Anti-CD276 nano antibody and application thereof

Abstract

The invention relates to an anti-CD276 nano antibody. The anti-CD276 nano antibody comprises a complementary determining region (CDR) and a framework region (FR), wherein the complementarity determining region CDR comprises complementarity determining regions CDR1-CDR3, and the complementarity determining regions CDR1-CDR3 are And the framework region FR comprises framework regions FR1-FR5. The anti-human CD276 nano antibody provided by the invention has the characteristic of immunoreaction with a CD276 antigen, is good in specificity and high in affinity, can be applied to preparation of a CD276 detection reagent or an anti-tumor drug and the like, and can be used as an antigen recognition structural domain of a CAR so as to construct a CAR-T cell; the compound has a remarkable killing effect on a plurality of tumor cell lines expressing CD276 antigens; the anti-human CD276 nano antibody provided by the invention can be used for detecting the expression of CD276 in tumor tissues.

Description

Technical Field

[0001] The present invention relates to the field of biological genes, and in particular to an anti-CD276 nano antibody and an application thereof. Background Art

[0002] CD276 (Recombinant cluster of differentiation 276), also known as B7-H3, is a type I transmembrane glycoprotein with a molecular weight of 45-66kDa. It has a similar molecular structure to B7-H1 (PD-L1) and is an important immune checkpoint molecule of the B7-CD28 family. CD276 mainly exists in membrane protein and soluble form, of which the soluble form is cleaved by metalloproteinases from membrane proteins. In addition, CD276 protein has also been found in exosomes and other extracellular vesicles. CD276 may play a dual role of co-stimulation and co-inhibition in the immune system: on the one hand, it has a dual role in co-stimulation and co-inhibition of CD4 + and CD8 + T cell co-stimulatory effect. As a co-stimulatory molecule, CD276 signal induces cellular immunity, promotes the secretion of cytokines such as IFN-γ, IL-8, and TNF-α, and enhances CD8 + On the other hand, it can inhibit Treg cells, thereby allowing tumors to escape immune responses. Its mechanism may be related to three major signaling pathways of NFAT, NFκB and AP-1 factors that regulate gene transcription through T cell surface receptors. A large number of studies have confirmed that CD276 is widely expressed in various types of cancer, and its correlation with poor tumor prognosis has also been confirmed. At present, the clinical application research of CD276 antibodies mainly includes antibody-drug conjugates (ADC) for tumors, antibody-mediated cytotoxicity (ADCC) drugs, bispecific and trispecific antibodies, chimeric antigen receptor T cells (CAR-T) therapy, etc.

[0003] Heavy chain antibodies are a new type of antibody found in camelids in 1989 that lacks the constant domain of heavy chain 1 (CH1) of the light chain. Nano antibodies are cloned from the variable domain of heavy chain of heavy chain antibody (VHH), with a crystal diameter of only 2.5nm and a molecular weight of 12-15kDa, making them the smallest known antibodies. Compared with traditional antibodies, nano antibodies have the following advantages: 1. Nano antibodies have a longer third antigen complementary determining region, maintain antigen binding ability similar to that of normal double-chain antibodies, and can better bind to antigen epitopes. 2. Inter-ring disulfide bonds are common inside nano antibodies, which are more stable at high temperatures and higher concentrations of organic solvents. 3. The second framework region of nano antibodies contains four conservative hydrophilic amino acid mutations, which makes them hydrophilic and not easy to aggregate, affecting antigen recognition. 4. Nano antibodies are small in size and can better penetrate dense tumor tissues, and are used in the treatment of solid tumors. 5. Nanobodies only have heavy chain variable regions. When multiple nanobodies are used in combination, there will be no cross-reaction between the heavy chain and the light chain, which will affect the therapeutic effect of the antibody. 6. Nanobodies come from the camel family and are highly similar to human antibodies. They have low immunogenicity and will not produce neutralizing antibodies during treatment.

[0004] Most of the antibodies against CD276 reported on the market are single chain antibodies (scFv) composed of the variable domain of heavy chain of heavy chain antibody (VHH) and the variable domain of light chain. The size of scFv antibody is almost twice that of VHH antibody, and in scFv, four residues in FR2 form a hydrophobic interface. Although this hydrophobic region promotes the connection between VH-VL, it reduces the solubility of scFv, resulting in a high tendency to aggregate. Compared with VHH antibody, scFv antibody is not easy to dissolve, which may affect the recognition of antigen. Another major disadvantage of scFv is that most of them are derived from rodents, because hybridoma technology has only been well developed in mice and rats. The sequence homology of mouse VL and VH with the corresponding regions of humans is only 53% and 51%, respectively, while VHH antibody shows a high sequence similarity with human VH (VH3 gene family), about 75-90% homology, so VHH antibody is more humanized and less immunogenic. Even after humanization, the variable regions of mouse scFv antibodies can still induce anti-idiotypic responses, which is not conducive to immunotherapy.

[0005] In summary, it is urgent to develop a new technical solution to solve the problems existing in the prior art. Summary of the invention

[0006] Based on the advantages of nano-antibodies, the present invention successfully constructed an alpaca phage antibody library immunized with the extracellular segment protein of human CD276, and screened out two new VHH sequences of nano-antibodies against CD276. Our experiments found that the nano-antibody can be used for both cell flow cytometry and tissue immunohistochemistry to detect human CD276 antigen, so it has the application prospect of detecting CD276 antigen; at the same time, the nano-antibody sequence can be prepared into chimeric antigen receptor T cells (CAR-T), and can effectively kill CD276-positive tumor cells, so it has the prospect of being applied in immunotherapy targeting CD276-positive cells.

[0007] The term "chimeric antigen receptor" as used herein refers to an artificial receptor that has been genetically engineered to give immune cells (such as T lymphocytes) new abilities to target specific antigens. These receptors are chimeric because they combine the antigen-specific recognition domain of an antibody with the T cell activation domain into one receptor.

[0008] The term "heavy chain CDR" involved in the present invention, i.e., the complementarity determining region of the heavy chain, refers to the core conserved structure of the antibody heavy chain variable region, which is the key region that determines the antibody binding to the antigen, including CDR1, CDR2 and CDR3.

[0009] The term "framework region FR" involved in the present invention refers to the sequence excluding the CDR region in the VHH antibody.

