胞的多次推注施用，或通过细胞的连续输注施用来施用。在一些实施方案中，细胞剂量或任何其他疗法(例如，淋巴细胞清除疗法、干预疗法和/或组合疗法)的施用是通过门诊递送进行的。
进一步，对于疾病的预防或治疗，适当的剂量可取决于要治疗的疾病类型、细胞的类型、疾病的严重程度和病程、是针对预防目的还是针对治疗目的而施用细胞、先前治疗、受试者的临床病史和对细胞的反应以及主治医师的决断。在一些实施方案中，适合将药物组合物和/或CAR-T细胞一次或在一系列治疗中施用至受试者。
在一些实施方案中，所述CAR-T细胞是作为组合治疗的一部分来施用，如与另一种或另外的治疗性干预(如抗体或工程化细胞或受体或药剂、如细胞毒性剂或治疗剂)同时或以任何顺序依序施用。在一些实施方案中，将所述CAR-T细胞与一种或多种另外的治疗剂共同施用或与另一种治疗性干预联合施用(同时或以任何顺序依次施用)。在一些实施方案中，所述另外的治疗剂是现有技术中本领域技术人员公知的可用于肿瘤干预治疗的任何干预制剂或药剂，在一些实施方案中，在所述一种或多种另外的治疗剂之前施用所述细胞。在一些实施方案中，在所述一种或多种另外的治疗剂之后施用所述细胞。在一些实施方案中，所述一种或多种另外的药剂包括细胞因子如IL-2，以例如增强持久性。在一些实施方案中，所述方法包括施用化学治疗剂。在一些实施方案中，所述方法包括在所述施用之前施用化学治疗剂(例如，调理性化学治疗剂)，例如以减小肿瘤负荷。在一些实施方案中，用免疫清除(例如，淋巴细胞清除)疗法预调理受试者可以改善细胞疗法的效果。
在一些实施方案中，所提供的方法和应用中涉及的施用CAR-T细胞或药物组合物的全部或一部分，所述细胞如表达本发明第二方面所述的嵌合抗原受体CAR或本发明第三方面所述的嵌合抗原受体的工程化免疫细胞，在一些实施方案中，向受试者施用特定量或数量的细胞，或特定量的含有所述特定量或数量的细胞的药物组合物。在一些实施方案中，向受试者施用一个或多个细胞剂量，其含有特定量或数量的细胞或特定量的含有所述特定量或数量的细胞的药物组合物。在一些实施方案中，根据所提供方法和/或用所提供的制品或组合物将一定剂量的细胞施用至受试者。在一些实施方案中，剂量的大小、量或时间安排根据所述受试者的年龄来确定。在一些实施方案中，剂量的大小、量或时间安排根据所述受试者的体重来确定。在一些实施方案中，剂量的大小、量或时间安排根据受试者的肿瘤的特定类型来确定。
本发明的第十八方面提供了一种检测CD7蛋白或其抗原片段的方法。
进一步，所述方法包括如下步骤：
(1)获取含有CD7蛋白或其抗原片段的样品；
(2)将步骤(1)收集的样品与本发明第一方面所述的纳米抗体、本发明第二方面所述的人源化的抗CD7纳米抗体、本发明第八方面所述的缀合物、本发明第十方面所述的试剂盒或本发明第十一方面所述的检测CD7蛋白或其抗原片段的试剂接触；
(3)检测抗体-抗原复合物的存在。
进一步，所述纳米抗体是被可用于检测的标记物标记的纳米抗体。
进一步，所述可用于检测的标记物包括荧光色素、亲和素、顺磁原子、放射性同位素。
进一步，所述荧光色素为荧光素、罗丹明、Texas红、藻红蛋白、藻蓝蛋白、别藻蓝蛋白、多甲藻黄素-叶绿素蛋白。
进一步，所述亲和素为生物素、卵白亲和素、链亲和素、卵黄亲和素、类亲和素。
进一步，所述放射性同位素为放射性碘、放射性铯、放射性铱、放射性钴。
本发明的第十九方面提供了一种本发明第七方面所述的经工程改造的宿主细胞的制备方法。
进一步，所述方法包括如下步骤：将本发明第六方面所述的重组表达载体引入到宿主细胞中。
进一步，所述引入的方法包括脂质转染法、微注射、电穿孔、DNA载体、RNA载体、逆转录病毒载体、慢病毒载体、痘病毒载体、单纯疱疹病毒载体、腺病毒载体、腺相关病毒载体。
本发明的第二十方面提供了一种用于诊断受试者是否患有表达CD7肿瘤的方法。
进一步，所述方法包括如下步骤：
(1)提供来自怀疑患有表达CD7肿瘤的受试者的样品；
(2)将样品与本发明第一方面所述的纳米抗体、本发明第二方面所述的人源化的抗CD7纳米抗体、本发明第八方面所述的缀合物、本发明第十方面所述的试剂盒或本发明第十一方面所述的检测CD7蛋白或其抗原片段的试剂接触；
(3)检测包含所述纳米抗体与抗原的复合物的形成，得到在来自所述受试者的样品中CD7的量，并将所述CD7在来自所述受试者的样品中的量与其在已知标准品或参照样品中的量进行比较，并确定来自所述受试者的样品的CD7水平是否落入与肿瘤相关的CD7水平内。
进一步，步骤(1)中所述样品包括尿液、血液、血清、血浆、唾液、腹水、循环细胞、循环肿瘤细胞、非组织缔合的细胞、组织或组织学制备物。
进一步，所述表达CD7肿瘤为T淋巴细胞系血液肿瘤。
进一步，所述肿瘤包括急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、NKT细胞白血病、外周T细胞淋巴瘤(NHL)、NKT细胞淋巴瘤、间变性大细胞淋巴瘤(ALCL)。
本发明的第二十一方面提供了本发明第一方面所述的纳米抗体或本发明第二方面所述的人源化的抗CD7纳米抗体在检测CD7蛋白或其抗原片段中的应用。
本发明的第二十二方面提供了本发明第一方面所述的纳米抗体或本发明第二方面所述的人源化的抗CD7纳米抗体在制备用于检测CD7蛋白或其抗原片段的试剂或试剂盒中的应用。
本发明的第二十三方面提供了本发明第五方面所述的核酸分子或本发明第六方面所述的重组表达载体在制备经工程改造的宿主细胞中的应用。
进一步，所述经工程改造的宿主细胞为本发明第七方面所述的经工程改造的宿主细胞。
本发明的第二十四方面提供了本发明第一方面所述的纳米抗体、本发明第二方面所述的人源化的抗CD7纳米抗体、本发明的第三方面所述的嵌合抗原受体、本发明的第四方面所述的嵌合抗原受体、本发明第五方面所述的核酸分子、本发明的第六方面所述的重组表达载体、本发明的第七方面所述的经工程改造的宿主细胞、本发明的第八方面所述的缀合物、本发明的第九方面所述的药物组合物或本发明的第十二方面所述的生物制剂在制备抗肿瘤药物中的应用。
进一步，所述抗肿瘤药物包括抗肿瘤免疫细胞治疗剂、抗肿瘤的基因治疗药物。
进一步，所述肿瘤为表达CD7的肿瘤。
进一步，所述肿瘤为T淋巴细胞系血液肿瘤。
进一步，所述肿瘤包括急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、NKT细胞白血病、外周T细胞淋巴瘤(NHL)、NKT细胞淋巴瘤、间变性大细胞淋巴瘤(ALCL)。
本发明的第二十五方面提供了本发明第一方面所述的纳米抗体、本发明的第二方面所述的人源化的抗CD7纳米抗体、本发明的第三方面所述的嵌合抗原受体、本发明的第四方面所述的嵌合抗原受体、本发明的第五方面所述的核酸分子、本发明的第六方面所述的重组表达载体、本发明的第七方面所述的经工程改造的宿主细胞、本发明的第八方面所述的缀合物、本发明的第九方面所述的药物组合物或本发明的第十二方面所述的生物制剂在制备用于制备预防和/或治疗肿瘤的免疫细胞的试剂盒中的应用。
进一步，所述肿瘤为表达CD7的肿瘤。
进一步，所述肿瘤为T淋巴细胞系血液肿瘤。
进一步，所述肿瘤包括急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、 NKT细胞白血病、外周T细胞淋巴瘤(NHL)、NKT细胞淋巴瘤、间变性大细胞淋巴瘤(ALCL)。
本发明的第二十六方面提供了本发明第一方面所述的纳米抗体、本发明的第二方面所述的人源化的抗CD7纳米抗体、本发明的第三方面所述的嵌合抗原受体、本发明的第四方面所述的嵌合抗原受体、本发明的第五方面所述的核酸分子、本发明的第六方面所述的重组表达载体、本发明的第七方面所述的经工程改造的宿主细胞、本发明的第八方面所述的缀合物或本发明的第九方面所述的药物组合物在制备用于预防和/或治疗肿瘤的生物制剂中的应用。
进一步，所述肿瘤为表达CD7的肿瘤。
进一步，所述肿瘤为T淋巴细胞系血液肿瘤。
进一步，所述肿瘤包括急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、NKT细胞白血病、外周T细胞淋巴瘤(NHL)、NKT细胞淋巴瘤、间变性大细胞淋巴瘤(ALCL)。
本发明的第二十七方面提供了本发明第七方面所述的经工程改造的宿主细胞在预防和/或治疗肿瘤中的应用。
进一步，所述肿瘤为表达CD7的肿瘤。
进一步，所述肿瘤为T淋巴细胞系血液肿瘤。
进一步，所述肿瘤包括急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、NKT细胞白血病、外周T细胞淋巴瘤(NHL)、NKT细胞淋巴瘤、间变性大细胞淋巴瘤(ALCL)。
本发明的第二十八方面提供了本发明的第八方面所述的缀合物在预防和/或治疗肿瘤中的应用。
进一步，所述肿瘤为表达CD7的肿瘤。
进一步，所述肿瘤为T淋巴细胞系血液肿瘤。
进一步，所述肿瘤包括急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、NKT细胞白血病、外周T细胞淋巴瘤(NHL)、NKT细胞淋巴瘤、间变性大细胞淋巴瘤(ALCL)。
本发明的第二十九方面提供了本发明第九方面所述的药物组合物在预防和/或治疗肿瘤中的应用。
进一步，所述肿瘤为表达CD7的肿瘤。
进一步，所述肿瘤为T淋巴细胞系血液肿瘤。
进一步，所述肿瘤包括急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、NKT细胞白血病、外周T细胞淋巴瘤(NHL)、NKT细胞淋巴瘤、间变性大细胞淋巴瘤(ALCL)。
本发明的第三十方面提供了本发明第十方面所述的试剂盒在制备用于预防和/或治疗肿瘤的免疫细胞中的应用。
进一步，所述免疫细胞为本发明第七方面所述的经工程改造的宿主细胞。
本发明的第三十一方面提供了本发明第十一方面所述的检测CD7蛋白或其抗原片段的试剂在检测CD7蛋白或其抗原片段中的应用。
本发明的第三十二方面提供了本发明的第十二方面所述的生物制剂在预防和/或治疗肿瘤中的应用。
进一步，所述肿瘤为表达CD7的肿瘤。
进一步，所述肿瘤为T淋巴细胞系血液肿瘤。
