B7-H3 nano antibody, preparation method and application thereof
阅读说明：本技术 B7-h3纳米抗体、其制备方法及用途 (B7-H3 nano antibody, preparation method and application thereof ) 是由 王春河 陈艺丽 刘鑫圆 闫帅 罗伟东 刘国键 李欢欢 林以均 王米娜 王琪 于 2019-10-09 设计创作，主要内容包括：本发明提供了一种B7-H3纳米抗体,其制备方法及用途。所述B7-H3纳米抗体包括框架区(FR)和互补决定区(CDR),所述互补决定区包含互补决定区1(CDR1)、互补决定区2(CDR2)和互补决定区3(CDR3)。所述B7-H3纳米抗体可以特异性地与B7-H3结合,灵敏地检测B7-H3分子,并有望作为治疗性抗体用于各种B7-H3分子高表达的恶性肿瘤的治疗。(The invention provides a B7-H3 nano antibody, a preparation method and application thereof. The B7-H3 nanobody includes a Framework Region (FR) and a Complementarity Determining Region (CDR) including complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR 3). The B7-H3 nano antibody can be specifically combined with B7-H3, sensitively detects B7-H3 molecules, and is expected to be used as a therapeutic antibody for treating various malignant tumors with high expression of B7-H3 molecules.)
1. A B7-H3 nanobody comprising a Framework Region (FR) and a Complementarity Determining Region (CDR) comprising a complementarity determining region 1(CDR1), a complementarity determining region 2(CDR2), and a complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is any one selected from SEQ ID NO 5, SEQ ID NO 12, SEQ ID NO 19, SEQ ID NO 24, SEQ ID NO 29, SEQ ID NO 34, SEQ ID NO 41, SEQ ID NO 46, SEQ ID NO 51, SEQ ID NO 58, SEQ ID NO 63 and SEQ ID NO 68;
the complementarity determining region 2(CDR2) is any one selected from SEQ ID NO 6, SEQ ID NO 13, SEQ ID NO 20, SEQ ID NO 25, SEQ ID NO 30, SEQ ID NO 35, SEQ ID NO 42, SEQ ID NO 47, SEQ ID NO 52, SEQ ID NO 59, SEQ ID NO 64 and SEQ ID NO 69; and
the complementarity determining region 3(CDR3) is any one selected from the group consisting of SEQ ID NO 7, SEQ ID NO 14, SEQ ID NO 21, SEQ ID NO 26, SEQ ID NO 31, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 43, SEQ ID NO 48, SEQ ID NO 53, SEQ ID NO 60, SEQ ID NO 65 and SEQ ID NO 70.
2. The B7-H3 nanobody of claim 1, wherein the B7-H3 nanobody comprises the following regions in order: framework region 1(FR1), complementarity determining region 1(CDR1), framework region 2(FR2), complementarity determining region 2(CDR2), framework region 3(FR3), complementarity determining region 3(CDR3), and framework region 4(FR4), wherein,
the framework region 1(FR1) is any one selected from SEQ ID NO 1, SEQ ID NO 8, SEQ ID NO 15, SEQ ID NO 22, SEQ ID NO 27, SEQ ID NO 32, SEQ ID NO 38, SEQ ID NO 44, SEQ ID NO 49, SEQ ID NO 54, SEQ ID NO 61 and SEQ ID NO 66;
the complementarity determining region 1(CDR1) is any one selected from SEQ ID NO 5, SEQ ID NO 12, SEQ ID NO 19, SEQ ID NO 24, SEQ ID NO 29, SEQ ID NO 34, SEQ ID NO 41, SEQ ID NO 46, SEQ ID NO 51, SEQ ID NO 58, SEQ ID NO 63 and SEQ ID NO 68;
the framework region 2(FR2) is any one selected from SEQ ID NO:2, SEQ ID NO:9, SEQ ID NO:16, SEQ ID NO:39, SEQ ID NO: 55;
the complementarity determining region 2(CDR2) is any one selected from SEQ ID NO 6, SEQ ID NO 13, SEQ ID NO 20, SEQ ID NO 25, SEQ ID NO 30, SEQ ID NO 35, SEQ ID NO 42, SEQ ID NO 47, SEQ ID NO 52, SEQ ID NO 59, SEQ ID NO 64 and SEQ ID NO 69;
the framework region 3(FR3) is selected from any one of SEQ ID NO 3, SEQ ID NO 10, SEQ ID NO 17, SEQ ID NO 23, SEQ ID NO 28, SEQ ID NO 33, SEQ ID NO 40, SEQ ID NO 45, SEQ ID NO 50, SEQ ID NO 56, SEQ ID NO 62 and SEQ ID NO 67;
the complementarity determining region 3(CDR3) is any one selected from SEQ ID NO 7, SEQ ID NO 14, SEQ ID NO 21, SEQ ID NO 26, SEQ ID NO 31, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 43, SEQ ID NO 48, SEQ ID NO 53, SEQ ID NO 60, SEQ ID NO 65 and SEQ ID NO 70; and
the framework region 4(FR4) is any one selected from the group consisting of SEQ ID NO:4, SEQ ID NO:11, SEQ ID NO:18 and SEQ ID NO: 57.