[0010] One object of the present invention is to provide an anti-CD276 nanobody, wherein the anti-CD276 nanobody comprises a complementary determining region CDR and a framework region FR;

[0011] in,

[0012] The complementary determining region CDR includes complementary determining regions CDR1-CDR3;

[0013] The complementary determining regions CDR1-CDR3 are shown in amino acid sequences 1-3 respectively;

[0014] The amino acid sequences 1-3 are shown as SEQ ID No.1-SEQ ID No.3.

[0015] Further, the framework region FR includes framework regions FR1-FR5;

[0016] in,

[0017] The framework regions FR1-FR5 are shown in amino acid sequences 4-8 respectively;

[0018] The amino acid sequences 4-8 are shown as SEQ ID No.4-SEQ ID No.8.

[0019] Furthermore, the amino acid sequence of the anti-CD276 nanobody is CD276-VHH1 or CD276-VHH2; the CD276-VHH1 and CD276-VHH2 are shown in SEQ ID No.9-SEQ ID No.10.

[0020] Furthermore, the anti-CD276 nanobody is selected from: animal-derived antibodies, chimeric antibodies, humanized antibodies or a combination thereof.

[0021] Another object of the present invention is to provide a nucleic acid molecule encoding the above-mentioned anti-CD276 nanobody.

[0022] Furthermore, the nucleic acid molecule comprises the nucleic acid sequence SEQ ID No.11 or SEQ ID No.12.

[0023] Another object of the present invention is to provide a vector, which comprises the above-mentioned anti-CD276 nanobody.

[0024] Furthermore, the vector is selected from a DNA molecule, an mRNA molecule or a cell.

[0025] Another object of the present invention is to provide the use of the carrier in antigen detection, adsorption of antigen reagents and immunotherapy.

[0026] Furthermore, the antigen detection is CD276 antigen detection.

[0027] Furthermore, the immunotherapy is immunotherapy targeting CD276 positive cells.

[0028] Phage display technology is the most commonly used method for producing nano-antibodies. cDNA is obtained from natural, immune or synthetic antibody libraries through genetic engineering. The DNA is inserted into the appropriate position of the phage coat protein, and a fusion protein is formed with the coat protein and expressed on the phage surface. Then, proteins that can bind to the antigen are screened. Using immune antibody libraries to screen antibodies has high specificity and is easier to obtain high-affinity antibodies.

[0029] Therefore, the present invention is based on the alpaca phage antibody display library immunized with the extracellular segment of human CD276 protein constructed by the inventor's team to screen anti-CD276 nanoantibodies, which can be well applied to immunotherapy against CD276-positive tumor cells.

[0030] The present invention discovered two new anti-human CD276 nanoantibody sequences, which are completely different from other nanoantibody sequences and can be used for the recognition of CD276 antigen, the internalization of CD276-positive cells, the construction of CAR-T cells and the killing of CD276-positive cells.

[0031] The present invention has the following beneficial effects:

[0032] The present invention discloses an anti-CD276 nano antibody, which includes heavy chain CDR1-CDR3. The present invention first expresses and purifies the CD276 extracellular segment polypeptide and makes it immunogenic, then couples the CD276 extracellular segment polypeptide to an ELISA plate to display the correct spatial structure of the protein, and uses phage display technology to screen the CD276 extracellular segment immune nano antibody gene library (camel heavy chain antibody phage display gene library) using this form of antigen, thereby obtaining a CD276-specific nano antibody gene, and transferring this gene to Escherichia coli to establish a nano antibody strain that can be efficiently expressed in Escherichia coli; our preliminary experiments found that the CD276 nano antibody screened by the method of the present invention has It has the characteristic of immune reaction with CD276 antigen, and has good specificity and high affinity, and can be used to prepare CD276 detection reagents or anti-tumor drugs, etc. For example, the anti-human CD276 nanoantibody provided by the present invention can be used as the antigen recognition domain of CAR to construct CAR-T cells, which has a significant killing effect on various tumor cell lines expressing CD276 antigen; the anti-human CD276 nanoantibody provided by the present invention can be internalized by cells positive for CD276 expression; in addition, the anti-human CD276 nanoantibody provided by the present invention can be used to detect the expression of CD276 in tumor tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 PCR gel electrophoresis results of VHH fragment amplification are shown;

[0034] in,

[0035] Figure 1 (a) shows the gel running results of the first round of PCR products;

[0036] Figure 1 (b) shows the gel running results of the second round of PCR products.

[0037] Figure 2 The results of library insertion rate detection are shown.

[0038] Figure 3 Antibody sequence alignment results are shown.

[0039] Figure 4 Phage-ELISA validation results are shown;

[0040] in,

[0041] Figure 4 (a) shows the reading value of the clones with positive ELISA color development in the first plate of 96-well plate at 450nm on the microplate reader, NC represents the negative control, and 1-10 represents 10 different clones;

[0042] Figure 4 (b) shows the reading values ​​of the clones that were positive for ELISA in the second 96-well plate at 450 nm on a microplate reader, NC represents the negative control, and 1-10 represent 10 different clones.

[0043] Figure 5 The flow cytometry results of the phage supernatant of the positive clones are shown;

[0044] in,

[0045] Figure 5 (a) shows the flow cytometry results of CD276-VHH1 and HepG2, Hela and Raji cells respectively;

[0046] Figure 5 (b) shows the flow cytometry results of CD276-VHH2 and HepG2, Hela and Raji cells, respectively.

[0047] Figure 6 Shown is a schematic diagram of constructing PMIGV-CD276-VHH1-mIgGFc and PMIGV-CD276-VHH2-mIgGFc vector plasmids;

[0048] in,

[0049] Figure 6 (a) shows a schematic diagram of constructing the PMIGV-CD276-VHH1-mIgGFc vector plasmid;

[0050] Figure 6 (b) shows a schematic diagram of constructing the PMIGV-CD276-VHH2-mIgGFc vector plasmid.

[0051] Figure 7 The nucleic acid electrophoresis diagram of the vector construction of Test Example 1 is shown;

[0052] in,

[0053] Figure 7 (a) shows the nucleic acid electrophoresis diagram of the first PCR;

[0054] Figure 7 (b) shows the nucleic acid electrophoresis diagram of the second PCR.

