A nanobody against cd276 and uses thereof

Abstract

The present application relates to a kind of anti-CD276 nanobody, the anti-CD276 nanobody includes complementary determining region CDR and framework region FR;Wherein, the complementary determining region CDR includes complementary determining region CDR1-CDR3;The framework region FR includes framework region FR1-FR5.The anti-human CD276 nanobody provided by the present application has the immunoreaction characteristic with CD276 antigen, and specificity is good, and affinity is high, can be applied to the preparation of CD276 detection reagent or antitumor drug etc., the anti-human CD276 nanobody provided by the present application can be used as the antigen recognition domain of CAR Then the construction of CAR-T cell, it has significant killing effect to the multiple tumor cell lines of expression CD276 antigen;The anti-human CD276 nanobody provided by the present application can be used to detect the expression of CD276 in tumor tissue.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological genes, in particular to a nano-antibody against CD276 and application thereof. BACKGROUND

[0002] CD276 (Recombinant cluster of differentiation 276), also known as B7-H3, is a type I transmembrane glycoprotein with a molecular weight of 45-66 kDa, which 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 the form of membrane protein and soluble form, wherein the soluble form is derived from the membrane protein by metalloproteinase cleavage. In addition, CD276 protein is also 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 co-stimulatory effect on CD4 + and CD8 + T cells, as a co-stimulatory molecule, CD276 signal induces cellular immunity, promotes the secretion of cytokines such as IFN-γ, IL-8, TNF-α, and enhances the cytotoxicity of CD8 + T cells; on the other hand, it can inhibit Treg cells, so that the tumor escapes the immune response, and the mechanism may be related to the three main signal pathways of NFAT, NFKB and AP-1 factors that regulate gene transcription on the surface receptors of T cells. A large number of studies have confirmed that CD276 is widely expressed in various types of cancer, and its correlation with poor prognosis of tumor has also been confirmed. At present, the clinical application research of CD276 antibody mainly includes antibody-drug conjugates (ADC) against tumor, antibody-dependent cell-mediated cytotoxicity (ADCC) drugs, bispecific and trispecific antibodies, chimeric antigen receptor T cell (CAR-T) therapy, etc.

[0003] Heavy chain antibody is a new type of antibody discovered in 1989 in Camelidae, which lacks light chain and constant domain 1 of heavy chain (CH1). Nanobody is cloned from the variable domain of heavy chain of heavy chain antibody (VHH), with a crystal diameter of only 2.5 nm and a molecular weight of 12-15 kDa, which is the smallest antibody known. Compared with traditional antibodies, nanobody has the following advantages: 1. Nanobody has a longer third antigenic complementarity determining region, which maintains similar antigen binding capacity as normal double-chain antibodies and can better bind to antigen epitopes. 2. Inter-loop disulfide bonds are commonly found in nanobodies, which are more stable in high temperature and higher concentration of organic solvents. 3. The second framework region of nanobody contains four conserved hydrophilic amino acid mutations, which makes it hydrophilic and less likely to aggregate and affect antigen recognition. 4. Nanobody has a small volume and can better penetrate dense tumor tissues, making it suitable for solid tumor treatment. 5. Nanobody only has a heavy chain variable region, so when multiple nanobodies are used together, there is no cross-reaction between heavy and light chains, which can affect the therapeutic effect of antibodies. 6. Nanobody is derived from Camelidae, which has a high degree of similarity to human antibodies and low immunogenicity, so it is less likely to produce neutralizing antibodies during treatment.

[0004] Most of the antibodies reported on the market for CD276 are single-chain antibodies (single chain fragment variable, scFv) composed of a variable domain of heavy chain of heavy chain antibody (VHH) and a light chain variable region. 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 of VH-VL, it reduces the solubility of scFv, resulting in a high tendency to aggregate. Compared with VHH antibodies, scFv antibodies are not easily dissolved, which may affect antigen recognition. Another major disadvantage of scFv is that most of them are derived from rodents, as 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 antibodies show high sequence similarity to human VH (VH3 gene family), with about 75-90% homology. Therefore, VHH antibodies have a higher degree of humanization and lower immunogenicity. Even after humanization, the variable region of murine scFv antibodies can still cause anti-idiotype reactions, which is not conducive to immunotherapy.

