CN110551218B - GD2 nano antibody and application thereof - Google Patents
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Abstract
The invention provides a GD2 nano antibody and application thereof, and the nano antibody can be applied to development of GD2 molecular detection reagents and therapeutic antibodies.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a GD2 nano antibody and application thereof, wherein the nano antibody can be applied to development of GD2 molecular detection reagents and therapeutic antibodies.
Background
The disialoganglioside (GD2) molecule is a carcinoembryonic antigen expressed in fetuses. GD2 molecule is also expressed in neural stem cells, mesenchymal stem cells, and breast cancer stem cells. After birth, it can be detected in melanocytes of peripheral neurons, the central nervous system and the skin. The biological role of GD2 is not yet very clear. GD2 molecules on the surface of neuroblastoma have been reported to mediate the interconnection between tumor cells by extracellular matrix proteins. The expression of GD2 antigen was higher and more homogeneous on the surface and inside the neuroblastoma tumor compared to other antigens, with a higher content of free GD2 antigen in the patient's serum. The GD2 molecule did not disappear when GD2 antigen was bound to the antibody. The national cancer center of America has GD2 antigen as the 12 th ranking tumor antigen according to the standards of antigen treatment function, immunogenicity, carcinogenicity, specificity, expression level, and antigen tumor cell expression ratio. Besides neuroblastoma, other malignant tumors such as melanoma, soft tissue sarcoma, osteosarcoma, desmoplastic small round cell tumor, small cell lung cancer also express GD2 molecule.
Nanobody technology is a revolution in antibody engineering by biomedical scientists based on traditional antibodies, using molecular biology techniques in combination with the concept of nanoparticle science, to develop the latest and smallest antibody molecules originally found in camel blood by the belgium scientist Hamers, R. 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 development of a detection reagent and a therapeutic antibody of GD2 by applying a nano antibody technology has wide prospects.
Disclosure of Invention
The present inventors have conducted extensive studies in view of the deficiencies of the prior art, and as a result, have completed the present invention.
Accordingly, in one aspect, the present invention provides a GD2 nanobody, which is a nanobody directed to an epitope of the ganglioside GD2 molecule, comprising a Framework Region (FR) and a Complementarity Determining Region (CDR), wherein,
complementarity determining region 1(CDR1) is selected from the group consisting of SEQ ID NO 5, SEQ ID NO 11, SEQ ID NO 19, SEQ ID NO 27, SEQ ID NO 33, SEQ ID NO 40, SEQ ID NO 47, and SEQ ID NO 61;
complementarity determining region 2(CDR2) is selected from the group consisting of SEQ ID NO 6, SEQ ID NO 12, SEQ ID NO 20, SEQ ID NO 28, SEQ ID NO 34, SEQ ID NO 41, SEQ ID NO 48, SEQ ID NO 54, and SEQ ID NO 62; and
complementarity determining region 3(CDR3) is selected from the group consisting of SEQ ID NO 7, SEQ ID NO 13, SEQ ID NO 21, SEQ ID NO 29, SEQ ID NO 35, SEQ ID NO 42, SEQ ID NO 49, SEQ ID NO 55, and SEQ ID NO 63.
Preferably, the invention provides a GD2 nanobody, which is a nanobody directed to an epitope of the ganglioside GD2 molecule, comprising, preferably consisting of, in order:
framework region 1(FR1) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:44, SEQ ID NO:51 and SEQ ID NO: 57;
complementarity determining region 1(CDR1) selected from the group consisting of SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:19, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:40, SEQ ID NO:47, and SEQ ID NO: 61;
framework region 2(FR2) selected from the group consisting of SEQ ID NO 2, SEQ ID NO 9, SEQ ID NO 16, SEQ ID NO 24, SEQ ID NO 38, SEQ ID NO 45, SEQ ID NO 52 and SEQ ID NO 58;
complementarity determining region 2(CDR2) selected from the group consisting of SEQ ID NO 6, SEQ ID NO 12, SEQ ID NO 20, SEQ ID NO 28, SEQ ID NO 34, SEQ ID NO 41, SEQ ID NO 48, SEQ ID NO 54, and SEQ ID NO 62;
framework region 3(FR3) selected from the group consisting of SEQ ID NO 3, SEQ ID NO 10, SEQ ID NO 17, SEQ ID NO 25, SEQ ID NO 32, SEQ ID NO 39, SEQ ID NO 46, SEQ ID NO 53 and SEQ ID NO 59;
complementarity determining region 3(CDR3) selected from the group consisting of SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:42, SEQ ID NO:49, SEQ ID NO:55, and SEQ ID NO: 63; and
framework region 4(FR4) selected from the group consisting of SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26 and SEQ ID NO: 60.
In the present invention, alternative examples of each framework region 1(FR1), each complementarity determining region 1(CDR1), each framework region 2(FR2), each complementarity determining region 2(CDR2), each framework region 3(FR3), each complementarity determining region 3(CDR3), and each framework region 4(FR4) have high sequence homology, for example, 80% or more.
More preferably, the invention provides a GD2 nanobody, which is a nanobody directed to an epitope of the ganglioside GD2 molecule, comprising, preferably consisting of, in order:
framework region 1(FR1) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:44, SEQ ID NO:51 and SEQ ID NO: 57;
complementarity determining region 1(CDR1) shown in SEQ ID NO: 5;
framework region 2(FR2) selected from the group consisting of SEQ ID NO 2, SEQ ID NO 9, SEQ ID NO 16, SEQ ID NO 24, SEQ ID NO 38, SEQ ID NO 45, SEQ ID NO 52 and SEQ ID NO 58;
complementarity determining region 2(CDR2) shown in SEQ ID NO: 6;
framework region 3(FR3) selected from the group consisting of SEQ ID NO 3, SEQ ID NO 10, SEQ ID NO 17, SEQ ID NO 25, SEQ ID NO 32, SEQ ID NO 39, SEQ ID NO 46, SEQ ID NO 53 and SEQ ID NO 59;
complementarity determining region 3(CDR3) shown in SEQ ID NO: 7; and
framework region 4(FR4) selected from the group consisting of SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26 and SEQ ID NO: 60.
More preferably, the invention provides a GD2 nanobody, which is a nanobody directed to an epitope of the ganglioside GD2 molecule, comprising, preferably consisting of, in order:
framework region 1(FR1) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:44, SEQ ID NO:51 and SEQ ID NO: 57;
complementarity determining region 1(CDR1) shown in SEQ ID NO: 11;
framework region 2(FR2) selected from the group consisting of SEQ ID NO 2, SEQ ID NO 9, SEQ ID NO 16, SEQ ID NO 24, SEQ ID NO 38, SEQ ID NO 45, SEQ ID NO 52 and SEQ ID NO 58;
complementarity determining region 2(CDR2) shown in SEQ ID NO: 12;
framework region 3(FR3) selected from the group consisting of SEQ ID NO 3, SEQ ID NO 10, SEQ ID NO 17, SEQ ID NO 25, SEQ ID NO 32, SEQ ID NO 39, SEQ ID NO 46, SEQ ID NO 53 and SEQ ID NO 59;
complementarity determining region 3(CDR3) shown in SEQ ID NO: 13; and
framework region 4(FR4) selected from the group consisting of SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26 and SEQ ID NO: 60.