[0055] Figure 8 The results of vector sequencing comparison are shown;

[0056] in,

[0057] Figure 8 (a) shows the sequencing comparison results of PMIGV-CD8 signal peptide-CD276-VHH1-mIgGFc vector;

[0058] Figure 8 (b) shows the sequencing alignment results of PMIGV-CD8 signal peptide-CD276-VHH2-mIgGFc vector.

[0059] Fig. 9 The results of restriction enzyme digestion verification of plasmids are shown;

[0060] in,

[0061] Fig. 9 (a) shows Figure 8 (a) XhoI and EcoRI double restriction enzyme digestion verification results of the CD276-VHH1 expression vector;

[0062] Fig. 9 (b) shows Figure 8 (b) XhoI and EcoRI double restriction enzyme digestion verification results of the CD276-VHH2 expression vector.

[0063] Fig.10 Western blot was used to verify the expression and concentration of CD276-VHH1-mIgGFc protein and CD276-VHH2-mIgGFc protein;

[0064] in,

[0065] Fig.10 (a) shows the expression of CD276-VHH1-mIgGFc protein before (1X) and after (10X) concentration;

[0066] Fig.10 (b) shows the expression of CD276-VHH2-mIgGFc protein before (1X) and after (10X) concentration.

[0067] Fig.11 Flow cytometry validation of CD276 expression in tumor cells is shown;

[0068] in,

[0069] Fig.11 (a) shows the expression of CD276 in Raji cells;

[0070] Fig.11 (b) shows the expression of CD276 in 293T cells;

[0071] Fig.11 (c) shows the expression of CD276 in Hela cells;

[0072] Fig.11 (d) shows the expression of CD276 in HepG2 cells;

[0073] Fig.11 (e) shows the expression of CD276 in KYSE270 cells;

[0074] Fig.11 (f) shows the expression of CD276 in KYSE30 cells.

[0075] Fig.12 The fluorescence intensity of CD276-positive tumor cells is shown;

[0076] in,

[0077] Fig.12 (a) shows the expression intensity of CD276 in HepG2, KYSE270, KYSE30, Hela and Raji tumor cells;

[0078] Fig.12 (b) shows the average fluorescence intensity of CD276 expression in HepG2, KYSE270, KYSE30, Hela and Raji tumor cells.

[0079] Fig.13 The binding of CD276 nanobody to CD276 positive cell lines is shown;

[0080] in,

[0081] Fig.13 (a) shows the binding of CD276-VHH1 and CD276-VHH2 to Raji cell line;

[0082] Fig.13 (b) shows the binding of CD276-VHH1 and CD276-VHH2 to HepG2 cell line;

[0083] Fig.13 (c) shows the binding of CD276-VHH1 and CD276-VHH2 to Hela cell line;

[0084] Fig.13 (d) shows the binding of CD276-VHH1 and CD276-VHH2 to KYSE270 cell line;

[0085] Fig.13 (e) shows the binding of CD276-VHH1 and CD276-VHH2 to the KYSE30 cell line.

[0086] Fig.14 Flow cytometry was used to detect the binding effect of CD276-VHH1-mIgGFc and CD276-VHH2-mIgGFc on Hela cells at different concentration gradients;

[0087] in,

[0088] Fig.14 (a) shows the binding of Hela to CD276-VHH1 and CD276-VHH2 primary antibodies, secondary antibodies, and commercial antibodies, respectively;

[0089] Fig.14 (b) shows the detection effect when CD276-VHH1 fusion protein was used as the primary antibody in volumes of 200 μl, 50 μl, 10 μl and 2 μl respectively;

[0090] Fig.14 (c) shows the detection effect when CD276-VHH2 fusion protein was used as the primary antibody in volumes of 200 μl, 50 μl, 10 μl and 2 μl, respectively.

[0091] Fig.15 A schematic diagram of the CAR lentiviral vector plasmid is shown;

[0092] in,

[0093] Fig.15 (a) shows a schematic diagram of the pEF-CD276-VHH1-CAR lentiviral vector plasmid;

[0094] Fig.15 (b) shows a schematic diagram of the pEF-CD276-VHH2-CAR lentiviral vector plasmid.

[0095] Fig.16 The electrophoresis diagram of the vector construction of Test Example 2 is shown.

[0096] Fig.17 Plasmid sequencing alignment is shown;

[0097] in,

[0098] Fig.17 (a) shows the sequencing alignment of pEF-CD276-VHH1-CAR plasmid;

[0099] Fig.17(b) shows the sequencing alignment of pEF-CD276-VHH2-CAR plasmid.

[0100] Fig.18 The results of CAR lentivirus titer determination are shown;

[0101] in,

[0102] Fig.18 (a) shows the determination of CD276-VHH1-CAR lentivirus titer by flow cytometry;

[0103] Fig.18 (b) shows the determination of CD276-VHH2-CAR lentivirus titer by flow cytometry.

[0104] Fig.19 The nuclear CD3 of lymphocytes in PBMC is shown + T cell magnetic bead sorting efficiency;

[0105] in,

[0106] Fig.19 (a) shows the CD3 + The proportion of T cells;

[0107] Fig.19 (b) shows the CD3 in PBMC after CD3 / CD28 magnetic bead sorting. + The proportion of T cells.

[0108] Fig. 20 The positive rate of CAR-T cells after lentiviral infection is shown;

[0109] in,

[0110] Fig. 20 (a) shows the positive rate of CAR-T cells after lentivirus infection in the CD276-VHH1 group;

[0111] Fig. 20 (b) shows the positive rate of CAR-T cells after lentivirus infection in the CD276-VHH2 group.

[0112] Fig.21 The killing effects of CD276-VHH1-CAR-T cells and CD276-VHH2-CAR-T cells on five tumor cells at different effector-target ratios are shown;

[0113] in,

[0114] Fig.21 (a) shows the killing effect on Raji cells;

[0115] Fig.21(b) shows the killing effect on HepG2 cells;

[0116] Fig.21 (c) shows the killing effect on KYSE30 cells;

[0117] Fig.21 (d) shows the killing effect on Hela cells;

[0118] Fig.21 (e) shows the killing effect on KYSE270 cells.