[0005] In summary, there is an urgent need to develop a new technical solution to solve the problems in the prior art. SUMMARY

[0006] Based on the advantages of nanobodies, the present application successfully constructed a llama phage antibody library immunized with human CD276 extracellular segment protein, and screened 2 new VHH sequences of anti-CD276 nanobodies from it. Our experiments found that the nanobodies can be used for cell flow cytometry and tissue immunohistochemistry to detect human CD276 antigen, so they have application prospects for detecting CD276 antigen; at the same time, the nanobody sequence can be prepared into chimeric antigen receptor T cells (CAR-T), and can effectively kill CD276 positive tumor cells, so it has application prospects in targeted immunotherapy of CD276 positive cells.

[0007] The term "chimeric antigen receptor" involved in the present application refers to an artificial receptor, which is genetically engineered to give immune cells (such as T lymphocytes) new ability to target specific antigens. These receptors are chimeric because they combine the domain that specifically recognizes antigens in antibodies and the T cell activation function domain into one receptor.

[0008] The term "heavy chain CDR" involved in the present application refers to the core conserved structure of the variable region of antibody heavy chain, which is the key region that determines the binding of antibodies to antigens, including CDR1, CDR2 and CDR3.

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

[0010] One object of the present application is to provide an anti-CD276 nanobody, which comprises a complementarity determining region CDR and a framework region FR.

[0011] wherein,

[0012] The complementarity determining region CDR comprises complementarity determining regions CDR1-CDR3.

[0013] The complementarity determining regions CDR1-CDR3 are respectively shown as amino acid sequences 1-3.

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

[0015] Further, the framework region FR comprises framework regions FR1-FR5.

[0016] wherein,

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

[0018] The amino acid sequence 4-8 is shown as SEQ ID No.4-SEQ ID No.8.

[0019] Further, 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.

[0020] Further, the anti-CD276 nanobody is selected from an animal-derived antibody, a chimeric antibody, a humanized antibody or a combination thereof.

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

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

[0023] Another object of the present application is to provide a vector comprising the anti-CD276 nanobody.

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

[0025] Another object of the present application is to provide the application of the vector in antigen detection, adsorption of antigen reagent and immunotherapy.

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

[0027] Further, the immunotherapy is immunotherapy against CD276 positive cells.

[0028] Phage display technology is the most commonly used method for producing nanobodies, which acquires cDNA from natural, immune or synthetic antibody library through genetic engineering. The DNA is inserted into a suitable position of phage coat protein to form a fusion protein with the coat protein, which is expressed on the surface of phage, and then the protein capable of binding to the antigen is screened. The antibody is screened by using immune antibody library, which has high specificity and is more likely to obtain high affinity antibody.

[0029] Therefore, the present application is based on the anti-CD276 nanobody screened from the human CD276 protein extracellular segment immunized llama phage antibody display library constructed by the inventors' team, which can be well applied to the immunotherapy against CD276 positive tumor cells.

[0030] The present application finds two completely new anti-human CD276 nanobody sequences, which are completely different from other nanobody sequences, and which can be used for recognition of CD276 antigen, endocytosis of CD276 positive cells, construction of CAR-T cells and killing of CD276 positive cells.