More preferably, the invention provides a GD2 nanobody, which is a nanobody directed to an epitope of the ganglioside GD2 molecule, comprising, preferably consisting of, in order:
framework region 1(FR1) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:44, SEQ ID NO:51 and SEQ ID NO: 57;
complementarity determining region 1(CDR1) shown in SEQ ID NO: 19;
framework region 2(FR2) selected from the group consisting of SEQ ID NO 2, SEQ ID NO 9, SEQ ID NO 16, SEQ ID NO 24, SEQ ID NO 38, SEQ ID NO 45, SEQ ID NO 52 and SEQ ID NO 58;
complementarity determining region 2(CDR2) shown in SEQ ID NO: 20;
framework region 3(FR3) selected from the group consisting of SEQ ID NO 3, SEQ ID NO 10, SEQ ID NO 17, SEQ ID NO 25, SEQ ID NO 32, SEQ ID NO 39, SEQ ID NO 46, SEQ ID NO 53 and SEQ ID NO 59;
complementarity determining region 3(CDR3) shown in SEQ ID NO: 21; and
framework region 4(FR4) selected from the group consisting of SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26 and SEQ ID NO: 60.
More preferably, the invention provides a GD2 nanobody, which is a nanobody directed to an epitope of the ganglioside GD2 molecule, comprising, preferably consisting of, in order:
framework region 1(FR1) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:44, SEQ ID NO:51 and SEQ ID NO: 57;
complementarity determining region 1(CDR1) shown in SEQ ID NO: 27;
framework region 2(FR2) selected from the group consisting of SEQ ID NO 2, SEQ ID NO 9, SEQ ID NO 16, SEQ ID NO 24, SEQ ID NO 38, SEQ ID NO 45, SEQ ID NO 52 and SEQ ID NO 58;
complementarity determining region 2(CDR2) shown in SEQ ID NO: 28;
framework region 3(FR3) selected from the group consisting of SEQ ID NO 3, SEQ ID NO 10, SEQ ID NO 17, SEQ ID NO 25, SEQ ID NO 32, SEQ ID NO 39, SEQ ID NO 46, SEQ ID NO 53 and SEQ ID NO 59;
complementarity determining region 3(CDR3) shown in SEQ ID NO: 29; and
framework region 4(FR4) selected from the group consisting of SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26 and SEQ ID NO: 60.
More preferably, the invention provides a GD2 nanobody, which is a nanobody directed to an epitope of the ganglioside GD2 molecule, comprising, preferably consisting of, in order:
framework region 1(FR1) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:44, SEQ ID NO:51 and SEQ ID NO: 57;
complementarity determining region 1(CDR1) shown in SEQ ID NO: 33;
framework region 2(FR2) selected from the group consisting of SEQ ID NO 2, SEQ ID NO 9, SEQ ID NO 16, SEQ ID NO 24, SEQ ID NO 38, SEQ ID NO 45, SEQ ID NO 52 and SEQ ID NO 58;
complementarity determining region 2(CDR2) shown in SEQ ID NO: 34;
framework region 3(FR3) selected from the group consisting of SEQ ID NO 3, SEQ ID NO 10, SEQ ID NO 17, SEQ ID NO 25, SEQ ID NO 32, SEQ ID NO 39, SEQ ID NO 46, SEQ ID NO 53 and SEQ ID NO 59;
complementarity determining region 3(CDR3) shown in SEQ ID NO: 35; and
framework region 4(FR4) selected from the group consisting of SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26 and SEQ ID NO: 60.
More preferably, the invention provides a GD2 nanobody, which is a nanobody directed to an epitope of the ganglioside GD2 molecule, comprising, preferably consisting of, in order:
framework region 1(FR1) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:44, SEQ ID NO:51 and SEQ ID NO: 57;
complementarity determining region 1(CDR1) shown in SEQ ID NO: 40;
framework region 2(FR2) selected from the group consisting of SEQ ID NO 2, SEQ ID NO 9, SEQ ID NO 16, SEQ ID NO 24, SEQ ID NO 38, SEQ ID NO 45, SEQ ID NO 52 and SEQ ID NO 58;
complementarity determining region 2(CDR2) shown in SEQ ID NO: 41;
framework region 3(FR3) selected from the group consisting of SEQ ID NO 3, SEQ ID NO 10, SEQ ID NO 17, SEQ ID NO 25, SEQ ID NO 32, SEQ ID NO 39, SEQ ID NO 46, SEQ ID NO 53 and SEQ ID NO 59;
complementarity determining region 3(CDR3) shown in SEQ ID NO: 42; and
framework region 4(FR4) selected from the group consisting of SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26 and SEQ ID NO: 60.
More preferably, the invention provides a GD2 nanobody, which is a nanobody directed to an epitope of the ganglioside GD2 molecule, comprising, preferably consisting of, in order:
framework region 1(FR1) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:44, SEQ ID NO:51 and SEQ ID NO: 57;
complementarity determining region 1(CDR1) shown in SEQ ID NO: 47;
framework region 2(FR2) selected from the group consisting of SEQ ID NO 2, SEQ ID NO 9, SEQ ID NO 16, SEQ ID NO 24, SEQ ID NO 38, SEQ ID NO 45, SEQ ID NO 52 and SEQ ID NO 58;
complementarity determining region 2(CDR2) shown in SEQ ID NO: 48;
framework region 3(FR3) selected from the group consisting of SEQ ID NO 3, SEQ ID NO 10, SEQ ID NO 17, SEQ ID NO 25, SEQ ID NO 32, SEQ ID NO 39, SEQ ID NO 46, SEQ ID NO 53 and SEQ ID NO 59;
complementarity determining region 3(CDR3) shown in SEQ ID NO: 49; and
framework region 4(FR4) selected from the group consisting of SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26 and SEQ ID NO: 60.
More preferably, the invention provides a GD2 nanobody, which is a nanobody directed to an epitope of the ganglioside GD2 molecule, comprising, preferably consisting of, in order:
framework region 1(FR1) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:44, SEQ ID NO:51 and SEQ ID NO: 57;
complementarity determining region 1(CDR1) shown in SEQ ID NO: 40;
framework region 2(FR2) selected from the group consisting of SEQ ID NO 2, SEQ ID NO 9, SEQ ID NO 16, SEQ ID NO 24, SEQ ID NO 38, SEQ ID NO 45, SEQ ID NO 52 and SEQ ID NO 58;
complementarity determining region 2(CDR2) shown in SEQ ID NO: 54;
framework region 3(FR3) selected from the group consisting of SEQ ID NO 3, SEQ ID NO 10, SEQ ID NO 17, SEQ ID NO 25, SEQ ID NO 32, SEQ ID NO 39, SEQ ID NO 46, SEQ ID NO 53 and SEQ ID NO 59;
complementarity determining region 3(CDR3) shown in SEQ ID NO: 55; and
framework region 4(FR4) selected from the group consisting of SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26 and SEQ ID NO: 60.