进一步，所述肿瘤包括急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、NKT细胞白血病、外周T细胞淋巴瘤(NHL)、NKT细胞淋巴瘤、间变性大细胞淋巴瘤(ALCL)。
相对于现有技术，本发明具有的优点和有益效果如下：
本发明提供了抗CD7的纳米抗体，所述纳米抗体与CD7具有较好的亲和力，将所述纳米抗体作为嵌合抗原受体的抗原结合区进行了嵌合抗原受体修饰并用于CAR-T细胞疗法中，相对于传统的基于单克隆抗体(scFv)构建得到的CAR-T细胞而言，本发明基于单纳米抗体或双纳米抗体构建得到的CAR-T细胞具有多种优势，不仅有效避免了传统的以scFv为基础设计的CAR-T所常有的表达困难、稳定性差等缺陷，而且明显增强了免疫细胞靶向识别肿瘤抗原的能力，加强了对肿瘤细胞的杀伤活性，应用前景广阔。
附图说明
图1为CD7抗原纯化SDS-PAGE结果图；
图2为羊驼免疫流程图；
图3为纳米抗体文库构建过程中第一轮PCR扩增结果图；
图4为纳米抗体文库构建过程中第二轮PCR扩增结果图；
图5为纳米抗体文库多样性检测PCR结果图；
图6为噬菌体淘选后阳性组与阴性组的比值结果图；
图7为单克隆筛选OD值统计结果图；
图8为单克隆筛选与鉴定统计结果图；
图9为单VHH CAR-T结构示意图；
图10为慢病毒包装流程图；
图11为CAR-T细胞培养流程图；
图12为单VHH CAR-T细胞流式细胞术检测代表性结果图；
图13为单VHH CAR-T细胞CD7+平均MFI结果统计图；
图14为单VHH CAR-T细胞杀伤比例统计结果图；
图15为单VHH K562细胞系平均MFI值统计结果图；
图16为单VHH专属性检测结果图；
图17为单VHH CAR-T细胞流式细胞术检测代表性结果图；
图18为单VHH CAR-T细胞CD7+平均MFI结果图；
图19为源化序列与DP-47、模板h-NbBcII10PGLA及原始序列比对结果图，其中，突出显示位点为突变位点；
图20为双VHH CAR-T结构示意图；
图21为双VHH CAR-T细胞流式细胞术检测代表性结果图；
图22为单VHH CAR-T细胞、双VHH CAR-T细胞CD7+平均MFI结果统计图；
图23为dVHH-D CAR-T细胞扩增曲线结果图；
图24为dVHH-D CAR-T细胞杀伤结果图；
图25为dVHH-E CAR-T细胞扩增曲线结果图；
图26为dVHH-E CAR-T细胞杀伤结果图。
具体实施方式
下面结合具体实施例，进一步阐述本发明，仅用于解释本发明，而不能理解为对本发明的限制。本领域的普通技术人员可以理解为：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。下述实施例中所使用的实验方法如无特殊说明，均为常规方法；下述实施例中所使用的实验方法如无特殊说明，均为常规方法；下述实施例中所用的试剂、材料等，如无特殊说明，均可从商业途径得到。
实施例1抗原制备
1、实验方法
利用RNA提取试剂盒提取T细胞中的RNA。参考SuperScript TMII Reverse Transcriptase使用说明书，利用random primers引物进行反转录，获得cDNA。以cDNA为模板，通过PCR获得抗原CD7的胞外区基因序列。将CD7胞外区基因序列连接入蛋白表达载体中进行表达，并进行Ni柱纯化，获得纯化的CD7-His蛋白。
2、实验结果
结果见图1，结果显示，经过SDS-PAGE鉴定，本发明成功制备了大小为17.2kDa，纯度＞90％的CD7抗原，可进行后续的羊驼免疫。
实施例2纳米抗体文库的构建
1、实验方法
(1)利用实施例1中本发明自主纯化的CD7-His蛋白进行羊驼免疫，具体羊驼免疫流程图见图2。每周进行1次免疫，共连续进行6次免疫；(3)最后一次免疫7天后采集外周血100mL，通过Ficoll密度梯 度离心法分离外周血单核细胞，并进行RNA的提取，并利用反转录试剂盒进行cDNA的制备；(4)利用SOE-PCR获得VHH片段，并将其连接入pMES4噬菌体展示载体，(5)将连接产物电转化至电转感受态细胞TG1中，所得菌库即为构建的CD7的单域重链抗体噬菌体展示文库，库容量为3.37×10 8；(6)文库构建完成后，为检测文库的插入效率，随机选择25个克隆利用引物MP57以及GⅢ进行了菌落PCR，并将PCR产物进行Sanger测序。
2、实验结果
第一轮PCR扩增结果图见图3，结果显示，第一轮PCR结束后，回收大小约为700bp的DNA片段，第二轮PCR扩增结果图见图4，结果显示，第二轮PCR结束后，回收大小约为400bp的DNA片段，文库构建完成后，为检测文库的插入效率，随机选择25个克隆利用引物MP57以及GⅢ进行了菌落PCR，并将PCR产物进行Sanger测序，结果显示插入率接近95％(见图5)。
实施例3纳米抗体的富集筛选
1、噬菌体纳米抗体文库的扩增
(1)取TG1大肠杆菌纳米抗体文库转接到2-YT液体培养基中，37℃、200rpm培养至OD值为0.5，然后加入辅助噬菌体VCSM13对细胞进行侵染。轻轻混合后在37℃下孵育30分钟。离心菌液以去除微量葡萄糖，再将沉淀重悬于同时加有氨苄和卡那霉素抗性的2-YT培养基中，37℃，200rpm摇培过夜，以扩增展示纳米抗体的噬菌体；(2)将过夜培养物转移到50mL离心管中，离心取上清液，并添加20％(wt/vol)PEG6000/2.5M NaCl溶液沉淀噬菌体。离心弃上清，PBS重悬沉淀，离心取上清至新的离心管中添加20％(wt/vol)PEG6000/2.5M NaCl溶液重新沉淀噬菌体。离心弃上清，并将沉淀重新悬浮于1mL PBS中。离心后将上清液转移到新的离心管中，添加甘油至终浓度为20％保存于-80℃中；(3)噬菌体纳米抗体库滴度测定，将噬菌体按10倍梯度进行稀释，取不同稀释倍数的噬菌体对对数生长期的TG1细菌进行侵染，37℃过夜培养，通过第二天的菌斑数目推算噬菌体纳米抗体库的滴度。
2、噬菌体富集筛选
(1)通过ELISA的方法对纳米抗体进行淘选，将重组CD7-His蛋白包被在酶标板上，在4℃下孵育过夜；(2)用250μL PBST清洗酶标板三次，添加200μL封闭液，于室温孵育酶标板2h；(3)在每个孔中加入相对应的噬菌体，室温孵育2h；(4)250μL PBST洗板15次；(5)每孔添加100μL浓度为0.25mg/mL的胰蛋白酶，室温700rpm孵育0.5h；(6)用AEBSF洗脱噬菌体；(7)将洗脱下的噬菌体进行滴度测定和噬菌体侵染扩增；(8)洗脱下的噬菌体数量阳性：阴性≥100时，停止淘选。
3、实验结果
淘选结果见表1和图6，淘选两轮后，阳性组与阴性组的比值达到了438倍，已达到了筛选单克隆的标准。所以淘选两轮后，停止淘选，进行下一步单克隆的筛选和鉴定。
表1噬菌体淘选结果
筛选次数 CD7 空白 比例
第一次筛选 8×10 6 2×10 5 40
第二次筛选 3.51×10 8 8×10 5 438
实施例4阳性单克隆的筛选与鉴定
1、实验方法
(1)从经过2-3轮筛选后得到的TG1大肠杆菌文库中挑选单个克隆进行扩大培养，并利用辅助噬菌体VCSM13进行侵染，进行单克隆噬菌体的制备。(2)用适量纳米抗体噬菌体与K562-CD7阳性细胞，室温孵育2h；(3)PBST清洗板子后，加入HA-HRP抗体，室温孵育1h；(4)PBST清洗板子后，加入100μL TMB单组份显色液，室温孵育30min后加入100μL终止液；(5)利用酶标仪检测450nm处的吸光度；(6)当样品孔的OD450值与空白对照比例大于2时判定为阳性克隆；(7)对阳性克隆进行菌液PCR，进行Sanger测序；(8)经过Sanger测序的单克隆，利用软件DNAMAN进行序列比对。并筛选出序列特异性的克隆。
2、实验结果
本实施例筛选了9个96孔板共864个单克隆，单克隆OD值结果如图7所示，根据计算原则进行计算，筛选到阳性克隆231个，序列特异性的克隆17个，如图8所示，所述序列特异性的纳米抗体的编号分别为VHH01-VHH20(不包含VHH02、VHH05和VHH11)，VHH01、VHH03、VHH04、VHH06、VHH07、VHH08、 VHH09、VHH10、VHH12、VHH13、VHH14、VHH15、VHH16、VHH17、VHH18、VHH19、VHH20其对应的可变区氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:9、SEQ ID NO:17、SEQ ID NO:25、SEQ ID NO:33、SEQ ID NO:41、SEQ ID NO:49、SEQ ID NO:57、SEQ ID NO:65、SEQ ID NO:73、SEQ ID NO:81、SEQ ID NO:89、SEQ ID NO:97、SEQ ID NO:105、SEQ ID NO:113、SEQ ID NO:121、SEQ ID NO:129所示，其分别对应的可变区核酸序列分别如SEQ ID NO:2、SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:26、SEQ ID NO:34、SEQ ID NO:42、SEQ ID NO:50、SEQ ID NO:58、SEQ ID NO:66、SEQ ID NO:74、SEQ ID NO:82、SEQ ID NO:90、SEQ ID NO:98、SEQ ID NO:106、SEQ ID NO:114、SEQ ID NO:122、SEQ ID NO:130所示。
实施例5单VHH CAR-T细胞的制备及体外功能验证
1、单VHH CAR结构构建
(1)将序列特异性的克隆进行单VHH CAR结构构建。首先，利用PCR的方法扩增阳性克隆株的VHH序列，第一轮PCR引物为：
NCAR-F1：5’-CTGCAGGAGTCTGGRGGAGG-3’
NCAR-R1：5’-TGAGGAGACGGTGACCTGGG-3’
第一轮PCR结束后，以第一轮PCR产物为模板进行第二轮PCR，第二轮PCR引物为：
NCAR-F2：5’-TTTCTGCTGATCCCCCAGGTGCAGCTGCAGGAGTCTGGRGGAGG-3’