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

[0032] The present application discloses an anti-CD276 nanobody, which comprises heavy chain CDR1-CDR3. The present application first expresses and purifies CD276 extracellular segment polypeptide, and makes it immunogenic, then couples the CD276 extracellular segment polypeptide on an enzyme-labeled plate to display the correct spatial structure of the protein. The antigen in this form is used to screen the CD276 extracellular segment immune nanobody gene library (camel heavy chain antibody phage display gene library) by phage display technology, so as to obtain CD276 specific nanobody gene. The gene is transferred into Escherichia coli, so as to establish a nanobody strain capable of being efficiently expressed in Escherichia coli. Our previous experiments found that the CD276 nanobody screened by the method of the present application has the characteristics of immunoreaction with CD276 antigen, and has good specificity and high affinity, and can be applied to the preparation of CD276 detection reagent or anti-tumor drugs, etc. For example, the anti-human CD276 nanobody provided by the present application can be used as the antigen recognition domain of CAR, and then the CAR-T cell is constructed, which has a significant killing effect on various tumor cell lines expressing CD276 antigen. The anti-human CD276 nanobody provided by the present application can be endocytosed by CD276 expression positive cells. In addition, the anti-human CD276 nanobody provided by the present application can be used for detecting the expression of CD276 in tumor tissues. BRIEF DESCRIPTION OF DRAWINGS

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

[0034] Among them,

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

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

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

[0038] Figure 3 The antibody sequence alignment results are shown.

[0039] Figure 4 The phage-ELISA verification results are shown;

[0040] wherein,

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

[0042] Figure 4 (b) shows the reading value of clones with positive ELISA coloration in the second 96-well plate at 450 nm of the microplate reader, NC represents the negative control, and 1-10 represents 10 different clones.

[0043] Figure 5 shows the flow results of positive clone phage supernatant;

[0044] wherein,

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

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

[0047] Figure 6 shows the schematic diagram of constructing PMIGV-CD276-VHH1-mIgGFc and PMIGV-CD276-VHH2-mIgGFc carrier plasmid;

[0048] wherein,

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

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

[0051] Figure 7 shows the nucleic acid electrophoretogram of carrier construction of Test Example 1;

[0052] wherein,

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

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

[0055] Figure 8 Sequencing alignment results of the vector are shown;

[0056] wherein,

[0057] Figure 8 (a) Sequencing alignment results of the PMIGV-CD8 signal peptide-CD276-VHH1-mIgGFc vector are shown;

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

[0059] Figure 9 Restriction enzyme verification plasmid results are shown;

[0060] wherein,

[0061] Figure 9 (a) Restriction enzyme verification results of the expression vector of CD276-VHH1 in (a) are shown; Figure 8 (a) Restriction enzyme verification results of the expression vector of CD276-VHH1 in (a) are shown;

[0062] Figure 9 (b) Restriction enzyme verification results of the expression vector of CD276-VHH2 in (b) are shown. Figure 8 (b) Restriction enzyme verification results of the expression vector of CD276-VHH2 in (b) are shown.

[0063] Figure 10 Western blot verification of the expression and concentration of CD276-VHH1-mIgGFc protein and CD276-VHH2-mIgGFc protein are shown;

[0064] wherein,

[0065] Figure 10 (a) Expression of CD276-VHH1-mIgGFc protein before concentration (1X) and after concentration (10X) is shown in (a);

[0066] Figure 10 (b) Expression of CD276-VHH2-mIgGFc protein before concentration (1X) and after concentration (10X) is shown in (b).

[0067] Figure 11 Flow cytometry verification of the expression of CD276 in tumor cells is shown;

[0068] wherein,

[0069] Figure 11 (a) Expression of CD276 in Raji cells is shown in (a);

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

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

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

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

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

[0075] Figure 12 shows the fluorescence intensity of CD276 positive tumor cells;

[0076] wherein,

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

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

[0079] Figure 13 shows the binding of CD276 Nanobody to CD276 positive cell lines;

[0080] wherein,

[0081] Figure 13 (a) shows the binding of CD276-VHH1, CD276-VHH2 to Raji cell line, respectively;

[0082] Figure 13 (b) shows the binding of CD276-VHH1, CD276-VHH2 to HepG2 cell line, respectively;