More preferably, the invention provides a GD2 nanobody, which is a nanobody directed to an epitope of the ganglioside GD2 molecule, comprising, preferably consisting of, in order:
framework region 1(FR1) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:44, SEQ ID NO:51 and SEQ ID NO: 57;
complementarity determining region 1(CDR1) shown in SEQ ID NO: 61;
framework region 2(FR2) selected from the group consisting of SEQ ID NO 2, SEQ ID NO 9, SEQ ID NO 16, SEQ ID NO 24, SEQ ID NO 38, SEQ ID NO 45, SEQ ID NO 52 and SEQ ID NO 58;
complementarity determining region 2(CDR2) shown in SEQ ID NO: 62;
framework region 3(FR3) selected from the group consisting of SEQ ID NO 3, SEQ ID NO 10, SEQ ID NO 17, SEQ ID NO 25, SEQ ID NO 32, SEQ ID NO 39, SEQ ID NO 46, SEQ ID NO 53 and SEQ ID NO 59;
complementarity determining region 3(CDR3) shown in SEQ ID NO: 63; and
framework region 4(FR4) selected from the group consisting of SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26 and SEQ ID NO: 60.
Still more preferably, the present invention provides a GD2 nanobody, which is a nanobody directed to an epitope of the ganglioside GD2 molecule, the amino acid sequence of which is selected from the group consisting of: SEQ ID NO 8, SEQ ID NO 14, SEQ ID NO 22, SEQ ID NO 30, SEQ ID NO 36, SEQ ID NO 43, SEQ ID NO 50, SEQ ID NO 56 and SEQ ID NO 64.
In yet another aspect, the present invention provides a DNA molecule encoding the GD2 nanobody described above.
In yet another aspect, the present invention provides an expression vector comprising the above-described DNA molecule.
In yet another aspect, the present invention provides a host cell comprising the above-described expression vector.
In yet another aspect, the present invention provides the use of the GD2 nanobody described above as a GD2 molecular detection reagent and/or a therapeutic antibody.
In another aspect, the present invention provides a method for preparing the GD2 nanobody, comprising: the method comprises the following steps:
1. coupling ganglioside GD2 to a KLH molecule;
2. constructing a nano antibody library aiming at GD2 molecules;
3. screening antibodies aiming at GD2 nano-particles;
4. detecting single positive clone by immunoblotting;
5. positive clones were further verified with phase Elisa;
6. and (3) expressing and purifying the GD2 nano antibody in a eukaryotic expression system.
In one embodiment, in step 1, ganglioside GD2 is coupled to KLH molecule using alkynyl-PEG 4-NHS.
Has the advantages that:
compared with the prior art, the invention has the following advantages: the GD2-KLH is coupled firstly, GD2 molecules are enabled to have immunogenicity, GD2-KLH molecules are coupled on an immune tube or GD2-KLH biotin labels are coupled on streptavidin magnetic beads, correct spatial structures of GD2 molecules are displayed, immune nano antibody gene libraries (camel heavy chain antibody phage display gene libraries) are screened by using the antigen in the form through a phage display technology, so that nano antibody genes with GD2 molecule specificity are obtained, the nano antibody genes are transferred into eukaryotic expression cells HEK293F, and therefore the nano antibody strain capable of being efficiently expressed in HEK293F is established.
Drawings
FIG. 1 shows a coupling route for GD2-PEG4-KLH in example 1.
FIG. 2 shows the results of the ion chromatography glycoform analysis in example 1. Wherein A is a KLH-Alkyne ion chromatographic analysis chart; b is KLH-GD2 ion chromatography diagram; c is GD2 ion chromatography diagram; d is a standard curve of the last peak (P3) when GD2 is a standard substance.
FIG. 3 is an Output phase immunoblot in example 3.
FIG. 4 is a graph of the binding activity of positive clones to KLH-GD2 molecules for Elisa tests at the level of phase in example 5.
FIG. 5 is a SDS-PAGE pattern of nanobody expression purification in example 6.
Detailed Description
The present invention is further described below by way of specific examples, but the present invention is not limited to the examples.
KLH (sigma, H7017), sodium phosphate (Hu test, 20040928), sodium chloride (Hu test, 10019308), polyvinylpyrrolidone (Wokay, XW252495411), sucrose (Hu test, 10021418), Alkyne-PEG4-NHS (Kaixin, 1393330-40-9), BTTAA (Abmole, M9021), GD2(OligoTech, GLY094), cyclophosphamide (Baxter Oncology GmbH), goat antibody M13(Abcam,24229)
Example 1: coupling of ganglioside GD2-KLH molecules (FIG. 1)
(1) 10mg/mL of carrier protein KLH is prepared, and the final buffer is 31mM sodium phosphate, 0.46M sodium chloride, 2% PVP polyvinylpyrrolidone, 41mM sucrose and pH 7.4; (2) taking 20mg of Alkyne-PEG4-NHS, and preparing 100mg/mL mother solution by DMSO; (3) taking 400ml of KLH mother liquor, and adjusting the pH to 8.0 by using 1N NaOH; (4) adding 40ml of Alkyne mother liquor into the solution, and incubating for 4 hours at room temperature; (5) ultrafiltering, concentrating, replacing with 50mM sodium phosphate buffer solution with pH of 8.0, and concentrating to obtain Alkyne-PEG 4-KLH; (6) synthesizing Az-fluorescein isothiocyanate (Az-FITC), and carrying out fluorescence analysis on Alkyne-KLH through the Az-FITC to confirm that the KLH carries alkynyl groups; (7) under ligand BTTAA, click chemical reaction is carried out through Cu (I) catalysis, GD2-N3 content change in solution is analyzed through ion chromatography, and the reaction process is indirectly verified; (8) after the reaction is finished, performing ultrafiltration concentration and replacing a buffer solution system; (9) 200g of KLH-GD2, KLH-Alkyne and 50g of GD2 were taken, respectively, and an appropriate amount of water and TFA were added thereto, respectively, to give a final volume of 600L and a final concentration of 2M TFA. Each system was heated at 100 ℃ for 3 hours and then lyophilized by centrifugation. Subsequently, 60L of ddH were added each2The O is redissolved. And an appropriate amount was taken for ion chromatography glycoform analysis (fig. 2). (10) Calculation was performed from the above standard curve: the GD2 content in KLH-GD2(22g) was: (118.347-46.35-3.9195)/30.434 ═ 2.2 g. The percentage is: 10 percent.
Example 2: construction of Nanobody library against GD2 molecule
(1) 1mg of GD2-KLH prepared in example 1 was mixed with an equal volume of Freund's adjuvant to immunize a dromedary camel 7 times, and the animals were given cyclophosphamide (Table 1) by intravenous injection in an amount of 15. mu.g/kg three days before the first immunization; (2) after 7 times of immunization, 200mL of camel peripheral blood lymphocytes are extracted, and total RNA is extracted; (3) obtaining cDNA through reverse transcription and amplifying VHH fragments through nested PCR; (4) annealing the VHH single-stranded DNA into a phage vector by using a kunkel reaction; (5) the kunkel product is transformed into an electrotransformation competent DH10B, a GD2 nanobody library is constructed and the library volume is determined, the size of the library volume is 1.1 × 10e 9.