NCAR-R2：5’-TAGGAGCCGGGGTGGGCGGCCGCGGTGCTGGGGTAGTTGAGGAGACGGTGACCTGGG-3’
(2)将第二轮PCR产物通过同源重组的方式，连接入载体Senl-S88BZ，载体用Not I单酶切。至此，包含有靶向CD7单VHH的CAR结构构建成功。共构建17个单VHH序列，分别命名为VHH01至VHH20(其中不包含02、05和11)，构建得到的单VHH CAR-T结构示意图如图9所示，其中，EF1α为延长因子1α的启动子，leader是信号肽的编码序列，VHH是抗CD7纳米抗体的编码序列，CD8H+TM为CD8铰链区和跨膜区，4-1BB和CD3ζ胞内信号区为胞内共刺激域，通过T2A肽连接表达tEGFR胞外区域，以便病毒转导后检测CAR的表达。
2、单VHH CAR-T细胞的制备
CAR-T细胞制备前，首先进行慢病毒的包装：(1)将目的质粒与三个辅助质粒(pMD2.G、pRSV-REV、pMDLg)在PEI-Pro作用下共转染293FT细胞；(2)包装6小时进行换液；(3)包装48小时后进行慢病毒收获；(4)收获的慢病毒原液进行超速离心浓缩，用DMEM高糖培养基重悬慢病毒颗粒，并进行分装备用；慢病毒包装流程如图10所示；
慢病毒包装完成后，进行CAR-T细胞的制备：(1)采集患者或健康供者外周血单个核细胞(PBMC)；(2)通过CD3磁珠进行αβT细胞分选；(3)分选完成的αβT细胞在TexMACS GMP培养基(MACS)中进行培养；(4)2天后进行慢病毒的转导；(5)继续培养到12-14天进行CAR-T细胞收获，获得靶向CD7的VHH NS CAR-T细胞(命名为VHH01-VHH20，其中不包括VHH02、VHH05和VHH11)，CAR-T细胞培养流程如图11所示；(6)培养过程中进行流式检测，测定CAR+细胞的比例及CD7+细胞平均MFI值。
3、单VHH CAR-T细胞的体外功能验证
为了验证本实施例制备得到的抗CD7VHH CAR-T细胞的体外生物学活性，培养过程中进行了体外杀伤实验的验证：首先收集靶细胞KG-1a-GFP-Luc，2000rpm离心5min，DPBS重悬计数，按1×10 5个/孔的数量加到96孔板中。然后根据E：T＝3：1向靶细胞中添加适量的效应细胞，混合后孵育4小时，加入相应的荧光素酶底物，并将96孔板用电化学发光酶标仪读取荧光素酶数值。根据数值变化，计算杀伤比例。另外，在17种单VHH CAR-T细胞培养的第12天，取17种单VHH CAR-T细胞、空白T细胞分别与CD7表达阳性细胞系KG-1a-GFP-Luc按照E：T＝3：1的比例进行细胞杀伤实验。
4、实验结果
单VHH CAR-T细胞流式细胞术检测代表性结果、及其CD7+平均MFI结果分别见图12和图13，结果显示，VHH01至VHH20(不包括VHH02、VHH05和VHH11)的CAR阳性率分别为：61.04％、96.2％、78.9％、83％、85％、72.72％、89％、95.6％、90.98％、68.38％、65％、84.1％、79.6％、54.4％、89.2％、72.6％、62.15％；空白T细胞CD7阳性细胞平均MFI值为2588，VHH01至VHH20(不包括VHH02、VHH05和VHH11)的CAR-T细胞CD7阳性细胞平均MFI值分别为：2257、694、1281、498、1566、2362、841、197、190、2010、1473、1077、1300、2054、1117、1391、2041；
体外功能验证的结果见图14，结果显示，空白T细胞的杀伤比例为13.65％，VHH01至VHH20(VHH02和、VHH05和VHH11)CAR-T细胞的杀伤比例分别为4.79％、75.7％、57％、62.19％、61.63％、68.52％、71.47％、91.36％、78.18％、1.18％、60.44％、56.77％、72.87％、8.58％、74.52％、61.71％、9.38％。
实施例6纳米抗体亲和力、专属性的检测
1、亲和力检测
将实施例4中经筛选鉴定得到的17种单VHH对应的VHH CAR结构转导人髓系白血病细胞系K562，在转导后第4天，利用CD7-His蛋白进行流式检测，统计阳性细胞的平均MFI值。根据各VHH与CD7亲和力的不同，阳性细胞平均MFI值会有差异，MFI值越高代表所对应的纳米抗体对抗原CD7的亲和力越高，共进行三次平行实验。
2、专属性检测
(1)将测序获得的不同克隆株的VHH片段克隆到原核表达载体PET-28a-SUMO中；(2)测序正确后提取质粒，然后转化至大肠杆菌菌株BL21中，并在IPTG诱导下进行蛋白表达(3)超声裂解菌体获得粗提蛋白；(4)经过镍柱离子亲和层析纯化纳米抗体；(5)以纯化后纳米抗体为一抗，以HIS-FITC抗体为二抗通过流式细胞术检测17个纳米抗体与K562和K562-CD7细胞系的结合情况。
3、实验结果
纳米抗体亲和力的检测结果见图15，结果显示，17个单VHH结构均与CD7-His发生了特异性结合，其中VHH03、VHH06、VHH10、VHH12的亲和力较强，表明了本发明实施例4中经筛选鉴定得到的17种单VHH与CD7均具有较好的亲和力；
纳米抗体专属性的检测结果见图16，结果显示，17个VHH均能与CD7发生特异性结合，表明了本发明实施例4中经筛选鉴定得到的17种单VHH具有较好的专属性。
实施例7人源化纳米抗体(hVHH06)的构建
1、实验方法
(1)以文献报道的通用人源化框架h-NbBcII10FGLA为参考(详见文献：Vincke,C.,et al.,General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold.J Biol Chem,2009.284(5):p.3273-3284)，并通过与DP-47进行比对，对VHH06关键位置的残基进行了人源化改造，改造后纳米抗体命名为hVHH06，三种结构(人源化序列与DP-47、模板h-NbBcII10PGLA及原始序列)的序列对比如图19所示，所述hVHH06的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:139、SEQ ID NO:141、SEQ ID NO:143所示，CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:140、SEQ ID NO:142、SEQ ID NO:144所示，所述hVHH06的氨基酸序列如SEQ ID NO:137所示，核苷酸序列如SEQ ID NO:138所示；(2)将hVHH06和VHH06按照前述方法同时进行慢病毒包装，并制备得到CAR-T细胞；(3)在CAR-T培养的第6天进行流式细胞术检测；(4)在CAR-T培养的第10天，以KG-1a-GFP-Luc为靶细胞，按照前述方法，设定效靶比为E：T＝5：1、10：1、20：1进行杀伤实验。
2、实验结果
流式细胞术检测的结果见图17，结果显示，VHH06和hVHH06的转导率分别为12.4％、21％，CD7阳性率分别为0.249％、0.157％；杀伤实验的结果见图18，结果显示，E：T＝5：1、10：1、20：1时，空白T细胞对KG-1a-GFP-Luc细胞的杀伤均值分别为27.40％、28.00％、26.80％；VHH06对KG-1a-GFP-Luc细胞的杀伤均值分别为69.90％、79.80％、79.70％，hVHH06对KG-1a-GFP-Luc细胞的杀伤均值分别为70.80％、81.20％、89.60％，由以上结果可知，人源化对抗体的性状未产生影响，表明了本实施例成功对VHH06进行了人源化改造。
实施例8双VHH CAR-T细胞的制备
1、双VHH CAR结构构建
(1)将实施例6中筛选到的功能较好的VHH(VHH03、VHH06、VHH10、VHH12)用于构建双VHH的CAR结构目的质粒，结构示意图如图20所示。首先，利用PCR的方法扩增阳性克隆株的VHH序列，第一轮PCR引物为：
dNCAR-F1：5’-CAGGTGCAGCTGCAGGAG-3’
dNCAR-R1：5’-TGAGGAGACGGTGACCTGG-3’
第一轮PCR结束后，以第一轮PCR产物为模板进行第二轮PCR，第二轮PCR引物为：
dNCAR-F2：5’-CCAGGTCACCGTCTCCTCAGGAGGAGGAGGATCCGGAGGAGGAGGATCTGGCGGCGGCGGCAGTGGCGGCGGCGGCTCCGGCGGCGGCGGCTCTCAGGTGCAGCTGCAGGAG-3’
dNCAR-R2：5’-TAGGAGCCGGGGTGGGCGGCCGCGGTGCTGGGGTAGTTGAGGAGACGGTGACCTGG-3’
然后，将第二轮PCR产物通过同源重组的方式，连接入载体VHH-XX(XX代表靶向CD7单VHH的CAR结构的序号)中，载体用Not I单酶切。其中，以VHH-06为载体，分别与VHH03、VHH12一起构建的双VHH结构分别命名为dVHH-B、dVHH-C。以VHH-10为载体，分别与VHH12、VHH10一起构建的双VHH结构分别命名为dVHH-D、dVHH-E。由VHH12为载体，与VHH12一起构建的双VHH结构命名为dVHH-F。
2、双VHH CAR-T细胞的制备
双VHH CAR-T细胞的制备流程与实施例5中单VHH CAR-T细胞的制备流程一致，慢病毒包装过程如图10所示，培养流程如图11所示。将5个双VHH结构和转导T细胞制备得到的CAR-T细胞、VHH10CAR-T细胞、VHH12CAR-T细胞培养6天后进行流式细胞术检测。
3、实验结果
本实施例共构建5个双VHH结构，分别命名为dVHH-B至dVHH-F(VHH-06+VHH-03、VHH-06+VHH-12、VHH-10+VHH-12、VHH-10+VHH-10、VHH-12+VHH-12)，结构如图20所示，EF1α为延长因子1α的启动子，leader是信号肽的编码序列，VHH是抗CD7纳米抗体的编码序列，CD8H+TM为CD8铰链区和跨膜区，4-1BB和CD3ζ胞内信号区为胞内共刺激域，通过T2A肽连接表达tEGFR胞外区域，以便病毒转导后检测CAR的表达；