3. The B7-H3 nanobody of claim 1, wherein the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:5,
the complementarity determining region 2(CDR2) is SEQ ID NO:6,
the complementarity determining region 3(CDR3) is SEQ ID NO. 7.
4. The B7-H3 nanobody of claim 1, wherein the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:12,
the complementarity determining region 2(CDR2) is SEQ ID NO:13,
the complementarity determining region 3(CDR3) is SEQ ID NO: 14.
5. The B7-H3 nanobody of claim 1, wherein the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:19,
the complementarity determining region 2(CDR2) is SEQ ID NO:20,
the complementarity determining region 3(CDR3) is SEQ ID NO: 21.
6. The B7-H3 nanobody of claim 1, wherein the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:24,
the complementarity determining region 2(CDR2) is SEQ ID NO:25,
the complementarity determining region 3(CDR3) is SEQ ID NO: 26.
7. The B7-H3 nanobody of claim 1, wherein the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:29,
the complementarity determining region 2(CDR2) is SEQ ID NO:30,
the complementarity determining region 3(CDR3) is SEQ ID NO: 31.
8. The B7-H3 nanobody of claim 1, wherein the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:34,
the complementarity determining region 2(CDR2) is SEQ ID NO:35,
the complementarity determining region 3(CDR3) is SEQ ID NO: 36.
9. The B7-H3 nanobody of claim 1, wherein the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:12,
the complementarity determining region 2(CDR2) is SEQ ID NO:13,
the complementarity determining region 3(CDR3) is SEQ ID NO: 37.
10. The B7-H3 nanobody of claim 1, wherein the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:41,
the complementarity determining region 2(CDR2) is SEQ ID NO:42,
the complementarity determining region 3(CDR3) is SEQ ID NO: 43.
11. The B7-H3 nanobody of claim 1, wherein the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:46,
the complementarity determining region 2(CDR2) is SEQ ID NO:47,
the complementarity determining region 3(CDR3) is SEQ ID NO: 48.
12. The B7-H3 nanobody of claim 1, wherein the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:51,
the complementarity determining region 2(CDR2) is SEQ ID NO:52,
the complementarity determining region 3(CDR3) is SEQ ID NO: 53.
13. The B7-H3 nanobody of claim 1, wherein the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:58,
the complementarity determining region 2(CDR2) is SEQ ID NO:59,
the complementarity determining region 3(CDR3) is SEQ ID NO: 60.
14. The B7-H3 nanobody of claim 1, wherein the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:63,
the complementarity determining region 2(CDR2) is SEQ ID NO:64,
the complementarity determining region 3(CDR3) is SEQ ID NO: 65.
15. The B7-H3 nanobody of claim 1, wherein the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:68,
the complementarity determining region 2(CDR2) is SEQ ID NO:69,
the complementarity determining region 3(CDR3) is SEQ ID NO: 70.
16. The B7-H3 nanobody of claim 1, wherein the B7-H3 nanobody has an amino acid sequence selected from any one of:
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 and 83.
17. A method of making the B7-H3 nanobody of claim 1, comprising the steps of:
1) constructing a B7-H3 molecular nano antibody library;
2) screening the B7-H3 nano antibody;
3) detecting a single positive clone by immunoblotting;
4) positive clones were further verified with phase Elisa; and
5) expressing and purifying the B7-H3 nano antibody in a eukaryotic expression system.
18. A DNA molecule encoding the B7-H3 nanobody of claim 1.
19. An expression vector comprising the DNA molecule of claim 18.
20. The use of the B7-H3 nanobody of claim 1 in the preparation of a B7-H3 molecular detection reagent.
21. The use of the B7-H3 nanobody of claim 1 in the preparation of a medicament for the treatment of malignant tumor highly expressed by B7-H3 molecules.