[ Table 1]
Camel immune arrangement table
| Number of immunizations | Time of immunization | Adjuvant type | Immunization dose (mg) | |
| 0 | Negative three days | Cyclophosphamide 7.5 | |
|
| 1 | Day one | Complete Freund's adjuvant | 1 | Subcutaneous injection |
| 2-7 | Once a week | Incomplete Freund's adjuvant | 1 | Subcutaneous injection |
Example 3: screening process for GD2 nano-antibody
(1) Liquid phase elutriation: 1) blocking 10e12 pfu input phase with a casein blocking buffer solution with a final concentration of 0.5%, and incubating for 1h at room temperature; 2) simultaneously, 3 multiplied by 100 mu l of streptavidin magnetic beads are washed three times by sterile PBS, the supernatant is discarded, and the mixture is sealed by 500 mu l of 1% casein sealing buffer solution for 1h at room temperature; 3) discarding the sealing liquid in the step 2), adding 1mL of sealed phase into the magnetic beads, fully and uniformly mixing, and rotating and uniformly mixing for 15min (negative screening); 4) taking the supernatant to a new sterile EP tube, adding KLH-biotn antigen with the final concentration of 50 mu g/mL, and rotating, uniformly mixing and combining for 2h at room temperature; 5) discarding the confining liquid in the step 2), transferring 1mL of phase-KLH-biotin mixed liquid into magnetic beads, fully and uniformly mixing, and rotating and uniformly mixing for 15 min; (negative selection); 6) taking the supernatant to a new sterile EP tube, adding GD2-KLH-biotn antigen with the final concentration of 100 mu g/mL, rotating and uniformly mixing at room temperature for 2 hours; 7) simultaneously taking 100 mu l of streptavidin magnetic beads, washing the streptavidin magnetic beads with sterile PBS for three times, discarding supernatant, and sealing the streptavidin magnetic beads with 500 mu l of 1% casein sealing buffer solution at room temperature for 1 h; 8) discarding the confining liquid in the step 2), adding 1mL of phase-GD 2-KLH-biotin mixture into the magnetic beads, fully and uniformly mixing, and rotating, uniformly mixing and combining for 15 min; 9) discarding the supernatant, washing with 1 × PBS for 10 times, and collecting magnetic beads; 10) adding 400 mu l of freshly prepared elution buffer 100mM Triethylamine into the magnetic beads in the step 9), and performing rotary elution for 10 min; 11) transferring 400. mu.l of the supernatant to a sterile EP tube, and adding 200. mu.l of PH6.4 Tris-HCl for neutralization, which is the output phase obtained by screening;
(2) solid phase elutriation: 1) coating the immune tube with 1mL of GD2-KLH antigen solution with the final concentration of 50 mu g/mL KLH/100 mu g/mL, and incubating overnight at 4 ℃; 2) on the next day, the uncoated immune tubes were washed with 1 × PBS for 3 times, spin-dried, and 4mL of 1% casein blocking buffer was added and spin-blocked at room temperature for 1 h; 3) blocking 5 × 10e12 pfu first round input phase with 1% final concentration of BSA, supplementing to 1mL final volume with 1 × PBS, spin blocking at room temperature for 1 h; 4) discarding sealing liquid in the immune tube without being coated with the antigen, putting the sealed phase into the immune tube without being coated with the antigen, and rotationally combining for 2 hours; (negative selection); 5) meanwhile, discarding the coating solution in the immune tube coated with KLH, washing with 1 XPBS for 3 times, spin-drying, adding 4mL of sealing buffer solution, and rotationally sealing at room temperature for 1 h; 6) removing a sealing liquid in an immune tube of KLH, transferring the negative screened phase into the immune tube, and carrying out rotary combination for 2 h; 7) meanwhile, discarding the coating solution in the GD 2-KLH-coated immune tube, washing with 1 XPBS for 3 times, spin-drying, adding 4mL of sealing buffer solution, and rotating and sealing at room temperature for 1 h; 8) discarding a sealing solution in an immune tube of GD2-KLH, transferring the negative screened phase into the immune tube, and carrying out rotary combination for 2 h; 9) discarding the supernatant, washing the immunotube with 1 XPBST for 10 times, and spin-drying; 10) adding 1ml of freshly prepared elution buffer solution 100mM Triethylamine into an immune tube, and standing and incubating at room temperature for 10 min; 11) 1mL of the eluate was transferred to a sterile EP tube and neutralized with 500. mu.l of pH6.4 Tris-HCl, which was the output phase obtained by the screening.
Example 4: detection of a single positive clone by immunoblotting (Mccafferty J, Griffiths A D, Winter G, et al. phase antibodies: filtration phase displaying antibodies variable domains [ J ]. Nature,1990, 348(6301):552-554.Huse W, Sastry L, Iverson S, et al. Generation of a large combinatorial library of the immunoglobulin recombinant in phase lambda [ J ]. Biotechnology,1989, 246(4935): 1275. 1281)
(1) Laying 2000pfu output phase each LB plate, culturing at 37 deg.C for 6-8 h; (2) coating a whatman 0.45 mu M NC membrane with 5mL of anti-M13 antibody of 2 mu g/mL, and incubating for 2.5h at room temperature; (3) discarding the coating solution, sealing the NC membrane by using 5mL of 1% casein blocking buffer solution, and incubating for 1h at room temperature; (4) discarding the blocking solution, washing with 1 × PBS for 3 times, and air drying; (5) pasting an NC film on an LB flat plate with a phase, punching holes for positioning, and culturing at room temperature overnight; (6) the membrane is uncovered on the next day and placed in a new culture dish, 1 XPBS is used for washing for 3 times, 50 mu g/mL GD2-KLH-biotin is added for incubation for 1h at room temperature; (7) washing 0.1% PBST for 8 times, adding Neutravidin-AP diluted at a ratio of 1:1000, and incubating at room temperature for 30 min; (8) washing 0.1% PBST for 8 times, adding 10ml AP chromogenic substrate (100 μ l BCIP +100 μ l NBT), and developing at room temperature for 10-30 min; (9) selecting positive clones, performing plaque PCR, and sequencing; (10) the sequences were aligned by DNAman sequence alignment software and clones with the same CDR1, CDR2, CDR3 were considered to be identical clones.
Example 5: positive clones were further verified by phase Elisa
(1) Amplification of phase: 1:100 inoculating XL1blue E.coli in LB liquid medium (containing 10. mu.g/mL tetracycline) to OD60 ═ 0.6; subpackaging Xl1blue into a 96-hole deep-hole plate, adding 600 mu l/hole, and adding 15 mu l of picked phase solution; amplifying overnight, centrifuging at 4000rpm for 30min, and taking the supernatant to be used as phase Elisa;
(2) elisa detection: 1) 50 μ g/mL GD2-KLH antigen was diluted to 100nM with 1 XPBS, coated with 50 μ l per well of ELISA plate, overnight at 4 ℃; 2) discarding the coating solution, spin-drying the ELISA plate, and washing with 1 × PBS for 3 times; 3) adding 200 mul of 1% casein blocking buffer solution into each hole, and blocking for 1h at room temperature; 4) discarding the confining liquid, adding 50 mu l of high-titer phage into each hole, and incubating for 2h at room temperature; 5) washing with 0.1% PBST for 6 times, and drying; 6) goat anti-M13-HRP antibody was added at a 1:5000 dilution, 50. mu.l/well and incubated at room temperature for 1 h. Washing with 0.1% PBST for 6 times, and drying; 7) 50 μ l of TMB developing solution was added to each well, and color development was performed at room temperature for 5 min. 50 μ l of 2M H was added2SO4Stopping the reaction, and measuring the OD450 value; 8) Positive was observed when the OD of the sample well was 3 times greater than the OD of the control well.