CAR+细胞的比例及CD7MFI值对应的结果图分别见图21和图22，结果显示，dVHH-B至dVHH-F的CAR阳性率分别为：22.59％、53.6％、68.68％、55.34％，VHH10的阳性率为72.2％，VHH12的阳性率为86.7％；空白T细胞CD7阳性细胞平均MFI值为10109，dVHH-B至dVHH-F的CAR-T细胞CD7阳性细胞平均MFI值分别为：708、797、648、577、1057，VHH10的CAR-T细胞CD7阳性细胞平均MFI值为2302，VHH12的CAR-T细胞CD7阳性细胞平均MFI值为1238。
实施例9双VHH CAR-T细胞(dVHH-D)的培养及体外功能实验验证
1、dVHH-D双VHH CAR-T细胞的制备
用实施例8中所述的CAR-T细胞的制备方法对dVHH-D、VHH10、VHH12结构进行体外培养，并对培养过程中的细胞扩增倍数进行统计。
2、dVHH-D双VHH CAR-T细胞的体外功能实验验证
为比较dVHH-D与单VHH CAR-T的体外功能，dVHH-D、VHH10、VHH12CAR-T细胞培养的第12天，CAR-T细胞以及空白T细胞分别与CD7表达阳性细胞系CCRF-CEM(白血病T淋巴瘤细胞)按照E：T＝2：1的比例进行细胞杀伤实验：首先收集靶细胞，2000rpm离心5min，DPBS重悬计数，进行CFSE染色，并按照1E5/孔的数量添加到96孔板中。然后根据不同效靶比(E：T＝0.5：1、1：1、2：1)向靶细胞中添加适量的效应细胞，混合后孵育4小时，流式细胞术检测细胞杀伤比例。
3、实验结果
dVHH-D扩增曲线见图23，结果显示，扩增到第14天，dVHH-D的平均扩增倍数为46.35，VHH-12的平均扩增倍数为26.3，VHH-10的平均扩增倍数为22.75，可见dVHH-D的扩增倍数要显著优于单VHH CAR-T；
dVHH-D体外功能验证的结果见图24，结果显示，在杀伤比例为0.5：1、1：1、2：1时，dVHH-D对CCRF-CEM的杀伤均值分别为86.925％、92.115％、94.465％，VHH10对CCRF-CEM的杀伤均值分别为45.55％、69.95％、85.85％，VHH12对CCRF-CEM的杀伤均值分别为58.65％、80.9％、82.95％，可见dVHH-D对CCRF-CEM具有较高的杀伤力。
实施例10双VHH CAR-T细胞(dVHH-E)的培养及体外功能实验验证
1、dVHH-E双VHH CAR-T细胞的制备
用实施例8中所述的CAR-T细胞的制备方法对dVHH-E、VHH10、VHH12结构进行体外培养，并对培养过程中的细胞扩增倍数进行统计。
2、dVHH-E双VHH CAR-T细胞的体外功能实验验证
用实施例9中描述的方法进行dVHH-E双VHH CAR-T体外功能实验验证。
3、实验结果
dVHH-E扩增曲线见图25，结果显示，扩增到第13天，dVHH-E的平均扩增倍数为45.875，VHH-12的平均扩增倍数为19.775，VHH-10的平均扩增倍数为21.36，可见dVHH-E的扩增倍数要显著优于单VHH CAR-T；
dVHH-E体外功能验证的结果见图26，结果显示，在杀伤比例为0.5：1、1：1、2：1时，dVHH-E对CCRF-CEM的杀伤均值分别为58.15％、84.385％、91.775％，VHH10对CCRF-CEM的杀伤均值分别为45.55％、69.95％、85.85％，VHH12对CCRF-CEM的杀伤均值分别为58.65％、80.9％、82.95％，可见dVHH-E表现出对CCRF-CEM相对较高的杀伤力。
实施例11 SPR方法测定抗体(dVHH-D、dVHH-E、VHH10、VHH12)亲和力
1、实验方法
本实施例中采用SPR方法(表面等离子共振法)测定抗体的亲和力，通过氨基偶联法在CM5芯片上固定实施例1中经制备得到的CD7-His蛋白，以抗体(dVHH-D、dVHH-E、VHH10、VHH12)作为分析物进行实验，检测各个抗体与CD7抗原之间的亲和力。
2、实验结果
结果显示，dVHH-D与CD7-His蛋白之间的亲和力常数为3.35E-09M，dVHH-E与CD7-His蛋白之间的亲和力常数为4.51E-09M，VHH10与CD7-His蛋白之间的亲和力常数为9.99E-08M，VHH12与CD7-His蛋白之间的亲和力常数为1.34E-09M(见表2)，上述结果进一步表明了dVHH-D、dVHH-E、VHH10、VHH12均能与CD7抗原发生特异性的结合，且具有较强的亲和力。
表2SPR方法测定抗体亲和力的结果统计
Figure PCTCN2022136785-appb-000001
上述实施例的说明只是用于理解本发明的方法及其核心思想，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。

Claims (126)

  1. 抗CD7纳米抗体，其特征在于，所述纳米抗体为VHH01、VHH03、VHH04、VHH06、VHH07、VHH08、VHH09、VHH10、VHH12、VHH13、VHH14、VHH15、VHH16、VHH17、VHH18、VHH19或VHH20中的任意一种；
    所述VHH01的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7所示或分别为与SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
    所述VHH03的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15所示或分别为与SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
    所述VHH04的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23所示或分别为与SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
    所述VHH06的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31所示或分别为与SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
    所述VHH07的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39所示或分别为与SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
    所述VHH08的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47所示或分别为与SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
    所述VHH09的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:55所示或分别为与SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:55具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
    所述VHH10的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63所示或分别为与SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
    所述VHH12的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71所示或分别为与SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
    所述VHH13的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79所示或分别为与SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
    所述VHH14的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87所示或分别为与SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
    所述VHH15的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95所示或分别为与SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95具有至少75％、76％、77％、78％、79％、 80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
    所述VHH16的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103所示或分别为与SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
    所述VHH17的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111所示或分别为与SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
    所述VHH18的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:115、SEQ ID NO:117、SEQ ID NO:119所示或分别为与SEQ ID NO:115、SEQ ID NO:117、SEQ ID NO:119具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