[0083] Figure 13 (c) shows the binding of CD276-VHH1, CD276-VHH2 to Hela cell line, respectively;

[0084] Figure 13 (d) shows the binding of CD276-VHH1, CD276-VHH2 to KYSE270 cell line, respectively;

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

[0086] Figure 14 shows the binding of CD276-VHH1-mIgGFc and CD276-VHH2-mIgGFc to Hela cells at different concentration gradients by flow cytometry;

[0087] wherein,

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

[0089] Figure 14 (b) shows the detection effect when CD276-VHH1 fusion protein is used as primary antibody at a volume of 200 μl, 50 μl, 10 μl and 2 μl, respectively;

[0090] Figure 14 (c) shows the detection effect when CD276-VHH2 fusion protein is used as primary antibody at a volume of 200 μl, 50 μl, 10 μl and 2 μl, respectively.

[0091] Figure 15 shows the schematic diagram of CAR lentiviral vector plasmid;

[0092] wherein,

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

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

[0095] Figure 16 shows the electropherogram of vector construction in Test Example 2.

[0096] Figure 17 shows the plasmid sequencing alignment map;

[0097] wherein,

[0098] Figure 17 (a) shows the pEF-CD276-VHH1-CAR plasmid sequencing alignment map;

[0099] Figure 17(b) shows pEF-CD276-VHH2-CAR plasmid sequencing alignment map.

[0100] Figure 18 CAR lentivirus titer determination results are shown;

[0101] wherein,

[0102] Figure 18 (a) shows flow cytometry determination of CD276-VHH1-CAR lentivirus titer;

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

[0104] Figure 19 Lymphocyte nuclei CD3 + T cell magnetic bead sorting efficiency;

[0105] wherein,

[0106] Figure 19 (a) shows the proportion of CD3 + T cells in PBMC before sorting;

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

[0108] Figure 20 CAR-T cell positive rate after lentivirus infection is shown;

[0109] wherein,

[0110] Figure 20 (a) shows the CAR-T cell positive rate after lentivirus infection of the CD276-VHH1 group;

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

[0112] Figure 21 CD276-VHH1-CAR-T cells and CD276-VHH2-CAR-T cells killing five kinds of tumor cells under different effector target ratios are shown;

[0113] wherein,

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

[0115] Figure 21(b) shows killing of HepG2 cells;

[0116] Figure 21 (c) shows killing of KYSE30 cells;

[0117] Figure 21 (d) shows killing of Hela cells;

[0118] Figure 21 (e) shows killing of KYSE270 cells.

[0119] Figure 22 shows secretion of IFN-gamma during CAR-T cell killing;

[0120] wherein,

[0121] Figure 22 (a) shows secretion of IFN-gamma during CAR-T killing of Raji cells;

[0122] Figure 22 (b) shows secretion of IFN-gamma during CAR-T killing of HepG2 cells;

[0123] Figure 22 (c) shows secretion of IFN-gamma during CAR-T killing of KYSE30 cells;

[0124] Figure 22 (d) shows secretion of IFN-gamma during CAR-T killing of Hela cells;

[0125] Figure 22 (e) shows secretion of IFN-gamma during CAR-T killing of KYSE270 cells;

[0126] Figure 22 (f) shows ELISA detection standard curve for IFN-gamma.

[0127] Figure 23 shows Raji cell CD276-VHH1 / VHH2 endocytosis (50 pg) results;

[0128] wherein,

[0129] Figure 23 (a) shows Raji cell CD276-VHH1 endocytosis (50 pg) results;

[0130] Figure 23 (b) shows Raji cell CD276-VHH2 endocytosis (50 pg) results.