Example 6: expression and purification of nano antibody in eukaryotic expression system
(1) Cloning VHH fragments of different clone strains obtained by the previous sequencing analysis into a PINfuse eukaryotic expression vector;
(2) extracting plasmids after the sequencing is correct; (3) HEK293F suspension cells were cultured in Freestyle 293 expression medium with density up to 1 × 10e6/ml, and were used for transfection when the survival rate was > 90%; (4) the mass ratio of PEI to plasmid is 3:1, and the transfection is carried out according to the proportion of 1 mug plasmid to 1ml HEK293F cell; (5) after 5 to 6 days after transfection, cell supernatants were collected, purified with protein A strain to obtain high-purity antibody protein, and the elution buffer was replaced with PBS using an ultrafiltration column.
By the method, the invention obtains 9 GD2 nano antibodies: t25; t30; t53; t63; t69; t101; t104; t308; and T334.
T25(SEQ ID NO:8):
(DVQLQESGGGLVQPGGSLRLSCAASGFTFRNYIMSWVRQAPGKGLERVSVINSGGG STYYADSVKGRFTISRDNAKNTLYLRLNSLKTEDTAMYYCALGDARSGGRAIFRGQGT QVTVSS)
FR1 shown in SEQ ID NO. 1:
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg
(DVQLQESGGGLVQPGGSLRLSCAASGFTFR)
FR2 shown in SEQ ID NO. 2:
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Arg Val Ser
(WVRQAPGKGLERVS)
FR3 shown in SEQ ID NO. 3:
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Arg Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala MET Tyr Tyr Cys Ala Leu
(RFTISRDNAKNTLYLRLNSLKTEDTAMYYCAL)
FR4 shown in SEQ ID NO. 4:
Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
(RGQGTQVTVSS)
CDR1 shown in SEQ ID NO. 5:
Asn Tyr Ile MET Ser
(NYIMS)
CDR2 shown in SEQ ID NO. 6:
Val Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly
(VINSGGGSTYYADSVKG)
CDR3 shown in SEQ ID NO. 7:
Gly Asp Ala Arg Ser Gly Gly Arg Ala Ile Phe
(GDARSGGRAIF)
T30(SEQ ID NO:14):
(DVQLQESGGGLVQPGGSLRLSCAASGFTFRRYDMSWVRQTPGKGLEWVSAINSVG GSTYYADSVKGRFTISRDNAKNTLYLQMNSLQTEDTGVYYCATDRPGSWYYRPNPRG QGTQVTVSS)
FR1 shown in SEQ ID NO. 1:
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg
(DVQLQESGGGLVQPGGSLRLSCAASGFTFR)
FR2 shown in SEQ ID NO: 9:
Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val Ser
(WVRQTPGKGLEWVS)
FR3 shown in SEQ ID NO. 10:
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln MET Asn Ser Leu Gln Thr Glu Asp Thr Gly Val Tyr Tyr Cys Ala Thr
(RFTISRDNAKNTLYLQMNSLQTEDTGVYYCAT)
FR4 shown in SEQ ID NO. 4:
Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
(RGQGTQVTVSS)
11, CDR1 shown in SEQ ID NO:
Arg Tyr Asp MET Ser
(RYDMS)
CDR2 shown in SEQ ID NO. 12:
Ala Ile Asn Ser Val Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly
(AINSVGGSTYYADSVKG)
13 CDR3 shown in SEQ ID NO:
Asp Arg Pro Gly Ser Trp Tyr Tyr Arg Pro Asn Pro
(DRPGSWYYRPNP)
T53(SEQ ID NO:22):
(DVQLQESGGGSVQAGGSLRLSCVGSASRNQMGWFRQAPGKEREEVAVIGLYGRTK YADSVKGRFTISKDNASKTLYLQMNSLKPEDTAMYYCATPRSAYGTIYAPRYDYWGQ GTQVTVSS)
FR1 shown in SEQ ID NO. 15:
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Val Gly Ser Ala Ser Arg
(DVQLQESGGGSVQAGGSLRLSCVGSASR)
FR2 shown in SEQ ID NO: 16:
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Glu Val Ala
(WFRQAPGKEREEVA)
FR3 shown in SEQ ID NO: 17:
Arg Phe Thr Ile Ser Lys Asp Asn Ala Ser Lys Thr Leu Tyr Leu Gln MET Asn Ser Leu Lys Pro Glu Asp Thr Ala MET Tyr Tyr Cys Ala Thr
(RFTISKDNASKTLYLQMNSLKPEDTAMYYCAT)
FR4 shown in SEQ ID NO: 18:
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
(WGQGTQVTVSS)
19 CDR1 shown in SEQ ID NO:
Asn Gln MET Gly
(NQMG)
20, CDR2 shown in SEQ ID NO:
Val Ile Gly Leu Tyr Gly Arg Thr Lys Tyr Ala Asp Ser Val Lys Gly
(VIGLYGRTKYADSVKG)
21, CDR3 shown in SEQ ID NO:
Pro Arg Ser Ala Tyr Gly Thr Ile Tyr Ala Pro Arg Tyr Asp Tyr
(PRSAYGTIYAPRYDY)
T63(SEQ ID NO:30):
(DVQLQESGGGLVQPGGSLRLSCAASGFTFSRYYMSWVRQAPGKGLEWVSGITSSDI RTYYADSVKDRFTISRDNAKNTLYLQLNSLKTEDTAMYYCAKDYNGSWSPRSQGTQV TVSS)
FR1 shown in SEQ ID NO: 23:
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
(DVQLQESGGGLVQPGGSLRLSCAASGFTFS)
FR2 shown in SEQ ID NO: 24:
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
(WVRQAPGKGLEWVS)
FR3 shown in SEQ ID NO: 25:
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala MET Tyr Tyr Cys Ala Lys
(RFTISRDNAKNTLYLQLNSLKTEDTAMYYCAK)
FR4 shown in SEQ ID NO: 26:
Gln Gly Thr Gln Val Thr Val Ser Ser
(QGTQVTVSS)
CDR1 shown in SEQ ID NO. 27:
Arg Tyr Tyr MET Ser
(RYYMS)
28 of CDR2 shown in SEQ ID NO:
Gly Ile Thr Ser Ser Asp Ile Arg Thr Tyr Tyr Ala Asp Ser Val Lys Asp
(GITSSDIRTYYADSVKD)
CDR3 shown in SEQ ID NO. 29:
Asp Tyr Asn Gly Ser Trp Ser Pro Arg Ser
(DYNGSWSPRS)
T69(SEQ ID NO:36):
(DVQLQESGGGLVQPGGSLRLSCAASGFPFSSFTMYWVRQAPGKGLEWVSAINSGGG TTYYSDSAKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVRTNPRNGLRGQGTQVT VSS)