    所述VHH19的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:123、SEQ ID NO:125、SEQ ID NO:127所示或分别为与SEQ ID NO:123、SEQ ID NO:125、SEQ ID NO:127具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
    所述VHH20的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135所示或分别为与SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列。
  2. 根据权利要求1所述的纳米抗体，其特征在于，所述VHH01的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8所示或分别为与SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
    所述VHH03的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16所示或分别为与SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
    所述VHH04的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24所示或分别为与SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
    所述VHH06的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32所示或分别为与SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
    所述VHH07的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40所示或分别为与SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
    所述VHH08的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48所示或分别为与SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
    所述VHH09的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56所示或分别为与SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
    所述VHH10的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64所示或分别为与SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
    所述VHH12的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72所示或分别为与SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
    所述VHH13的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80所示或分别为与SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
    所述VHH14的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:88所示或分别为与SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:88具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
    所述VHH15的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96所示或分别为与SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
    所述VHH16的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104所示或分别为与SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
    所述VHH17的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:112所示或分别为与SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:112具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
    所述VHH18的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:116、SEQ ID NO:118、SEQ ID NO:120所示或分别为与SEQ ID NO:116、SEQ ID NO:118、SEQ ID NO:120具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
    所述VHH19的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:124、SEQ ID NO:126、SEQ ID NO:128所示或分别为与SEQ ID NO:124、SEQ ID NO:126、SEQ ID NO:128具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列；
    所述VHH20的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:132、SEQ ID NO:134、SEQ ID NO:136所示或分别为与SEQ ID NO:132、SEQ ID NO:134、SEQ ID NO:136具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列。
  3. 根据权利要求2所述的纳米抗体，其特征在于，所述VHH01、VHH03、VHH04、VHH06、VHH07、VHH08、VHH09、VHH10、VHH12、VHH13、VHH14、VHH15、VHH16、VHH17、VHH18、VHH19、VHH20的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:9、SEQ ID NO:17、SEQ ID NO:25、SEQ ID NO:33、SEQ ID NO:41、SEQ ID NO:49、SEQ ID NO:57、SEQ ID NO:65、SEQ ID NO:73、SEQ ID NO:81、SEQ ID NO:89、SEQ ID NO:97、SEQ ID NO:105、SEQ ID NO:113、SEQ ID NO:121、SEQ ID NO:129所示或分别为与SEQ ID NO:1、SEQ ID NO:9、SEQ ID NO:17、SEQ ID NO:25、SEQ ID NO:33、SEQ ID NO:41、SEQ ID NO:49、SEQ ID NO:57、SEQ ID NO:65、SEQ ID NO:73、SEQ ID NO:81、SEQ ID NO:89、SEQ ID NO:97、SEQ ID NO:105、SEQ ID NO:113、SEQ ID NO:121、SEQ ID NO:129具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列。
  4. 根据权利要求3所述的纳米抗体，其特征在于，所述VHH01、VHH03、VHH04、VHH06、VHH07、 VHH08、VHH09、VHH10、VHH12、VHH13、VHH14、VHH15、VHH16、VHH17、VHH18、VHH19、VHH20的核苷酸序列分别如SEQ ID NO:2、SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:26、SEQ ID NO:34、SEQ ID NO:42、SEQ ID NO:50、SEQ ID NO:58、SEQ ID NO:66、SEQ ID NO:74、SEQ ID NO:82、SEQ ID NO:90、SEQ ID NO:98、SEQ ID NO:106、SEQ ID NO:114、SEQ ID NO:122、SEQ ID NO:130所示或分别为与SEQ ID NO:2、SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:26、SEQ ID NO:34、SEQ ID NO:42、SEQ ID NO:50、SEQ ID NO:58、SEQ ID NO:66、SEQ ID NO:74、SEQ ID NO:82、SEQ ID NO:90、SEQ ID NO:98、SEQ ID NO:106、SEQ ID NO:114、SEQ ID NO:122、SEQ ID NO:130具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列。