Technical Field
The invention relates to a nano antibody aiming at B7-H3 polypeptide molecular epitope, a preparation method of the nano antibody and application of the nano antibody.
Background
B7-H3 (also named CD276) is a B7 family member, has a sequence similar to the extracellular region of PD-L1(B7-H1), and is a phylogenetically conserved protein with multiple biological functions. B7-H3 is a type I transmembrane protein whose extracellular domain consists of a pair of immunoglobulin variable domains (IgV) and immunoglobulin constant domains (IgC) in mice (subtype 2IgB 7-H3); in humans, two identical pairs of immunoglobulins (subtype 4IgB 7-H3) are produced by exon replication, which is more commonly expressed. B7-H3 was expressed more widely at the mRNA level and was detected in non-lymphoid organs such as liver, heart, prostate and lymphoid organs such as spleen and thymus, but protein expression was tightly regulated. B7-H3 was structurally expressed on non-immune resting fibroblasts, endothelial cells, osteoblasts and amniotic fluid stem cells. Induced B7-H3 is capable of being expressed on immune cells, particularly antigen presenting cells. And regulatory T cells (Treg), IFN-gamma, Lipopolysaccharide (LPS) or anti-CD40 can induce dendritic cells to express B7-H3 protein by in vitro stimulation. Furthermore, monocytes and monocyte-derived DCs up-regulate B7-H3 expression levels upon LPS stimulation or cytokine-induced differentiation, respectively; B7-H3 protein was also detected on NK cells, B cells and a small number of T cells following PMA/Ionomycin stimulation. Unlike tight regulation in normal cells, B7-H3 exhibits aberrant overexpression in a variety of malignancies, including melanoma, leukemia, breast, prostate, ovarian, pancreatic, rectal, and the like. The high expression of B7-H3 is also related to poor prognosis and poor clinical curative effect, and the development of an antibody drug targeting B7-H3 becomes a promising tumor treatment strategy.
The nano antibody technology is an antibody engineering revolution carried out by biomedical scientists on the basis of traditional antibodies by applying molecular biology technology combined with the concept of nanoparticle science, so as to develop the latest and smallest antibody molecules, which are originally discovered in camel blood by the scientist Hamers, R in Belgium. While the common antibody proteins consist of two heavy chains and two light chains, the novel antibodies found in camelid blood have only two heavy chains, these "heavy chain antibodies" bind tightly to targets such as antigens like normal antibodies, but do not aggregate together into clumps like single chain antibodies. The nanometer antibody based on the 'heavy chain antibody' has molecular weight of only 1/10 of common antibody, flexible chemical property, high stability, high solubility, easy expression, high penetrability of tumor tissue and easy coupling with other molecules. Therefore, the application of the nano-antibody technology to research and develop the B7-H3 therapeutic antibody has wide prospect.
Disclosure of Invention
In view of the above problems, the present invention provides, in one aspect, a B7-H3 nanobody comprising a Framework Region (FR) and a Complementarity Determining Region (CDR) comprising a complementarity determining region 1(CDR1), a complementarity determining region 2(CDR2), and a complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is any one selected from
the complementarity determining region 2(CDR2) is any one selected from
the complementarity determining region 3(CDR3) is any one selected from
In a specific embodiment, the B7-H3 nanobody comprises the following regions in order: framework region 1(FR1), complementarity determining region 1(CDR1), framework region 2(FR2), complementarity determining region 2(CDR2), framework region 3(FR3), complementarity determining region 3(CDR3), and framework region 4(FR4), wherein,
the framework region 1(FR1) is any one selected from the group consisting of
the complementarity determining region 1(CDR1) is any one selected from
the framework region 2(FR2) is any one selected from SEQ ID NO:2, SEQ ID NO:9, SEQ ID NO:16, SEQ ID NO:39, SEQ ID NO: 55;
the complementarity determining region 2(CDR2) is any one selected from
the framework region 3(FR3) is selected from any one of
the complementarity determining region 3(CDR3) is any one selected from
the framework region 4(FR4) is any one selected from the group consisting of SEQ ID NO:4, SEQ ID NO:11, SEQ ID NO:18 and SEQ ID NO: 57.