FR1 shown in SEQ ID NO: 31:
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser
(DVQLQESGGGLVQPGGSLRLSCAASGFPFS)
FR2 shown in SEQ ID NO: 24:
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
(WVRQAPGKGLEWVS)
FR3 shown in SEQ ID NO: 32:
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln MET Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg
(RFTISRDNAKNTVYLQMNSLKPEDTAVYYCVR)
FR4 shown in SEQ ID NO. 4:
Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
(RGQGTQVTVSS)
33, CDR1 shown in SEQ ID NO:
Ser Phe Thr MET Tyr
(SFTMY)
CDR2 shown in SEQ ID NO: 34:
Ala Ile Asn Ser Gly Gly Gly Thr Thr Tyr Tyr Ser Asp Ser Ala Lys Gly
(AINSGGGTTYYSDSAKG)
35 of the CDR3 shown in SEQ ID NO:
Thr Asn Pro Arg Asn Gly Leu
(TNPRNGL)
T101(SEQ ID NO:43):
(DVQLQESGGGSVQTGGSLRLSCKASAYTYYSYCMGWIRQAPGKEREGVAGIDRDGS TSYTDSVKGRFTISKDNAKRTLYLQMDSLKPEDTALYYCAAETRPGYCNTDYRRAPSY NYRGQGTQVTVSS)
FR1 shown in SEQ ID NO: 37:
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu Arg Leu Ser Cys Lys Ala Ser Ala Tyr Thr Tyr Tyr
(DVQLQESGGGSVQTGGSLRLSCKASAYTYY)
FR2 shown in SEQ ID NO: 38:
Trp Ile Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
(WIRQAPGKEREGV)
FR3 shown in SEQ ID NO: 39:
Phe Thr Ile Ser Lys Asp Asn Ala Lys Arg Thr Leu Tyr Leu Gln MET Asp Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ala
(FTISKDNAKRTLYLQMDSLKPEDTALYYCAA)
FR4 shown in SEQ ID NO. 4:
Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
(RGQGTQVTVSS)
40 of CDR1 shown in SEQ ID NO:
Ser Tyr Cys MET Gly
(SYCMG)
41 CDR2 shown in SEQ ID NO:
Ala Gly Ile Asp Arg Asp Gly Ser Thr Ser Tyr Thr Asp Ser Val Lys Gly Arg
(AGIDRDGSTSYTDSVKGR)
42, CDR3 shown in SEQ ID NO:
Glu Thr Arg Pro Gly Tyr Cys Asn Thr Asp Tyr Arg Arg Ala Pro Ser Tyr Asn Tyr
(ETRPGYCNTDYRRAPSYNY)
T104(SEQ ID NO:50):
(DVQLQESGGGSVQAGGSLRLSCVASGSIYSNRCAGWFRQVPGKAREGVAAIYTGGA STYYADSVKGRFTISQDNAKNTLYLQMDSLNPEDTAIYYCAASRPRYGFTSCGLSQISY THWGQGTQVTVSS)
FR1 shown in SEQ ID NO: 44:
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Ile Tyr Ser
(DVQLQESGGGSVQAGGSLRLSCVASGSIYS)
FR2 shown in SEQ ID NO: 45:
Trp Phe Arg Gln Val Pro Gly Lys Ala Arg Glu Gly Val Ala
(WFRQVPGKAREGVA)
FR3 shown in SEQ ID NO: 46:
Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln MET Asp Ser Leu Asn Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala
(RFTISQDNAKNTLYLQMDSLNPEDTAIYYCAA)
FR4 shown in SEQ ID NO: 18:
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
(WGQGTQVTVSS)
47 CDR1 shown in SEQ ID NO:
Asn Arg Cys Ala Gly
(NRCAG)
48, CDR2 shown in SEQ ID NO:
Ala Ile Tyr Thr Gly Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly
(AIYTGGASTYYADSVKG)
CDR3 shown in SEQ ID NO. 49:
Ser Arg Pro Arg Tyr Gly Phe Thr Ser Cys Gly Leu Ser Gln Ile Ser Tyr Thr His
(SRPRYGFTSCGLSQISYTH)
T308(SEQ ID NO:56):
(DVQLQESGGGSVQAGGSLRLSCAASGYTYSSYCMGWFRQAPGKEREGVATIDIDGS TTYADSVKGRFTISKDNAKNTLYLQMNSLKPEDSAMYYCAADRVWGSCRRTSGNFGY WGQGTQVTVSS)
FR1 shown in SEQ ID NO: 51:
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser
(DVQLQESGGGSVQAGGSLRLSCAASGYTYS)
FR2 shown in SEQ ID NO: 52:
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
(WFRQAPGKEREGV)
FR3 shown in SEQ ID NO: 53:
Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln MET Asn Ser Leu Lys Pro Glu Asp Ser Ala MET Tyr Tyr Cys Ala Ala
(RFTISKDNAKNTLYLQMNSLKPEDSAMYYCAA)
FR4 shown in SEQ ID NO: 18:
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
(WGQGTQVTVSS)
40 of CDR1 shown in SEQ ID NO:
Ser Tyr Cys MET Gly
(SYCMG)
54, CDR2 shown in SEQ ID NO:
Ala Thr Ile Asp Ile Asp Gly Ser Thr Thr Tyr Ala Asp Ser Val Lys Gly
(ATIDIDGSTTYADSVKG)
CDR3 shown in SEQ ID NO: 55:
Asp Arg Val Trp Gly Ser Cys Arg Arg Thr Ser Gly Asn Phe Gly Tyr
(DRVWGSCRRTSGNFGY)
T334(SEQ ID NO:64):
(DVQLQESGGGLVQPGGSLRLSCAASGFSFSSYPMSWVRQPPGKELEWVSGIRSDGG NTHYADSVKGRFTISRDNAKNTLYLQLNSLKTEDTAMYYCTKGYNRPTTQLGGGQGT QVTVSS)
FR1 shown in SEQ ID NO: 57:
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser
(DVQLQESGGGLVQPGGSLRLSCAASGFSFS)
FR2 shown in SEQ ID NO: 58:
Trp Val Arg Gln Pro Pro Gly Lys Glu Leu Glu Trp Val
(WVRQPPGKELEWV)
FR3 shown in SEQ ID NO: 59:
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala MET Tyr Tyr Cys Thr Lys
(RFTISRDNAKNTLYLQLNSLKTEDTAMYYCTK)
FR4 shown in SEQ ID NO: 60:
Gly Gly Gln Gly Thr Gln Val Thr Val Ser Ser
(GGQGTQVTVSS)
61 and CDR1 shown in SEQ ID NO:
Ser Tyr Pro MET Ser
(SYPMS)
CDR2 shown in SEQ ID NO: 62:
Ser Gly Ile Arg Ser Asp Gly Gly Asn Thr His Tyr Ala Asp Ser Val Lys Gly
(SGIRSDGGNTHYADSVKG)
63, CDR3 shown in SEQ ID NO:
Gly Tyr Asn Arg Pro Thr Thr Gln Leu Gly
(GYNRPTTQLG)。
SEQUENCE LISTING
<110> Dashi pharmaceutical industry (Guangdong) Co., Ltd
Shanghai Maishi Biotechnology Co.,Ltd.
Shenzhen Chuangshi biomedical Co.,Ltd.