  5. 人源化的抗CD7纳米抗体，其特征在于，所述人源化的抗CD7纳米抗体为以通用人源化框架h-NbBcII10FGLA为参考，并通过与DP-47进行比对，对权利要求1所述的纳米抗体关键位置的残基进行人源化改造后得到的。
  6. 根据权利要求5所述的人源化的抗CD7纳米抗体，其特征在于，所述人源化的抗CD7纳米抗体为hVHH01、hVHH03、hVHH04、hVHH06、hVHH07、hVHH08、hVHH09、hVHH10、hVHH12、hVHH13、hVHH14、hVHH15、hVHH16、hVHH17、hVHH18、hVHH19或hVHH20中的任意一种。
  7. 根据权利要求6所述的人源化的抗CD7纳米抗体，其特征在于，所述hVHH06的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:139、SEQ ID NO:141、SEQ ID NO:143所示或分别为与SEQ ID NO:139、SEQ ID NO:141、SEQ ID NO:143具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列。
  8. 根据权利要求7所述的人源化的抗CD7纳米抗体，其特征在于，所述hVHH06的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:140、SEQ ID NO:142、SEQ ID NO:144所示或分别为与SEQ ID NO:140、SEQ ID NO:142、SEQ ID NO:144具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列。
  9. 根据权利要求7所述的人源化的抗CD7纳米抗体，其特征在于，所述hVHH06的氨基酸序列如SEQ ID NO:137所示或与SEQ ID NO:137具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列。
  10. 根据权利要求9所述的人源化的抗CD7纳米抗体，其特征在于，所述hVHH06的核苷酸序列如SEQ ID NO:138所示或与SEQ ID NO:138具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列。
  11. 基于单纳米抗体的嵌合抗原受体，其特征在于，所述嵌合抗原受体包含权利要求1所述的纳米抗体或权利要求5所述的人源化的抗CD7纳米抗体中的任意一种。
  12. 根据权利要求11所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包含跨膜结构域。
  13. 根据权利要求12所述的嵌合抗原受体，其特征在于，所述跨膜结构域包括下列分子的跨膜结构域：CD8α、CD28、IgG1、IgG4、4-1BB、PD-1、CD34、OX40、CD3ε、IL-2受体、IL-7受体、IL-11受体。
  14. 根据权利要求13所述的嵌合抗原受体，其特征在于，所述跨膜结构域为CD8α跨膜结构域。
  15. 根据权利要求12所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包含胞内信号传导结构域。
  16. 根据权利要求15所述的嵌合抗原受体，其特征在于，所述胞内信号传导结构域包括下列分子的胞内信号传导结构域：CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、TCRζ、CD4、CD5、CD8、CD21、CD22、CD79a、CD79b、CD278、FcεRI、DAP10、DAP12、CD66d。
  17. 根据权利要求16所述的嵌合抗原受体，其特征在于，所述胞内信号传导结构域为CD3ζ胞内信号传导结构域。
  18. 根据权利要求15所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包含铰链区。
  19. 根据权利要求18所述的嵌合抗原受体，其特征在于，所述铰链区包括下列分子的铰链区：CD8α、CD28、IgG1、IgG4、4-1BB、PD-1、CD34、OX40、CD3ε、IL-2受体、IL-7受体、IL-11受体。
  20. 根据权利要求19所述的嵌合抗原受体，其特征在于，所述铰链区为CD8α铰链区。
  21. 根据权利要求18所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包含信号肽。
  22. 根据权利要求21所述的嵌合抗原受体，其特征在于，所述信号肽包括下列分子的信号肽：T细胞受体的α链及β链、CD3ζ、CD3ε、CD4、CD5、CD8、CD9、CD28、CD16、CD22、CD33、CD37、CD45、CD64、CD80、CD86、CD134、CD137、CD154、GITR、GM-CSF。
  23. 根据权利要求21所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包含共刺激信号结构域。
  24. 根据权利要求23所述的嵌合抗原受体，其特征在于，所述共刺激信号结构域包括下列分子的共刺激信号结构域：4-1BB(CD137)、CD27、CD19、CD4、CD28、ICOS(CD278)、CD8α、CD8β、BAFFR、HVEM、LIGHT、KIRDS2、SLAMF7、NKp30、NKp46、CD40、CDS、ICAM-1、B7-H3、OX40、DR3、GITR、CD30、TIM1、CD2、CD7、CD226。
  25. 根据权利要求24所述的嵌合抗原受体，其特征在于，所述共刺激信号结构域为4-1BB共刺激信号结构域。
  26. 根据权利要求23所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包含EF1α、T2A、tEGFR。
  27. 根据权利要求26所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包含tEGFR信号肽。
  28. 根据权利要求27所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体为EF1α、信号肽、权利要求1所述的纳米抗体或权利要求5所述的人源化的抗CD7纳米抗体中的任意一种、CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次串联得到。
  29. 基于双纳米抗体的嵌合抗原受体，其特征在于，所述嵌合抗原受体包含权利要求1所述的纳米抗体或权利要求5所述的人源化的抗CD7纳米抗体中的任意两种。
  30. 根据权利要求29所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包含跨膜结构域。
  31. 根据权利要求30所述的嵌合抗原受体，其特征在于，所述跨膜结构域包括下列分子的跨膜结构域：CD8α、CD28、IgG1、IgG4、4-1BB、PD-1、CD34、OX40、CD3ε、IL-2受体、IL-7受体、IL-11受体。
  32. 根据权利要求31所述的嵌合抗原受体，其特征在于，所述跨膜结构域为CD8α跨膜结构域。
  33. 根据权利要求30所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包含胞内信号传导结构域。
  34. 根据权利要求33所述的嵌合抗原受体，其特征在于，所述胞内信号传导结构域包括下列分子的胞内信号传导结构域：CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、TCRζ、CD4、CD5、CD8、CD21、CD22、CD79a、CD79b、CD278、FcεRI、DAP10、DAP12、CD66d。
  35. 根据权利要求34所述的嵌合抗原受体，其特征在于，所述胞内信号传导结构域为CD3ζ胞内信号传导结构域。
  36. 根据权利要求33所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包含铰链区。
  37. 根据权利要求36所述的嵌合抗原受体，其特征在于，所述铰链区包括下列分子的铰链区：CD8α、CD28、IgG1、IgG4、4-1BB、PD-1、CD34、OX40、CD3ε、IL-2受体、IL-7受体、IL-11受体。
  38. 根据权利要求37所述的嵌合抗原受体，其特征在于，所述铰链区为CD8α铰链区。
  39. 根据权利要求36所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包含信号肽。
  40. 根据权利要求39所述的嵌合抗原受体，其特征在于，所述信号肽包括下列分子的信号肽：T细胞受体的α链及β链、CD3ζ、CD3ε、CD4、CD5、CD8、CD9、CD28、CD16、CD22、CD33、CD37、CD45、CD64、CD80、CD86、CD134、CD137、CD154、GITR、GM-CSF。
  41. 根据权利要求39所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包含共刺激信号结构域。
  42. 根据权利要求41所述的嵌合抗原受体，其特征在于，所述共刺激信号结构域包括下列分子的共刺激信号结构域：4-1BB(CD137)、CD27、CD19、CD4、CD28、ICOS(CD278)、CD8α、CD8β、BAFFR、HVEM、LIGHT、KIRDS2、SLAMF7、NKp30、NKp46、CD40、CDS、ICAM-1、B7-H3、OX40、DR3、GITR、CD30、TIM1、CD2、CD7、CD226。
  43. 根据权利要求42所述的嵌合抗原受体，其特征在于，所述共刺激信号结构域为4-1BB共刺激信号结构域。