[0119] Fig. 22 The secretion of IFN-γ during CAR-T cell killing is shown;

[0120] in,

[0121] Fig. 22 (a) shows the secretion of IFN-γ during CAR-T killing of Raji cells;

[0122] Fig. 22 (b) shows the secretion of IFN-γ during CAR-T killing of HepG2 cells;

[0123] Fig. 22 (c) shows the secretion of IFN-γ during CAR-T killing of KYSE30 cells;

[0124] Fig. 22 (d) shows the secretion of IFN-γ during the killing of Hela cells by CAR-T;

[0125] Fig. 22 (e) shows the secretion of IFN-γ during CAR-T killing of KYSE270 cells;

[0126] Fig. 22 (f) shows the standard curve of IFN-γ ELISA detection.

[0127] Fig.23 The results of CD276-VHH1 / VHH2 internalization (50 μg) in Raji cells are shown;

[0128] in,

[0129] Fig.23 (a) shows the results of CD276-VHH1 internalization (50 μg) in Raji cells;

[0130] Fig.23 (b) shows the results of CD276-VHH2 internalization (50 μg) in Raji cells.

[0131] Fig.24The results of CD276-VHH1 / VHH2 endocytosis (50 μg) in Hela cells are shown;

[0132] in,

[0133] Fig.24 (a) shows the results of CD276-VHH1 internalization (50 μg) in Hela cells;

[0134] Fig.24 (b) shows the results of CD276-VHH2 internalization (50 μg) in Hela cells.

[0135] Fig.25 The results of CD276-VHH1 / VHH2 endocytosis (50 μg) in HCT116 cells are shown;

[0136] in,

[0137] Fig.25 (a) shows the results of CD276-VHH1 internalization (50 μg) in HCT116 cells;

[0138] Fig.25 (b) shows the results of CD276-VHH2 internalization (50 μg) in HCT116 cells.

[0139] Fig.26 The binding of commercial CD276 antibody (Proteintech Cat No: 66481-1-Ig) to esophageal cancer tissue is shown; it can be used as a positive control.

[0140] Fig. 27 Binding of CD276-VHH1 to esophageal cancer is shown.

[0141] Fig.28 Binding of CD276-VHH2 to esophageal cancer is shown.

[0142] Fig.29 A negative control showing the binding of Nanobodies to esophageal cancer tissues. DETAILED DESCRIPTION

[0143] In order to more clearly illustrate the technical solution of the present invention, the following examples are listed. Unless otherwise stated, the raw materials, reactions and post-treatment methods shown in the examples are common raw materials on the market and technical methods well known to those skilled in the art.

[0144] Example

[0145] An anti-CD276 nanobody, comprising a complementary determining region CDR and a framework region FR;

[0146] in,

[0147] The complementary determining region CDR includes complementary determining regions CDR1-CDR3;

[0148] The complementary determining regions CDR1-CDR3 are shown in amino acid sequences 1-3 respectively;

[0149] The amino acid sequences 1-3 are shown as SEQ ID No.1-SEQ ID No.3;

[0150] The framework region FR includes framework regions FR1-FR5;

[0151] The framework regions FR1-FR5 are shown in amino acid sequences 4-8 respectively;

[0152] The amino acid sequences 4-8 are shown as SEQ ID No.4-SEQ ID No.8;

[0153] The amino acid sequence of the anti-CD276 Nanobody is CD276-VHH1 or CD276-VHH2;

[0154] The CD276-VHH1 and CD276-VHH2 are shown as SEQ ID No.9-SEQ ID No.10.

[0155] A nucleic acid molecule encoding the above-mentioned anti-CD276 nanobody comprises the nucleic acid sequence SEQ ID No.11 or SEQ ID No.12.

[0156] The method for preparing the above-mentioned anti-CD276 nanobody comprises the following steps:

[0157] S1. Construction of phage library

[0158] RNA was extracted from alpaca PBMC and reverse transcribed using the Novezan RNA reverse transcription kit (HiScriptⅡQSelect RT SuperMix for qPCR R232). The reaction solution preparation system and the RNA reverse transcription preparation system are shown in Tables 1 and 2, respectively.

[0159] Table 1 Reaction solution preparation system

[0160] Reagents Usage Oligo dT Primer (50μM) 1μl dNTP Mix 1μl Total RNA 5μg <![CDATA[ddH2O]]> Up to 10 μl

[0161] Table 2 RNA reverse transcription preparation system

[0162] Reagents Usage Reaction solution 10μl 5×PrimeScript II Buffer 4μl RNase Inhibitor 0.5μl PrimeScript II RTase 1μl <![CDATA[ddH2O]]> Up to 20 μl

[0163] Using cDNA as a template, 2*Phanta Max Master Mix from Novezan Biotech was used to amplify VHH in the first round by PCR. The PCR amplification VHH reaction system is shown in Table 3.

[0164] Table 3 PCR amplification VHH reaction system

[0165] Reagents Usage 2*Phanta Max Master Mix 10μl Primer1 0.4μl Primer2 0.4μl cDNA 0.6μl <![CDATA[ddH2O]]> Up to 20 μl

[0166] After the reaction was completed, 20 μl of the PCR product was subjected to 1% agarose gel electrophoresis, and the VHH product was purified by gel recovery at 600 bp. The PCR gel electrophoresis results were as follows: Figure 1 shown.

[0167] Figure 1 PCR gel electrophoresis results of VHH fragment amplification are shown;

[0168] in,

[0169] Figure 1 (a) shows the results of the first round of PCR product gel running, M (Marker) is Novozymes DL5000 DNA Marker, and the bands are 5k, 3k, 2k, 1.5k, 1k, 750bp, 500bp, 250bp, and 100bp respectively;

[0170] Figure 1 (b) shows the results of the second round of PCR product running. Figure 1 (a).

[0171] VHH and phage vector were connected via Goldengate, and the reaction system is shown in Table 4.

[0172] Table 4 VHH and phagemid vector connection system

[0173]

[0174] The ligation product was electroporated, voltage: 2477V; time: 4.6ms; storage capacity: 1.1x10 8 cfu. The library positive rate was detected using the primers on the vector and sequenced. The library insertion rate test results were as follows: Figure 2 shown.

[0175] Figure 2 The results of the library insertion rate detection are shown; the results show that the library insertion rate is 100%.

[0176] Several clones were randomly selected from the positive clones detected by colony PCR and sent for sequencing. The light chain antibody fragments in the sequencing results were translated into amino acid sequences and then sequenced for comparison to detect sequence diversity in the bacterial library. The antibody sequence comparison results were as follows: Figure 3 shown.