<120> GD2 nano antibody and application thereof
<130> DI19-1475-XC37
<160> 64
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> FR1
<400> 1
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg
20 25 30
<210> 2
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> FR2
<400> 2
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Arg Val Ser
1 5 10
<210> 3
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> FR3
<400> 3
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Arg
1 5 10 15
Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Ala Leu
20 25 30
<210> 4
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> FR4
<400> 4
Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 5
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> CDR1
<400> 5
Asn Tyr Ile Met Ser
1 5
<210> 6
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> CDR2
<400> 6
Val Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 7
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> CDR3
<400> 7
Gly Asp Ala Arg Ser Gly Gly Arg Ala Ile Phe
1 5 10
<210> 8
<211> 120
<212> PRT
<213> Artificial sequence
<220>
<223> T25
<400> 8
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Tyr
20 25 30
Ile Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Arg Val
35 40 45
Ser Val Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Arg Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Leu Gly Asp Ala Arg Ser Gly Gly Arg Ala Ile Phe Arg Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 9
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> FR2
<400> 9
Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val Ser
1 5 10
<210> 10
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> FR3
<400> 10
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Gln Thr Glu Asp Thr Gly Val Tyr Tyr Cys Ala Thr
20 25 30
<210> 11
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> CDR1
<400> 11
Arg Tyr Asp Met Ser
1 5
<210> 12
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> CDR2
<400> 12
Ala Ile Asn Ser Val Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 13
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> CDR3
<400> 13
Asp Arg Pro Gly Ser Trp Tyr Tyr Arg Pro Asn Pro
1 5 10
<210> 14
<211> 121
<212> PRT
<213> Artificial sequence
<220>
<223> T30
<400> 14
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr
20 25 30
Asp Met Ser Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Asn Ser Val Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Gln Thr Glu Asp Thr Gly Val Tyr Tyr Cys
85 90 95
Ala Thr Asp Arg Pro Gly Ser Trp Tyr Tyr Arg Pro Asn Pro Arg Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 15
<211> 28
<212> PRT
<213> Artificial sequence
<220>
<223> FR1
<400> 15
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Gly Ser Ala Ser Arg
20 25
<210> 16
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> FR2
<400> 16
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Glu Val Ala
1 5 10
<210> 17
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> FR3
<400> 17
Arg Phe Thr Ile Ser Lys Asp Asn Ala Ser Lys Thr Leu Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Thr
20 25 30
<210> 18
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> FR4
<400> 18
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 19
<211> 4
<212> PRT
<213> Artificial sequence
<220>
<223> CDR1
<400> 19
Asn Gln Met Gly
1
<210> 20
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> CDR2
<400> 20
Val Ile Gly Leu Tyr Gly Arg Thr Lys Tyr Ala Asp Ser Val Lys Gly
1 5 10 15
<210> 21
<211> 15
<212> PRT
<213> Artificial sequence
<220>
<223> CDR3
<400> 21
Pro Arg Ser Ala Tyr Gly Thr Ile Tyr Ala Pro Arg Tyr Asp Tyr
1 5 10 15
<210> 22
<211> 120
<212> PRT
<213> Artificial sequence
<220>
<223> T53
<400> 22
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Gly Ser Ala Ser Arg Asn Gln Met Gly
20 25 30
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Glu Val Ala Val Ile
35 40 45
Gly Leu Tyr Gly Arg Thr Lys Tyr Ala Asp Ser Val Lys Gly Arg Phe
50 55 60
Thr Ile Ser Lys Asp Asn Ala Ser Lys Thr Leu Tyr Leu Gln Met Asn
65 70 75 80
Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Thr Pro Arg
85 90 95
Ser Ala Tyr Gly Thr Ile Tyr Ala Pro Arg Tyr Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 23
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> FR1
<400> 23
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
20 25 30
<210> 24
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> FR2
<400> 24
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
1 5 10
<210> 25
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> FR3
<400> 25
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln
1 5 10 15
Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Ala Lys
20 25 30
<210> 26
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> FR4
<400> 26
Gln Gly Thr Gln Val Thr Val Ser Ser
1 5
<210> 27
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> CDR1
<400> 27
Arg Tyr Tyr Met Ser
1 5
<210> 28
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> CDR2
<400> 28
Gly Ile Thr Ser Ser Asp Ile Arg Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Asp
<210> 29
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> CDR3
<400> 29
Asp Tyr Asn Gly Ser Trp Ser Pro Arg Ser
1 5 10
<210> 30
<211> 117
<212> PRT
<213> Artificial sequence
<220>
<223> T63
<400> 30
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Thr Ser Ser Asp Ile Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Lys Asp Tyr Asn Gly Ser Trp Ser Pro Arg Ser Gln Gly Thr Gln
100 105 110
Val Thr Val Ser Ser
115
<210> 31
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> FR1
<400> 31
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser
20 25 30
<210> 32
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> FR3
<400> 32
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg
20 25 30
<210> 33
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> CDR1
<400> 33
Ser Phe Thr Met Tyr
1 5
<210> 34
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> CDR2
<400> 34
Ala Ile Asn Ser Gly Gly Gly Thr Thr Tyr Tyr Ser Asp Ser Ala Lys
1 5 10 15
Gly
<210> 35
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> CDR3
<400> 35
Thr Asn Pro Arg Asn Gly Leu
1 5
<210> 36
<211> 116
<212> PRT
<213> Artificial sequence
<220>
<223> T69
<400> 36
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Ser Phe
20 25 30
Thr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Asn Ser Gly Gly Gly Thr Thr Tyr Tyr Ser Asp Ser Ala
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Arg Thr Asn Pro Arg Asn Gly Leu Arg Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser
115
<210> 37
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> FR1
<400> 37
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Lys Ala Ser Ala Tyr Thr Tyr Tyr
20 25 30
<210> 38
<211> 13
<212> PRT
<213> Artificial sequence
<220>
<223> FR2
<400> 38
Trp Ile Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
1 5 10
<210> 39
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> FR3
<400> 39
Phe Thr Ile Ser Lys Asp Asn Ala Lys Arg Thr Leu Tyr Leu Gln Met
1 5 10 15
Asp Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ala
20 25 30
<210> 40
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> CDR1
<400> 40
Ser Tyr Cys Met Gly
1 5
<210> 41
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> CDR2
<400> 41
Ala Gly Ile Asp Arg Asp Gly Ser Thr Ser Tyr Thr Asp Ser Val Lys
1 5 10 15
Gly Arg
<210> 42
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> CDR3
<400> 42
Glu Thr Arg Pro Gly Tyr Cys Asn Thr Asp Tyr Arg Arg Ala Pro Ser
1 5 10 15
Tyr Asn Tyr
<210> 43
<211> 127
<212> PRT
<213> Artificial sequence
<220>
<223> T101
<400> 43
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Lys Ala Ser Ala Tyr Thr Tyr Tyr Ser Tyr
20 25 30
Cys Met Gly Trp Ile Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Asp Arg Asp Gly Ser Thr Ser Tyr Thr Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Arg Thr Leu Tyr Leu
65 70 75 80
Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala
85 90 95
Ala Glu Thr Arg Pro Gly Tyr Cys Asn Thr Asp Tyr Arg Arg Ala Pro
100 105 110
Ser Tyr Asn Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 44
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> FR1
<400> 44