  44. 根据权利要求41所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包含EF1α、T2A、tEGFR。
  45. 根据权利要求44所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包含tEGFR信号肽。
  46. 根据权利要求29所述的嵌合抗原受体，其特征在于，所述权利要求1所述的纳米抗体或权利要求5所述的人源化的抗CD7纳米抗体中的任意两种纳米抗体通过连接肽Linker进行连接。
  47. 根据权利要求46所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体为EF1α、信号肽、权利 要求1所述的纳米抗体或权利要求5所述的人源化的抗CD7纳米抗体中的任意两种、CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次串联得到。
  48. 根据权利要求47所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体为EF1α、信号肽、权利要求1所述的纳米抗体中的任意一种、Linker、权利要求1所述的纳米抗体中的任意一种、CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次串联得到。
  49. 根据权利要求48所述的嵌合抗原受体，其特征在于，所述权利要求1所述的纳米抗体中的任意一种为VHH03、VHH06、VHH10或VHH12。
  50. 根据权利要求49所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体为EF1α、信号肽、权利要求1中所述的VHH06、Linker、权利要求1中所述的VHH03、CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次串联得到。
  51. 根据权利要求49所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体为EF1α、信号肽、权利要求1中所述的VHH06、Linker、权利要求1中所述的VHH12、CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次串联得到。
  52. 根据权利要求49所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体为EF1α、信号肽、权利要求1中所述的VHH10、Linker、权利要求1中所述的VHH10、CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次串联得到。
  53. 根据权利要求49所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体为EF1α、信号肽、权利要求1中所述的VHH10、Linker、权利要求1中所述的VHH12、CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次串联得到。
  54. 根据权利要求49所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体为EF1α、信号肽、权利要求1中所述的VHH12、Linker、权利要求1中所述的VHH12、CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次串联得到。
  55. 核酸分子，其特征在于，所述核酸分子包含编码权利要求1所述的纳米抗体、权利要求5所述的人源化的抗CD7纳米抗体、权利要求11所述的嵌合抗原受体或权利要求29所述的嵌合抗原受体的核苷酸序列。
  56. 根据权利要求55所述的核酸分子，其特征在于，所述核苷酸序列如SEQ ID NO:2、SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:26、SEQ ID NO:34、SEQ ID NO:42、SEQ ID NO:50、SEQ ID NO:58、SEQ ID NO:66、SEQ ID NO:74、SEQ ID NO:82、SEQ ID NO:90、SEQ ID NO:98、SEQ ID NO:106、SEQ ID NO:114、SEQ ID NO:122、SEQ ID NO:130所示或与SEQ ID NO:2、SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:26、SEQ ID NO:34、SEQ ID NO:42、SEQ ID NO:50、SEQ ID NO:58、SEQ ID NO:66、SEQ ID NO:74、SEQ ID NO:82、SEQ ID NO:90、SEQ ID NO:98、SEQ ID NO:106、SEQ ID NO:114、SEQ ID NO:122、SEQ ID NO:130具有至少75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的核苷酸序列。
  57. 重组表达载体，其特征在于，所述重组表达载体包含权利要求55所述的核酸分子。
  58. 根据权利要求57所述的重组表达载体，其特征在于，所述表达载体包括DNA载体、RNA载体、质粒、转座子载体、CRISPR/Cas9载体、或病毒载体。
  59. 根据权利要求58所述的重组表达载体，其特征在于，所述病毒载体包含慢病毒载体、腺病毒载体、逆转录病毒载体。
  60. 经工程改造的宿主细胞，其特征在于，所述经工程改造的宿主细胞表达权利要求1所述的纳米抗体、权利要求5所述的人源化的抗CD7纳米抗体、权利要求11所述的嵌合抗原受体或权利要求29所述的嵌合抗原受体；
    所述经工程改造的宿主细胞还包括经工程改造的宿主细胞群体。
  61. 根据权利要求60所述的经工程改造的宿主细胞，其特征在于，所述经工程改造的宿主细胞包含权利要求57所述的重组表达载体。
  62. 根据权利要求61所述的经工程改造的宿主细胞，其特征在于，所述经工程改造的宿主细胞包含经工程改造的免疫细胞。
  63. 根据权利要求62所述的经工程改造的宿主细胞，其特征在于，所述经工程改造的免疫细胞包含T细胞、NK细胞、iNKT细胞、CTL细胞、单核细胞、巨噬细胞、树突状细胞、NKT细胞或其任意组合。
  64. 一种缀合物，其特征在于，所述缀合物包含权利要求1所述的纳米抗体或权利要求5所述的人源化的抗CD7纳米抗体、以及连接于所述纳米抗体的修饰部分，所述修饰部分包括可检测的标记、治疗剂。
  65. 根据权利要求64所述的缀合物，其特征在于，所述可检测的标记包括酶、放射性核素、荧光染料、发光物质、生物素。
  66. 根据权利要求64所述的缀合物，其特征在于，所述治疗剂包括具有抗肿瘤活性的药物或细胞毒剂。
  67. 一种药物组合物，其特征在于，所述药物组合物包含权利要求1所述的纳米抗体、权利要求5所述的人源化的抗CD7纳米抗体、权利要求11所述的嵌合抗原受体、权利要求29所述的嵌合抗原受体、权利要求55所述的核酸分子、权利要求57所述的重组表达载体、权利要求60所述的经工程改造的宿主细胞或权利要求64所述的缀合物。
  68. 一种试剂盒，其特征在于，所述试剂盒包含权利要求1所述的纳米抗体、权利要求5所述的人源化的抗CD7纳米抗体、权利要求11所述的嵌合抗原受体、权利要求29所述的嵌合抗原受体、权利要求55所述的核酸分子、权利要求57所述的重组表达载体、权利要求60所述的经工程改造的宿主细胞或权利要求64所述的缀合物。
  69. 一种检测CD7蛋白或其抗原片段的试剂，其特征在于，所述检测CD7蛋白或其抗原片段的试剂包含权利要求1所述的纳米抗体、权利要求5所述的纳米抗体或权利要求64所述缀合物。
  70. 一种生物制剂，其特征在于，所述生物制剂含有权利要求60所述的经工程改造的宿主细胞和/或权利要求67所述的药物组合物。
  71. 一种刺激对哺乳动物中靶细胞群或组织的免疫应答的方法，其特征在于，所述方法包括如下步骤：给哺乳动物施用有效量的权利要求60所述的经工程改造的宿主细胞、权利要求64所述的缀合物、权利要求67所述的药物组合物或权利要求70所述的生物制剂。
  72. 一种调节受试者体内的免疫应答的方法，其特征在于，所述方法包括如下步骤：向受试者施用有效量的权利要求60所述的经工程改造的宿主细胞、权利要求64所述的缀合物、权利要求67所述的药物组合物或权利要求70所述的生物制剂。
  73. 一种生产权利要求1所述的纳米抗体或权利要求5所述的人源化的抗CD7纳米抗体的方法，其特征在于，所述方法包括如下步骤：培养权利要求60所述的经工程改造的宿主细胞，从培养物中分离出权利要求1所述的纳米抗体或权利要求5所述的人源化的抗CD7纳米抗体。
  74. 一种特异性地抑制CD7活性的方法，其特征在于，所述方法包括如下步骤：将权利要求55所述的核酸分子导入到生物体细胞中，通过表达权利要求1所述的纳米抗体或权利要求5所述的人源化的抗CD7纳米抗体抑制CD7的活性。
  75. 一种预防和/或治疗CD7相关联的疾病或病症的方法，其特征在于，所述方法包括如下步骤：将有效量的权利要求1所述的纳米抗体、权利要求5所述的人源化的抗CD7纳米抗体、权利要求55所述的核酸分子、权利要求57所述的重组表达载体、权利要求60所述的经工程改造的宿主细胞、权利要求64所述的缀合物、权利要求67所述的药物组合物或权利要求70所述的生物制剂给予具有与CD7相关联的疾病或病症的受试者。
  76. 根据权利要求75所述的方法，其特征在于，所述CD7相关联的疾病或病症包括表达CD7的肿瘤。
  77. 根据权利要求76所述的方法，其特征在于，所述肿瘤为T淋巴细胞系血液肿瘤。
  78. 根据权利要求77所述的方法，其特征在于，所述肿瘤包括急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、NKT细胞白血病、外周T细胞淋巴瘤(NHL)、NKT细胞淋巴瘤、间变性大细胞淋巴瘤(ALCL)。