[0177] Figure 3 The antibody sequence alignment results are shown; the results show that these sequences are different and are alpaca antibody heavy chain variable region sequences.

[0178] S2. Preparation of phage antibody library and selection of nanobodies

[0179] Use helper phage to amplify the glycerol bacteria and detect the titer to obtain the phage antibody library. Use an immunotube to coat the antigen and block it with PBST, then add 1×10 12 The phages were panned, and pH 2.0 Gly-HCl and pH 9.5 Tris-HCl were used as eluents. The steps of screening and panning were repeated three times, and the titers of phages after three rounds of amplification and panning were shown in Table 5. Through the above operations, the phage antibody library was enriched.

[0180] Table 5 Panning and amplification results

[0181]

[0182]

[0183] S3. Validation of Nanobodies

[0184] The phages from the third round of panning were used to infect TG1 and single clones were selected for phage-ELISA verification. The verification results were as follows: Figure 4 The ELISA-positive phage supernatant was incubated with tumor cells expressing CD276 for flow cytometry detection. The flow cytometry results are shown in Figure 5 shown.

[0185] The flow cytometry-positive samples were sequenced and compared with the alpaca sequence, and finally two nanoantibody sequences, CD276-VHH1 and CD276-VHH2, were obtained, whose amino acid sequences are shown in SEQ ID No.9 and SEQ ID No.10.

[0186] Figure 4 Phage-ELISA validation results are shown;

[0187] in,

[0188] Figure 4(a) shows the reading value of the clones with positive ELISA color development in the first plate of 96-well plate at 450nm on the microplate reader, NC represents the negative control, and 1-10 represents 10 different clones;

[0189] Figure 4 (b) shows the reading values ​​of the clones that were positive for ELISA in the second 96-well plate at 450 nm on a microplate reader, NC represents the negative control, and 1-10 represent 10 different clones.

[0190] The results showed that after three rounds of enrichment, ELISA-positive phage clones were screened.

[0191] Figure 5 The flow cytometry results of the phage supernatant of the positive clones are shown;

[0192] in,

[0193] Figure 5 (a) shows the flow cytometry results of CD276-VHH1 and HepG2, Hela and Raji cells respectively;

[0194] Figure 5 (b) shows the flow cytometry results of CD276-VHH2 and HepG2, Hela and Raji cells, respectively.

[0195] The results showed that the two positive clone phages screened could specifically bind to tumor cells expressing CD276 positively, but not to negative cells.

[0196] Test Example 1 (as a flow cytometry antibody to detect CD276-positive tumor cell lines)

[0197] Test method:

[0198] Vector plasmid construction: The CD276-VHH1 and CD276-VHH2 nanoantibody sequences obtained by screening in the example were subjected to gene synthesis and amplified by PCR to obtain sufficient DNA fragments. The construction of the vector plasmid is as follows: Figure 6 shown.

[0199] Vector construction: After separating the target bands by gel electrophoresis, the DNA fragments encoding each nanobody were constructed into a eukaryotic expression vector containing a mouse-IgG-Fc (mIgGFc) fragment. The nucleic acid electrophoresis results of the vector construction were as follows: Figure 7 As shown in Figure 2, the sequencing results after the vector construction was completed were as follows: Figure 8 shown.

[0200] Enzyme digestion verification: Use Xho1 and EcoR1 to double digest the plasmid, and perform nucleic acid electrophoresis after digestion. The results are as follows Fig. 9As shown, the target recombinant plasmid was obtained. The target plasmid was transiently transferred into 293T cells by PEI transfection method for expression, and the supernatant was collected after 48 hours for concentration and preliminary purification to obtain the recombinant anti-CD276 nanobody.

[0201] Western blot verification: The recombinant anti-CD276 nanobody was concentrated and purified and then verified by Western blot. The verification results are as follows Fig.10 shown.

[0202] CD276 expression verification: CD276 positive cell lines were selected and their CD276 expression was verified using commercial antibodies. The verification results were as follows: Figure 11-12 shown.

[0203] Binding detection of nano-antibodies to CD276-positive cell lines: The obtained recombinant anti-CD276 nano-antibodies were used as the primary antibody of flow cytometry, coupled with the secondary antibody of FITC-anti-mouse-IgG, and co-incubated with tumor cells expressing CD276. The binding of the recombinant anti-CD276 nano-antibodies to CD276-positive cell lines was detected by flow cytometry. The results are as follows: Fig.13 shown.

[0204] Detection of the binding ability of nanoantibodies to tumor cells expressing CD276: Hela cells were used to detect the binding ability of CD276-VHH1 and CD276-VHH2 nanoantibodies to tumor cells expressing CD276 at different concentration gradients. The results are as follows Fig.14 shown.

[0205] Figure 6 Shown is a schematic diagram of constructing PMIGV-CD276-VHH1-mIgGFc and PMIGV-CD276-VHH2-mIgGFc vector plasmids;

[0206] in,

[0207] Figure 6 (a) shows a schematic diagram of constructing the PMIGV-CD276-VHH1-mIgGFc vector plasmid;

[0208] Figure 6 (b) shows a schematic diagram of constructing the PMIGV-CD276-VHH2-mIgGFc vector plasmid.

[0209] Figure 7 The nucleic acid electrophoresis diagram of the vector construction of Test Example 1 is shown;

[0210] in,

[0211] Figure 7 (a) shows the nucleic acid electrophoresis diagram of the first PCR;

[0212] Figure 7 (b) shows the nucleic acid electrophoresis diagram of the second PCR.

[0213] The results showed that CD276-VHH1 and CD276-VHH2 fragments with correct band sizes were successfully PCR-generated.

[0214] Figure 8 The results of vector sequencing comparison are shown;

[0215] in,

[0216] Figure 8 (a) shows the sequencing comparison results of PMIGV-CD8 signal peptide-CD276-VHH1-mIgGFc vector;

[0217] Figure 8 (b) shows the sequencing alignment results of PMIGV-CD8 signal peptide-CD276-VHH2-mIgGFc vector.

[0218] The results showed that the constructed vector sequence was correct.