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Ile Tyr Ser
20 25 30
<210> 45
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> FR2
<400> 45
Trp Phe Arg Gln Val Pro Gly Lys Ala Arg Glu Gly Val Ala
1 5 10
<210> 46
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> FR3
<400> 46
Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln
1 5 10 15
Met Asp Ser Leu Asn Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala
20 25 30
<210> 47
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> CDR1
<400> 47
Asn Arg Cys Ala Gly
1 5
<210> 48
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> CDR2
<400> 48
Ala Ile Tyr Thr Gly Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 49
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> CDR3
<400> 49
Ser Arg Pro Arg Tyr Gly Phe Thr Ser Cys Gly Leu Ser Gln Ile Ser
1 5 10 15
Tyr Thr His
<210> 50
<211> 128
<212> PRT
<213> Artificial sequence
<220>
<223> T104
<400> 50
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Ile Tyr Ser Asn Arg
20 25 30
Cys Ala Gly Trp Phe Arg Gln Val Pro Gly Lys Ala Arg Glu Gly Val
35 40 45
Ala Ala Ile Tyr Thr Gly Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Asn Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Ala Ser Arg Pro Arg Tyr Gly Phe Thr Ser Cys Gly Leu Ser Gln
100 105 110
Ile Ser Tyr Thr His Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 51
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> FR1
<400> 51
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser
20 25 30
<210> 52
<211> 13
<212> PRT
<213> Artificial sequence
<220>
<223> FR2
<400> 52
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
1 5 10
<210> 53
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> FR3
<400> 53
Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr Cys Ala Ala
20 25 30
<210> 54
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> CDR2
<400> 54
Ala Thr Ile Asp Ile Asp Gly Ser Thr Thr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 55
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> CDR3
<400> 55
Asp Arg Val Trp Gly Ser Cys Arg Arg Thr Ser Gly Asn Phe Gly Tyr
1 5 10 15
<210> 56
<211> 124
<212> PRT
<213> Artificial sequence
<220>
<223> T308
<400> 56
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Thr Ile Asp Ile Asp Gly Ser Thr Thr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Asp Arg Val Trp Gly Ser Cys Arg Arg Thr Ser Gly Asn Phe Gly
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 57
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> FR1
<400> 57
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser
20 25 30
<210> 58
<211> 13
<212> PRT
<213> Artificial sequence
<220>
<223> FR2
<400> 58
Trp Val Arg Gln Pro Pro Gly Lys Glu Leu Glu Trp Val
1 5 10
<210> 59
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> FR3
<400> 59
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln
1 5 10 15
Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Thr Lys
20 25 30
<210> 60
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> FR4
<400> 60
Gly Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 61
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> CDR1
<400> 61
Ser Tyr Pro Met Ser
1 5
<210> 62
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> CDR2
<400> 62
Ser Gly Ile Arg Ser Asp Gly Gly Asn Thr His Tyr Ala Asp Ser Val
1 5 10 15
Lys Gly
<210> 63
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> CDR3
<400> 63
Gly Tyr Asn Arg Pro Thr Thr Gln Leu Gly
1 5 10
<210> 64
<211> 119
<212> PRT
<213> Artificial sequence
<220>
<223> T334
<400> 64
Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Ser Tyr
20 25 30
Pro Met Ser Trp Val Arg Gln Pro Pro Gly Lys Glu Leu Glu Trp Val
35 40 45
Ser Gly Ile Arg Ser Asp Gly Gly Asn Thr His Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Thr Lys Gly Tyr Asn Arg Pro Thr Thr Gln Leu Gly Gly Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
Claims (7)
1. A GD2 nanobody, which is a nanobody directed to an epitope of the ganglioside GD2 molecule, comprising a Framework Region (FR) and a Complementarity Determining Region (CDR), wherein said Complementarity Determining Region (CDR) comprises complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), complementarity determining region 3(CDR3),
wherein the complementarity determining region 1(CDR1) is shown in SEQ ID NO:5, the complementarity determining region 2(CDR2) is shown in SEQ ID NO:6, and the complementarity determining region 3(CDR3) is shown in SEQ ID NO: 7; or
Wherein the complementarity determining region 1(CDR1) is shown in SEQ ID NO:11, the complementarity determining region 2(CDR2) is shown in SEQ ID NO:12, and the complementarity determining region 3(CDR3) is shown in SEQ ID NO: 13; or
Wherein the complementarity determining region 1(CDR1) is shown in SEQ ID NO:19, the complementarity determining region 2(CDR2) is shown in SEQ ID NO:20, and the complementarity determining region 3(CDR3) is shown in SEQ ID NO: 21; or
Wherein the complementarity determining region 1(CDR1) is shown in SEQ ID NO:27, the complementarity determining region 2(CDR2) is shown in SEQ ID NO:28, and the complementarity determining region 3(CDR3) is shown in SEQ ID NO: 29; or
Wherein the complementarity determining region 1(CDR1) is shown in SEQ ID NO:33, the complementarity determining region 2(CDR2) is shown in SEQ ID NO:34, and the complementarity determining region 3(CDR3) is shown in SEQ ID NO: 35; or
Wherein the complementarity determining region 1(CDR1) is shown in SEQ ID NO:40, the complementarity determining region 2(CDR2) is shown in SEQ ID NO:41, and the complementarity determining region 3(CDR3) is shown in SEQ ID NO: 42; or
Wherein the complementarity determining region 1(CDR1) is shown in SEQ ID NO:47, the complementarity determining region 2(CDR2) is shown in SEQ ID NO:48, and the complementarity determining region 3(CDR3) is shown in SEQ ID NO: 49; or
Wherein the complementarity determining region 1(CDR1) is shown in SEQ ID NO:40, the complementarity determining region 2(CDR2) is shown in SEQ ID NO:54, and the complementarity determining region 3(CDR3) is shown in SEQ ID NO: 55; or
Wherein the complementarity determining region 1(CDR1) is shown in SEQ ID NO:61, the complementarity determining region 2(CDR2) is shown in SEQ ID NO:62, and the complementarity determining region 3(CDR3) is shown in SEQ ID NO: 63.
2. The GD2 nanobody according to claim 1, comprising sequentially a framework region 1(FR1), a complementarity determining region 1(CDR1), a framework region 2(FR2), a complementarity determining region 2(CDR2), a framework region 3(FR3), a complementarity determining region 3(CDR3), and a framework region 4(FR4),
wherein said framework region 1(FR1) is selected from the group consisting of SEQ ID NO 1, SEQ ID NO 15, SEQ ID NO 23, SEQ ID NO 31, SEQ ID NO 37, SEQ ID NO 44, SEQ ID NO 51 and SEQ ID NO 57;
the framework region 2(FR2) is selected from the group consisting of SEQ ID NO 2, SEQ ID NO 9, SEQ ID NO 16, SEQ ID NO 24, SEQ ID NO 38, SEQ ID NO 45, SEQ ID NO 52 and SEQ ID NO 58;
the framework region 3(FR3) is selected from the group consisting of SEQ ID NO 3, SEQ ID NO 10, SEQ ID NO 17, SEQ ID NO 25, SEQ ID NO 32, SEQ ID NO 39, SEQ ID NO 46, SEQ ID NO 53 and SEQ ID NO 59; and
the framework region 4(FR4) is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26 and SEQ ID NO: 60.
3. The GD2 nanobody according to claim 1, having an amino acid sequence selected from any one of the following: SEQ ID NO 8, SEQ ID NO 14, SEQ ID NO 22, SEQ ID NO 30, SEQ ID NO 36, SEQ ID NO 43, SEQ ID NO 50, SEQ ID NO 56 and SEQ ID NO 64.
4. A DNA molecule encoding the GD2 nanobody of any one of claims 1-3.
5. An expression vector comprising the DNA molecule of claim 4.
6. A host cell comprising the expression vector of claim 5.
7. Use of the GD2 nanobody of any one of claims 1-3 in the preparation of GD2 molecular detection and/or therapeutic agents.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910874671.3A CN110551218B (en) | 2019-09-17 | 2019-09-17 | GD2 nano antibody and application thereof |
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|---|---|
| CN110551218A (en) | 2019-12-10 |
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