  79. 一种检测CD7蛋白或其抗原片段的方法，其特征在于，所述方法包括如下步骤：
    (1)获取含有CD7蛋白或其抗原片段的样品；
    (2)将步骤(1)收集的样品与权利要求1所述的纳米抗体、权利要求5所述的人源化的抗CD7纳米抗体、权利要求64所述的缀合物、权利要求68所述的试剂盒或权利要求69所述的检测CD7蛋白或其抗原片段的试剂接触；
    (3)检测抗体-抗原复合物的存在。
  80. 根据权利要求79所述的方法，其特征在于，所述纳米抗体是被可用于检测的标记物标记的纳米抗体。
  81. 根据权利要求80所述的方法，其特征在于，所述可用于检测的标记物包括荧光色素、亲和素、顺磁原子、放射性同位素。
  82. 根据权利要求81所述的方法，其特征在于，所述荧光色素为荧光素、罗丹明、Texas红、藻红蛋 白、藻蓝蛋白、别藻蓝蛋白、多甲藻黄素-叶绿素蛋白。
  83. 根据权利要求81所述的方法，其特征在于，所述亲和素为生物素、卵白亲和素、链亲和素、卵黄亲和素、类亲和素。
  84. 根据权利要求81所述的方法，其特征在于，所述放射性同位素为放射性碘、放射性铯、放射性铱、放射性钴。
  85. 一种权利要求60所述的经工程改造的宿主细胞的制备方法，其特征在于，所述方法包括如下步骤：将权利要求57所述的重组表达载体引入到宿主细胞中。
  86. 根据权利要求85所述的方法，其特征在于，所述引入的方法包括脂质转染法、微注射、电穿孔、DNA载体、RNA载体、逆转录病毒载体、慢病毒载体、痘病毒载体、单纯疱疹病毒载体、腺病毒载体、腺相关病毒载体。
  87. 一种用于诊断受试者是否患有表达CD7肿瘤的方法，其特征在于，所述方法包括如下步骤：
    (1)提供来自怀疑患有表达CD7肿瘤的受试者的样品；
    (2)将样品与权利要求1所述的纳米抗体、权利要求5所述的人源化的抗CD7纳米抗体、权利要求64所述的缀合物、权利要求68所述的试剂盒或权利要求69所述的检测CD7蛋白或其抗原片段的试剂接触；
    (3)检测包含所述纳米抗体与抗原的复合物的形成，得到在来自所述受试者的样品中CD7的量，并将所述CD7在来自所述受试者的样品中的量与其在已知标准品或参照样品中的量进行比较，并确定来自所述受试者的样品的CD7水平是否落入与肿瘤相关的CD7水平内。
  88. 根据权利要求87所述的方法，其特征在于，步骤(1)中所述样品包括尿液、血液、血清、血浆、唾液、腹水、循环细胞、循环肿瘤细胞、非组织缔合的细胞、组织或组织学制备物。
  89. 根据权利要求87所述的方法，其特征在于，所述表达CD7肿瘤为T淋巴细胞系血液肿瘤。
  90. 根据权利要求89所述的方法，其特征在于，所述肿瘤包括急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、NKT细胞白血病、外周T细胞淋巴瘤(NHL)、NKT细胞淋巴瘤、间变性大细胞淋巴瘤(ALCL)。
  91. 权利要求1所述的纳米抗体或权利要求5所述的人源化的抗CD7纳米抗体在检测CD7蛋白或其抗原片段中的应用。
  92. 权利要求1所述的纳米抗体或权利要求5所述的人源化的抗CD7纳米抗体在制备用于检测CD7蛋白或其抗原片段的试剂或试剂盒中的应用。
  93. 权利要求55所述的核酸分子或权利要求57所述的重组表达载体在制备经工程改造的宿主细胞中的应用。
  94. 根据权利要求93所述的应用，其特征在于，所述经工程改造的宿主细胞为权利要求60所述的经工程改造的宿主细胞。
  95. 权利要求1-4中任一项所述的纳米抗体、权利要求5-10中任一项所述的人源化的抗CD7纳米抗体、权利要求11-28中任一项所述的嵌合抗原受体、权利要求29-54中任一项所述的嵌合抗原受体、权利要求55或56所述的核酸分子、权利要求57-59中任一项所述的重组表达载体、权利要求60-63中任一项所述的经工程改造的宿主细胞、权利要求64-66中任一项所述的缀合物、权利要求67所述的药物组合物或权利要求70所述的生物制剂在制备抗肿瘤药物中的应用。
  96. 根据权利要求95所述的应用，其特征在于，所述抗肿瘤药物包括抗肿瘤免疫细胞治疗剂、抗肿瘤的基因治疗药物。
  97. 根据权利要求96所述的应用，其特征在于，所述肿瘤为表达CD7的肿瘤。
  98. 根据权利要求97所述的应用，其特征在于，所述肿瘤为T淋巴细胞系血液肿瘤。
  99. 根据权利要求98所述的应用，其特征在于，所述肿瘤包括急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、NKT细胞白血病、外周T细胞淋巴瘤(NHL)、NKT细胞淋巴瘤、间变性大细胞淋巴瘤(ALCL)。
  100. 权利要求1-4中任一项所述的纳米抗体、权利要求5-10中任一项所述的人源化的抗CD7纳米抗体、权利要求11-28中任一项所述的嵌合抗原受体、权利要求29-54中任一项所述的嵌合抗原受体、权利要求55或56所述的核酸分子、权利要求57-59中任一项所述的重组表达载体、权利要求60-63中任一项所述的经工程改造的宿主细胞、权利要求64-66中任一项所述的缀合物、权利要求67所述的药物组合物或权利要求70所述的生物制剂在制备用于制备预防和/或治疗肿瘤的免疫细胞的试剂盒中的应用。
  101. 根据权利要求100所述的应用，其特征在于，所述肿瘤为表达CD7的肿瘤。
  102. 根据权利要求101所述的应用，其特征在于，所述肿瘤为T淋巴细胞系血液肿瘤。
  103. 根据权利要求102所述的应用，其特征在于，所述肿瘤包括急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、NKT细胞白血病、外周T细胞淋巴瘤(NHL)、NKT细胞淋巴瘤、间变性大细胞淋巴瘤(ALCL)。
  104. 权利要求1-4中任一项所述的纳米抗体、权利要求5-10中任一项所述的人源化的抗CD7纳米抗体、权利要求11-28中任一项所述的嵌合抗原受体、权利要求29-54中任一项所述的嵌合抗原受体、权利要求55或56所述的核酸分子、权利要求57-59中任一项所述的重组表达载体、权利要求60-63中任一项所述的经工程改造的宿主细胞、权利要求64-66中任一项所述的缀合物或权利要求67所述的药物组合物在制备用于预防和/或治疗肿瘤的生物制剂中的应用。
  105. 根据权利要求104所述的应用，其特征在于，所述肿瘤为表达CD7的肿瘤。
  106. 根据权利要求105所述的应用，其特征在于，所述肿瘤为T淋巴细胞系血液肿瘤。
  107. 根据权利要求106所述的应用，其特征在于，所述肿瘤包括急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、NKT细胞白血病、外周T细胞淋巴瘤(NHL)、NKT细胞淋巴瘤、间变性大细胞淋巴瘤(ALCL)。
  108. 权利要求60-63中任一项所述的经工程改造的宿主细胞在预防和/或治疗肿瘤中的应用。
  109. 根据权利要求108所述的应用，其特征在于，所述肿瘤为表达CD7的肿瘤。
  110. 根据权利要求109所述的应用，其特征在于，所述肿瘤为T淋巴细胞系血液肿瘤。
  111. 根据权利要求110所述的应用，其特征在于，所述肿瘤包括急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、NKT细胞白血病、外周T细胞淋巴瘤(NHL)、NKT细胞淋巴瘤、间变性大细胞淋巴瘤(ALCL)。
  112. 权利要求64-66中任一项所述的缀合物在预防和/或治疗肿瘤中的应用。
  113. 根据权利要求112所述的应用，其特征在于，所述肿瘤为表达CD7的肿瘤。
  114. 根据权利要求113所述的应用，其特征在于，所述肿瘤为T淋巴细胞系血液肿瘤。
  115. 根据权利要求114所述的应用，其特征在于，所述肿瘤包括急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、NKT细胞白血病、外周T细胞淋巴瘤(NHL)、NKT细胞淋巴瘤、间变性大细胞淋巴瘤(ALCL)。
  116. 权利要求67所述的药物组合物在预防和/或治疗肿瘤中的应用。
  117. 根据权利要求116所述的应用，其特征在于，所述肿瘤为表达CD7的肿瘤。
  118. 根据权利要求117所述的应用，其特征在于，所述肿瘤为T淋巴细胞系血液肿瘤。
  119. 根据权利要求118所述的应用，其特征在于，所述肿瘤包括急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、NKT细胞白血病、外周T细胞淋巴瘤(NHL)、NKT细胞淋巴瘤、间变性大细胞淋巴瘤(ALCL)。
  120. 权利要求68所述的试剂盒在制备用于预防和/或治疗肿瘤的免疫细胞中的应用。
  121. 根据权利要求120所述的应用，其特征在于，所述免疫细胞为权利要求60所述的经工程改造的宿主细胞。
  122. 权利要求69所述的检测CD7蛋白或其抗原片段的试剂在检测CD7蛋白或其抗原片段中的应用。
  123. 权利要求70所述的生物制剂在预防和/或治疗肿瘤中的应用。
  124. 根据权利要求123所述的应用，其特征在于，所述肿瘤为表达CD7的肿瘤。
  125. 根据权利要求124所述的应用，其特征在于，所述肿瘤为T淋巴细胞系血液肿瘤。
  126. 根据权利要求125所述的应用，其特征在于，所述肿瘤包括急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、NKT细胞白血病、外周T细胞淋巴瘤(NHL)、NKT细胞淋巴瘤、间变性大细胞淋巴瘤(ALCL)。
PCT/CN2022/136785 2022-03-30 2022-12-06 抗cd7纳米抗体、衍生物及其在肿瘤治疗中的应用 WO2023185072A1 (zh)

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