[0219] Fig. 9 The results of restriction enzyme digestion verification of plasmids are shown;

[0220] in,

[0221] Fig. 9 (a) shows Figure 8 (a) XhoI and EcoRI double restriction enzyme digestion verification results of the CD276-VHH1 expression vector;

[0222] Fig. 9 (b) shows Figure 8 (b) XhoI and EcoRI double restriction enzyme digestion verification results of the CD276-VHH2 expression vector.

[0223] The results showed that the constructed vector structure was correct.

[0224] Fig.10 Western blot was used to verify the expression and concentration of CD276-VHH1-mIgGFc protein and CD276-VHH2-mIgGFc protein;

[0225] in,

[0226] Fig.10 (a) shows the expression of CD276-VHH1-mIgGFc protein before (1X) and after (10X) concentration;

[0227] Fig.10 (b) shows the expression of CD276-VHH2-mIgGFc protein before (1X) and after (10X) concentration.

[0228] The results showed that CD276-VHH1-mIgGFc protein and CD276-VHH2-mIgGFc protein were successfully expressed and concentrated.

[0229] Fig.11 Flow cytometry validation of CD276 expression in tumor cells is shown;

[0230] in,

[0231] Fig.11 (a) shows the expression of CD276 in Raji cells;

[0232] Fig.11 (b) shows the expression of CD276 in 293T cells;

[0233] Fig.11 (c) shows the expression of CD276 in Hela cells;

[0234] Fig.11 (d) shows the expression of CD276 in HepG2 cells;

[0235] Fig.11 (e) shows the expression of CD276 in KYSE270 cells;

[0236] Fig.11 (f) shows the expression of CD276 in KYSE30 cells.

[0237] The results showed that CD276 was expressed in Hela, HepG2, KYSE270 and KYSE30 tumor cells.

[0238] Fig.12 The fluorescence intensity of CD276-positive tumor cells is shown;

[0239] in,

[0240] Fig.12 (a) shows the expression intensity of CD276 in HepG2, KYSE270, KYSE30, Hela and Raji tumor cells;

[0241] Fig.12 (b) shows the mean florescence intensity (MFI) of CD276 in HepG2, KYSE270, KYSE30, Hela and Raji tumor cells.

[0242] The results showed that CD276 was expressed at different levels in the indicated tumor cells.

[0243] Fig.13 The binding of CD276 nanobody to CD276 positive cell lines is shown;

[0244] in,

[0245] Fig.13 (a) shows the binding of CD276-VHH1 and CD276-VHH2 to Raji cell line;

[0246] Fig.13 (b) shows the binding of CD276-VHH1 and CD276-VHH2 to HepG2 cell line;

[0247] Fig.13 (c) shows the binding of CD276-VHH1 and CD276-VHH2 to Hela cell line;

[0248] Fig.13 (d) shows the binding of CD276-VHH1 and CD276-VHH2 to KYSE270 cell line;

[0249] Fig.13 (e) shows the binding of CD276-VHH1 and CD276-VHH2 to the KYSE30 cell line.

[0250] The results show that the two nanoantibodies CD276-VHH1 and CD276-VHH2 screened by the present invention can be used for flow cytometry detection of CD276 expression in cells.

[0251] Fig.14 Flow cytometry was used to detect the binding effect of CD276-VHH1-mIgGFc and CD276-VHH2-mIgGFc on Hela cells at different concentration gradients;

[0252] in,

[0253] Fig.14 (a) shows the binding of Hela to CD276-VHH1 and CD276-VHH2 primary antibodies, secondary antibodies, and commercial antibodies, respectively;

[0254] Fig.14 (b) shows the detection effect when CD276-VHH1 fusion protein was used as the primary antibody in volumes of 200 μl, 50 μl, 10 μl and 2 μl respectively;

[0255] Fig.14(c) shows the detection effect when CD276-VHH2 fusion protein was used as the primary antibody in volumes of 200 μl, 50 μl, 10 μl and 2 μl, respectively.

[0256] The results show that the binding rates of the two nanobodies screened by the present invention to Hela cells vary with the amount of antibodies used.

[0257] From the above results, it can be seen that the two anti-CD276 nanoantibody sequences CD276-VHH1 and CD276-VHH2 prepared in the examples can recognize CD276-positive tumor cell lines. Therefore, the present invention can be used for the production of flow cytometry antibodies for the recognition of CD276-positive tumor cell lines.

[0258] Test Example 2 (as the antigen recognition domain on CAR involved in CAR-T cell killing of tumor cells)

[0259] Test method:

[0260] Vector construction: The gene fragment of the anti-CD276 nanobody of Test Example 1 was amplified by PCR to obtain a sufficient amount of DNA fragments. After separating the target bands by gel electrophoresis, the DNA fragments encoding each nanobody were constructed into a eukaryotic expression vector containing a CAR structure. The vector construction schematic diagram is shown in FIG. Fig.15 As shown, the results of nucleic acid electrophoresis are as follows Fig.16 shown.

[0261] Plasmid extraction: The target plasmid can be obtained by connecting, transforming and extracting the plasmid. The sequencing results are as follows: Fig.17 shown.

[0262] Titer determination: The virus is packaged and produced using the third-generation lentiviral packaging system. The titer is determined after virus production. The results are as follows: Fig.18 shown.

[0263] CAR-T cell killing test on CD276 tumor cell line: PBMCs were isolated from human peripheral blood, and human CD3 + T cells, sorted CD3 + T cell ratio Fig.19 As shown. Using lentivirus to infect human CD3 + T cells were used to prepare CAR-T cells, and the positive rate of CAR-T cells was detected using the prepared CD276-mIgGFc extracellular segment protein. The positive rate results are as follows Fig. 20As shown. According to the positive rate of CAR-T cells, the experimental scheme was designed to kill CD276 tumor cell lines. The present invention killed five cell lines, namely CD276 positive cell lines Hela, HepG2, KYSE30, KYSE270 and CD276 negative cell line Raji. CAR-T cells and tumor cells were incubated at a ratio of 0.5:1, 1:1, 2:1, 4:1, and 8:1, respectively. After 24 hours, the secretion of LDH was detected by Elisa to determine the killing results. The results are shown in Fig.21 In addition, IFN-γ secretion was also tested, and the results are shown in Fig. 22 shown.