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

[0132] in,

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

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

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

[0136] in,

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

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

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

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

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

[0142] Figure 29 A negative control showing the binding of Nanobodies to esophageal cancer tissue is shown. DETAILED DESCRIPTION

[0143] In order to more clearly illustrate the technical solution of the present invention, the following examples are given. 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 complementarity determining region (CDR) and a framework region (FR);

[0146] wherein,

[0147] the complementarity determining regions CDR include complementarity determining regions CDR1-CDR3;

[0148] the complementarity determining regions CDR1-CDR3 are respectively shown as amino acid sequences 1-3;

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

[0150] the framework regions FR include framework regions FR1-FR5;

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

[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 anti-CD276 nanobody described above, including nucleic acid sequences SEQ ID No. 11 or SEQ ID No. 12.

[0156] a preparation method of the anti-CD276 nanobody described above, including the following steps:

[0157] S1, construction of a phage library

[0158] RNA extraction is performed from llama PBMC, and RNA reverse transcription is performed using a Novozyme RNA reverse transcription kit (HiScript II QSelect RT SuperMix for qPCR R232), and the reaction liquid preparation system and the RNA reverse transcription preparation system are respectively shown as Table 1 and Table 2.

[0159] Table 1 reaction liquid preparation system

[0160] Reagent Amount used Oligo dT Primer (50 μΜ) 1 μΐ dNTP Mix 1 μΐ Total RNA 5 μg ddH2O Up to 10 μΐ

[0161] Table 2 RNA reverse transcription preparation system

[0162] Reagent Amount used Reaction solution 10 μΐ 5x PrimeScript II Buffer 4 μΐ RNase Inhibitor 0.5 μΐ PrimeScript II RTase 1 μΐ ddH2O Up to 20 μΐ

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

[0164] Table 3 PCR amplification reaction system of VHH

[0165] Reagent Amount used 2*Phanta Max Master Mix 10 μΐ Primer 1 0.4 μΐ Primer 2 0.4 μΐ cDNA 0.6 μΐ ddH2O Up to 20 μΐ

[0166] After the reaction, 20 μl of PCR product was taken for 1% agarose gel electrophoresis, and the VHH product was obtained by gel recovery and purification at 600 bp. The results of PCR gel electrophoresis are shown in Figure 1 .

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

[0168] Among them,

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

[0170] Figure 1 (b) shows the results of running the second round of PCR product, Marker is the same as Figure 1 (a).

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

[0172] Table 4 VHH and phagemid vector ligation system

[0173]

[0174] The ligation product was subjected to electroporation, voltage: 2477V; time: 4.6ms; library capacity: 1.1x10 8 cfu. The positive rate of the library was detected using the primer on the vector and sequencing, and the results of library insertion rate detection are shown in Figure 2 .

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

[0176] Several clones were randomly picked from the positive clones of colony PCR detection for sequencing. After the light chain antibody fragment in the sequencing results was translated into amino acid sequence, sequence alignment was performed to detect sequence diversity in the bacterial library. The antibody sequence alignment results are shown in Figure 3 .

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

[0178] S2, Preparation of phage antibody library and panning of nanobodies

[0179] The phage antibody library was prepared by amplifying the glycerol bacteria using helper phage and detecting the titer. The antigen was coated on the immunotube and blocked with PBST, 1x10 12 of phage was added for panning, and Gly-Hcl at pH 2.0 and Tris-Hcl at pH 9.5 were used for neutralization as eluent. The steps of screening and panning were repeated three times, and the phage titer determination after three rounds of amplification and panning is shown in Table 5. The phage antibody library was enriched by the above operation.

[0180] Table 5 Panning and amplification results

[0181]

[0182]

[0183] S3, Verification of nanobodies

[0184] The phage infected TG1 after the third round of panning was picked for single clone phage-ELISA verification, and the verification results are shown in Figure 4 . The phage supernatant positive in ELISA was incubated with CD276-expressing tumor cells for flow cytometry detection, and the flow cytometry detection results are shown in Figure 5 .

[0185] The flow cytometry positive sample was sequenced and aligned with the llama sequence, and finally two nanobody sequences CD276-VHH1 and CD276-VHH2 were obtained, and the amino acid sequences are shown in SEQ ID No. 9 and SEQ ID No. 10.