In one embodiment, the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:5,
the complementarity determining region 2(CDR2) is SEQ ID NO:6,
the complementarity determining region 3(CDR3) is SEQ ID NO. 7.
In one embodiment, the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:12,
the complementarity determining region 2(CDR2) is SEQ ID NO:13,
the complementarity determining region 3(CDR3) is SEQ ID NO: 14.
In one embodiment, the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:19,
the complementarity determining region 2(CDR2) is SEQ ID NO:20,
the complementarity determining region 3(CDR3) is SEQ ID NO: 21.
In one embodiment, the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:24,
the complementarity determining region 2(CDR2) is SEQ ID NO:25,
the complementarity determining region 3(CDR3) is SEQ ID NO: 26.
In one embodiment, the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:29,
the complementarity determining region 2(CDR2) is SEQ ID NO:30,
the complementarity determining region 3(CDR3) is SEQ ID NO: 31.
In one embodiment, the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:34,
the complementarity determining region 2(CDR2) is SEQ ID NO:35,
the complementarity determining region 3(CDR3) is SEQ ID NO: 36.
In one embodiment, the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:12,
the complementarity determining region 2(CDR2) is SEQ ID NO:13,
the complementarity determining region 3(CDR3) is SEQ ID NO: 37.
In one embodiment, the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:41,
the complementarity determining region 2(CDR2) is SEQ ID NO:42,
the complementarity determining region 3(CDR3) is SEQ ID NO: 43.
In one embodiment, the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:46,
the complementarity determining region 2(CDR2) is SEQ ID NO:47,
the complementarity determining region 3(CDR3) is SEQ ID NO: 48.
In one embodiment, the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:51,
the complementarity determining region 2(CDR2) is SEQ ID NO:52,
the complementarity determining region 3(CDR3) is SEQ ID NO: 53.
In one embodiment, the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:58,
the complementarity determining region 2(CDR2) is SEQ ID NO:59,
the complementarity determining region 3(CDR3) is SEQ ID NO: 60.
In one embodiment, the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:63,
the complementarity determining region 2(CDR2) is SEQ ID NO:64,
the complementarity determining region 3(CDR3) is SEQ ID NO: 65.
In one embodiment, the B7-H3 nanobody comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), wherein,
the complementarity determining region 1(CDR1) is SEQ ID NO:68,
the complementarity determining region 2(CDR2) is SEQ ID NO:69,
the complementarity determining region 3(CDR3) is SEQ ID NO: 70.
In a specific embodiment, the B7-H3 nanobody has an amino acid sequence selected from any one of:
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 and 83.
In another aspect, the present invention provides a method for preparing the aforementioned B7-H3 nanobody, comprising the following steps:
1) constructing a B7-H3 molecular nano antibody library;
2) screening the B7-H3 nano antibody;
3) detecting a single positive clone by immunoblotting;
4) positive clones were further verified with phase Elisa; and
5) expressing and purifying the B7-H3 nano antibody in a eukaryotic expression system.
In still another aspect, the present invention provides a DNA molecule encoding the above B7-H3 nanobody.
In a further aspect, the present invention provides an expression vector comprising the above-described DNA molecule.
In another aspect, the invention provides application of the B7-H3 nano antibody in preparation of a B7-H3 molecular detection reagent.
In another aspect, the invention provides an application of the B7-H3 nano antibody in preparing a medicament for treating malignant tumor with high expression of B7-H3 molecules.
Effects of the invention
The B7-H3 nano antibody can be specifically combined with B7-H3, sensitively detects B7-H3 molecules, and is expected to be used as a therapeutic antibody for treating various malignant tumors with high expression of B7-H3 molecules.
Drawings
FIG. 1 is an Output phase immunoblot in example 3.
FIG. 2 shows the results of further validation of positive clones by phase Elisa in example 4.
FIG. 3 is a SDS-PAGE pattern of the purification of the # 1-13 nanobody in example 5.
Detailed Description
Specific processes for preparing nanobodies of the present application are described below by way of specific examples, however, it will be understood by those skilled in the art that the scope of the present application should not be limited by these examples, but encompasses various equivalent modifications that may be made by those skilled in the art.
Complete/incomplete freund's adjuvant (sigma, F5506), streptavidin magnetic beads (invitrogen,65002), casein (Thermo,37528), PROTRAN BA85(Whatman,10401116), microplate (greiner,650061), goat anti-M13 HRP (GE, 279421).
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