[0264] Fig.15 A schematic diagram of the CAR lentiviral vector plasmid is shown;

[0265] in,

[0266] Fig.15 (a) shows a schematic diagram of the pEF-CD276-VHH1-CAR lentiviral vector plasmid;

[0267] Fig.15 (b) shows a schematic diagram of the pEF-CD276-VHH2-CAR lentiviral vector plasmid.

[0268] Fig.16 The electrophoresis diagram of the vector construction of Test Example 2 is shown; the results show that restriction enzyme digestion and PCR produced DNA fragments with the correct band size.

[0269] Fig.17 Plasmid sequencing alignment is shown;

[0270] in,

[0271] Fig.17 (a) shows the sequencing alignment of pEF-CD276-VHH1-CAR plasmid;

[0272] Fig.17 (b) shows the sequencing alignment of pEF-CD276-VHH2-CAR plasmid.

[0273] The results showed that the plasmid with the correct sequence was successfully constructed.

[0274] Fig.18 The results of CAR lentivirus titer determination are shown;

[0275] in,

[0276] Fig.18 (a) shows the determination of CD276-VHH1-CAR lentivirus titer by flow cytometry;

[0277] Fig.18 (b) shows the determination of CD276-VHH2-CAR lentivirus titer by flow cytometry.

[0278] The results showed that the titer of lentiviral CD276-VHH1-CAR was 2.6×10 5 IU / mL; the titer of CD276-VHH2-CAR was 1.07×10 6 IU / mL; can be used for subsequent T cell infection.

[0279] Fig.19 The nuclear CD3 of lymphocytes in PBMC is shown + T cell magnetic bead sorting efficiency;

[0280] in,

[0281] Fig.19 (a) shows the CD3 + The proportion of T cells;

[0282] Fig.19 (b) shows the CD3 in PBMC after CD3 / CD28 magnetic bead sorting. + The proportion of T cells.

[0283] The results showed that CD3 + The proportion of T cells was 76.2%, and CD3 + The proportion of T cells was 99.9%, and CD3 + T cells.

[0284] Fig. 20 The positive rate of CAR-T cells after lentiviral infection is shown;

[0285] in,

[0286] Fig. 20 (a) shows the positive rate of CAR-T cells after lentivirus infection in the CD276-VHH1 group;

[0287] Fig. 20 (b) shows the positive rate of CAR-T cells after lentivirus infection in the CD276-VHH2 group.

[0288] The results showed that T cells expressing CD276-VHH1 and CD276-VHH2 CAR were successfully cultured.

[0289] Fig.21 The killing effects of CD276-VHH1-CAR-T cells and CD276-VHH2-CAR-T cells on five tumor cells at different effector-target ratios are shown;

[0290] in,

[0291] Fig.21 (a) shows the killing effect on Raji cells;

[0292] Fig.21 (b) shows the killing effect on HepG2 cells;

[0293] Fig.21 (c) shows the killing effect on KYSE30 cells;

[0294] Fig.21 (d) shows the killing effect on Hela cells;

[0295] Fig.21 (e) shows the killing effect on KYSE270 cells.

[0296] The results showed that the CAR-T cells constructed with the two nanoantibody sequences screened by the present invention have the ability to kill CD276-positive tumor cells.

[0297] Fig. 22 The secretion of IFN-γ during CAR-T cell killing is shown;

[0298] in,

[0299] Fig. 22 (a) shows the secretion of IFN-γ during CAR-T killing of Raji cells;

[0300] Fig. 22 (b) shows the secretion of IFN-γ during CAR-T killing of HepG2 cells;

[0301] Fig. 22 (c) shows the secretion of IFN-γ during CAR-T killing of KYSE30 cells;

[0302] Fig. 22 (d) shows the secretion of IFN-γ during the killing of Hela cells by CAR-T;

[0303] Fig. 22 (e) shows the secretion of IFN-γ during CAR-T killing of KYSE270 cells;

[0304] Fig. 22 (f) shows the standard curve of IFN-γ ELISA detection.

[0305] The results showed that CAR-T cells secreted a large amount of IFN-γ when killing positive tumor cells, and the amount secreted was proportional to the killing effect.

[0306] It can be seen from the above test results that the two nanobodies CD276-VHH1 and CD276-VHH2 of the present invention can both be used as the antigen recognition domain of CAR to kill tumor cells.

[0307] Test Example 3 (Nanoantibodies will be internalized by CD276-positive cells and can be used for the preparation of drugs such as ADC)

[0308] Test method:

[0309] Cell preparation: Prepare CD276 positive cells: Hela, HCT116; CD276 negative cells: Raji cells. Collect 1-5×10 cells of each type. 5 Collect 6 tubes of each cell type into 1.5 ml EP tubes.

[0310] Antibody preparation: approximately 500 μl of each VHH-mIgGFc protein.

[0311] Antibody incubation plan: Collect cells into EP tubes and centrifuge to remove supernatant. Add 25μl of corresponding protein and incubate at 4℃ for 30min. Terminate incubation with PBS, wash away unbound antibodies, add 500μl of complete culture medium pre-cooled at 4℃ or pre-heated at 37℃ to resuspend cells, incubate at 4℃ and 37℃ for 4h / 6h, and detect once. After washing with PBS, add anti-mouse fluorescent secondary antibody and incubate at 4℃ for 30min. Terminate incubation with PBS and wash with PBS, then detect on flow cytometer. The results of protein endocytosis of Raji cells, Hela cells, and HCT116 cells are shown below. Figure 23-25 As shown; the internalization ratios of CD276-VHH1 and CD276-VHH2 in different cells are shown in Figure 6.

[0312] Table 6 Internalization ratio of CD276-VHH1 and CD276-VHH2 in different cells

[0313]

[0314] Fig.23 The results of CD276-VHH1 / VHH2 internalization (50 μg) in Raji cells are shown;

[0315] in,

[0316] Fig.23 (a) shows the results of CD276-VHH1 internalization (50 μg) in Raji cells;

[0317] Fig.23 (b) shows the results of CD276-VHH2 internalization (50 μg) in Raji cells.

[0318] The results showed that Raji cells that do not express CD276 internalized less CD276-VHH1 and CD276-VHH2 antibodies.

[0319] Fig.24 The results of CD276-VHH1 / VHH2 endocytosis (50 μg) in Hela cells are shown;

[0320] in,

[0321] Fig.24 (a) shows the results of CD276-VHH1 internalization (50 μg) in Hela cells;

[0322] Fig.24 (b) shows the results of CD276-VHH2 internalization (50 μg) in Hela cells.