[0186] Figure 4 The phage-ELISA verification results are shown;

[0187] Among them,

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

[0189] Figure 4 (b) shows the reading value of ELISA color-positive clones in the second plate 96-well plate at 450 nm of the microplate reader, NC represents negative control, 1-10 represents 10 different clones.

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

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

[0192] wherein,

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

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

[0195] The results show that the two positive clone phages screened can specifically bind to CD276-positive tumor cells, but not to negative cells.

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

[0197] Test method:

[0198] Vector plasmid construction: the CD276-VHH1 and CD276-VHH2 nanobody sequences obtained by screening in the examples are genetically synthesized, and a sufficient amount of DNA fragments are obtained by PCR amplification, and the construction of the vector plasmid is as shown in Figure 6 .

[0199] Vector construction: after separating the target band by gel electrophoresis, the DNA fragments encoding each nanobody are respectively constructed into a eukaryotic expression vector containing a mouse-IgG-Fc (mIgGFc) fragment, and the nucleic acid electrophoresis result of the vector construction is as shown in Figure 7 , and the sequencing result after the vector construction is completed is as shown in Figure 8 .

[0200] Enzymatic digestion verification: the large plasmid is verified by Xho1 and EcoR1 double enzyme digestion, and the nucleic acid electrophoresis detection is performed after enzyme digestion, and the result is as shown in Figure 9The target recombinant plasmid was obtained. The target plasmid was transfected into 293T cells by PEI transfection method for expression, and the supernatant was collected after 48h, concentrated and preliminarily purified to obtain the recombinant anti-CD276 nanobody.

[0201] Western blot verification: After the recombinant anti-CD276 nanobody was concentrated and purified, Western blot verification was carried out, and the verification results were as shown in Figure 10

[0202] CD276 expression verification: Select CD276 positive cell lines, and use commercial antibodies to verify the expression of CD276, and the verification results are as shown in Figure 11-12

[0203] Binding detection of nanobody and CD276 positive cell line: The obtained recombinant anti-CD276 nanobody was used as the first antibody of flow cytometry, and the second antibody coupled with FITC-anti-mouse-IgG was used to co-incubate with CD276 expressing tumor cells, and the binding of the recombinant anti-CD276 nanobody and the CD276 positive cell line was detected by machine, and the results are as shown in Figure 13

[0204] Binding ability detection of nanobody to CD276 expressing tumor cells: Hela cells were selected to detect the binding ability of CD276-VHH1 and CD276-VHH2 nanobodies to CD276 expressing tumor cells under different concentration gradients, and the results are as shown in Figure 14

[0205] Figure 6 The schematic diagram of constructing PMIGV-CD276-VHH1-mIgGFc and PMIGV-CD276-VHH2-mIgGFc carrier plasmid is shown;

[0206] Among them,

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

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

[0209] Figure 7 The nucleic acid electrophoretogram of the carrier construction of test example 1 is shown;

[0210] Among them,

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

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

[0213] The results show that the CD276-VHH1 and CD276-VHH2 fragments with correct band sizes are successfully PCR-ed.

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

[0215] Among them,

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

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

[0218] The results show that the constructed vector sequence is correct.

[0219] Figure 9 The enzyme digestion verification plasmid results are shown;

[0220] Among them,

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

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

[0223] The results show that the constructed vector structure is correct.

[0224] Figure 10 The Western blot verification of the expression and concentration of CD276-VHH1-mIgGFc protein and CD276-VHH2-mIgGFc protein is shown;

[0225] Among them,

[0226] Figure 10 (a) shows the expression of CD276-VHH1-mIgGFc protein without concentration (1X) and after concentration (10X);

[0227] Figure 10 (b) shows the expression of CD276-VHH2-mIgGFc protein without concentration (1X) and after concentration (10X).