[0323] The results showed that Hela cells expressing CD276 had a higher level of internalization of CD276-VHH1 and CD276-VHH2 antibodies.

[0324] Fig.25 The results of CD276-VHH1 / VHH2 endocytosis (50 μg) in HCT116 cells are shown;

[0325] in,

[0326] Fig.25 (a) shows the results of CD276-VHH1 internalization (50 μg) in HCT116 cells;

[0327] Fig.25 (b) shows the results of CD276-VHH2 internalization (50 μg) in HCT116 cells.

[0328] The results showed that HCT116 cells expressing CD276 had a higher level of internalization of CD276-VHH1 and CD276-VHH2 antibodies.

[0329] It can be seen from the above test results that the two nanobodies CD276-VHH1 and CD276-VHH2 of the present invention can be internalized by CD276-positive cells and can be used to produce ADC and other drugs that need to enter cells to exert their effects.

[0330] Test Example 4 (Recognition and binding of nanoantibodies to esophageal cancer tissue)

[0331] Test method:

[0332] Dewax the paraffin sections to make them water-free: sequentially place the sections into environmentally friendly dewaxing liquid I for 10 min - environmentally friendly dewaxing liquid II for 10 min - environmentally friendly dewaxing liquid III for 10 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - anhydrous ethanol III for 5 min - and wash with distilled water.

[0333] Antigen retrieval: The retrieval conditions are shown in Table 7. During the retrieval process, the buffer should be prevented from evaporating excessively and the slides should not be dried. After the retrieval is completed, cool naturally. Place the slides in PBS (pH 7.4) and wash them on a decolorizing shaker for 3 times, 5 minutes each time.

[0334] Circle serum blocking: After the slices are slightly dried, use a histochemical pen to draw a circle around the tissue, add BSA (if the primary antibody is from goat, use 10% donkey serum to block, and if the primary antibody is from other sources, use 3% BSA to block), and block for 30 minutes.

[0335] Add primary antibody: Add the prepared primary antibody dropwise and incubate the slices flat in a humidified box at 4°C overnight.

[0336] Add secondary antibody: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker for 3 times, 5 minutes each time. Add the corresponding secondary antibody and incubate at room temperature for 50 minutes in the dark.

[0337] DAPI counterstaining of cell nuclei: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker for 3 times, 5 minutes each time. Add DAPI staining solution and incubate at room temperature for 10 minutes in the dark.

[0338] Quenching tissue autofluorescence: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker for 3 times, 5 minutes each time. Add autofluorescence quencher B solution for 5 minutes and rinse with running water for 10 minutes.

[0339] Sealing: Sealing with anti-fluorescence fading mounting medium.

[0340] Image collection: DAPI excitation wavelength 330-380nm, emission wavelength 420nm; 488 excitation wavelength 465-495nm, emission wavelength 515-555nm; CY3 excitation wavelength 510-560nm, emission wavelength 590nm; CY5 excitation wavelength 608-648nm, emission wavelength 672-712nm.

[0341] Interpretation of results: DAPI channel cell nucleus is blue, 488 channel positive is green, the results are as follows Figure 26-29 shown.

[0342] Table 7 Antigen retrieval conditions

[0343]

[0344] Fig.26The binding of commercial CD276 antibody (Proteintech Cat No: 66481-1-Ig) to esophageal cancer tissue is shown; it can be used as a positive control.

[0345] Fig. 27 Binding of CD276-VHH1 to esophageal cancer is shown.

[0346] Fig.28 Binding of CD276-VHH2 to esophageal cancer is shown.

[0347] Fig.29 A negative control showing the binding of Nanobodies to esophageal cancer tissues.

[0348] The above results indicate that the CD276-VHH1 and CD276-VHH2 nanoantibodies screened by the present invention can be used for immunohistochemical detection of CD276 expression in human esophageal cancer tissues.

[0349] Based on the results of test examples 1-4, it can be clearly seen that the anti-human CD276 nanoantibody provided by the present invention can bind to the CD276 antigen and can sensitively detect CD276 antigen molecules; it can be used as the antigen recognition domain of CAR to construct CAR-T cells, and has a significant killing effect on various tumor cell lines expressing CD276 antigens; and can be used to prepare ADC and other drugs that need to enter CD276 positive cells to exert their effects. Therefore, it can be applied to the diagnosis and treatment of malignant tumors and has excellent market promotion prospects.

[0350] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0351] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. An anti-CD276 nanobody, characterized in that The anti-CD276 nanobody includes a complementary determining region CDR and a framework region FR; in, The complementary determining region CDR includes complementary determining regions CDR1-CDR3; The complementary determining regions CDR1-CDR3 are shown in amino acid sequences 1-3 respectively; The amino acid sequences 1-3 are shown as SEQ ID No.1-SEQ ID No.

3.

2. The anti-CD276 nanobody according to claim 1, characterized in that The framework region FR includes framework regions FR1-FR5; in, The framework regions FR1-FR5 are shown in amino acid sequences 4-8 respectively; The amino acid sequences 4-8 are shown as SEQ ID No.4-SEQ ID No.

8.

3. The anti-CD276 nanobody according to claim 1, characterized in that The amino acid sequence of the anti-CD276 Nanobody is CD276-VHH1 or CD276-VHH2; The CD276-VHH1 and CD276-VHH2 are shown as SEQ ID No.9-SEQ ID No.

10.

4. The anti-CD276 nanobody according to claim 1, characterized in that The anti-CD276 nanobody is selected from: animal-derived antibodies, chimeric antibodies, humanized antibodies or a combination thereof.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-CD276 Nanobody according to any one of claims 1-4.

6. The nucleic acid molecule according to claim 5, characterized in that The nucleic acid molecule comprises the nucleic acid sequence SEQ ID No.11 or SEQ ID No.

12.

7. A carrier, characterized in that The carrier comprises the anti-CD276 Nanobody according to any one of claims 1-4.

8. The carrier according to claim 7, characterized in that: The vector is selected from a DNA molecule, an mRNA molecule or a cell.

9. Use of the carrier according to any one of claims 7 to 8 in antigen detection, adsorption of antigen reagents and immunotherapy.

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