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

[0229] Figure 11 shows the flow cytometry verification of the expression of CD276 in tumor cells;

[0230] wherein,

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

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

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

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

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

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

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

[0238] Figure 12 shows the fluorescence intensity of CD276 positive tumor cells;

[0239] wherein,

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

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

[0242] The results show that the expression of CD276 in the indicated tumor cells is different.

[0243] Figure 13 The binding of CD276 nanobodies to CD276 positive cell lines is shown;

[0244] wherein,

[0245] Figure 13 (a) shows the binding of CD276-VHH1, CD276-VHH2 to Raji cell line, respectively;

[0246] Figure 13 (b) shows the binding of CD276-VHH1, CD276-VHH2 to HepG2 cell line, respectively;

[0247] Figure 13 (c) shows the binding of CD276-VHH1, CD276-VHH2 to Hela cell line, respectively;

[0248] Figure 13 (d) shows the binding of CD276-VHH1, CD276-VHH2 to KYSE270 cell line, respectively;

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

[0250] The results show that the two nanobodies CD276-VHH1, CD276-VHH2 screened by the application can be used for flow detection of the expression of CD276 in cells.

[0251] Figure 14 The binding effect of CD276-VHH1-mIgGFc and CD276-VHH2-mIgGFc to Hela cells under different concentration gradients is shown by flow cytometry;

[0252] wherein,

[0253] Figure 14 (a) shows the binding of Hela to CD276-VHH1 and CD276-VHH2 primary antibody, secondary antibody and commercialized antibody, respectively;

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

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

[0256] The results show that the binding rate of the two nanobodies screened by the application to Hela cells changes with the amount of the antibody used.

[0257] From the above results, it can be seen that the CD276-VHH1 and CD276-VHH2 nanobodies prepared in the examples can both recognize CD276-positive tumor cell lines, so the application can be used for the production of flow antibodies for the recognition of CD276-positive tumor cell lines.

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

[0259] Test method:

[0260] Vector construction: the gene fragments of the anti-CD276 nanobodies of Test Example 1 are amplified by PCR to obtain sufficient DNA fragments, and after the target band is separated by gel electrophoresis, the DNA fragments encoding each nanobody are constructed into a eukaryotic expression vector containing a CAR structure, as shown in the vector construction schematic Figure 15 , and the nucleic acid electrophoresis result is shown in Figure 16 .

[0261] Plasmid extraction: the target plasmid can be obtained by ligation, transformation and plasmid extraction, and the sequencing result is shown in Figure 17 .

[0262] Titer determination: virus packaging and production are performed by a third-generation lentivirus packaging system, and titer determination is performed after virus production, and the results are shown in Figure 18 .

[0263] CAR-T cell killing test of CD276 tumor cell lines: PBMCs were isolated from human peripheral blood, and human CD3 + T cells were isolated and activated from the PBMCs, and the sorted CD3 + T cell ratio is shown in Figure 19 . Human CD3 + T cells were infected with lentivirus to prepare CAR-T cells, and the CD276-mIgGFc extracellular segment protein prepared was used to detect the positive rate of the CAR-T cells, and the positive rate result is shown in Figure 20The killing of CD276 tumor cell lines was designed according to the positive rate of CAR-T cells. The present application killed five cell lines, namely CD276 positive cell lines Hela, HepG2, KYSE30, KYSE270 and CD276 negative cell line Raji. CAR-T cells were incubated with tumor cells 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, and the results are shown in Figure 21 In addition, the secretion of IFN-γ was detected, and the results are shown in Figure 22

[0264] Figure 15 The schematic diagram of the CAR lentivirus vector plasmid is shown;

[0265] wherein,

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

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

[0268] Figure 16 The electropherogram of the vector construction of Test Example 2 is shown; the results show that the DNA fragments obtained by enzyme digestion and PCR have correct band sizes.

[0269] Figure 17 The plasmid sequencing alignment map is shown;

[0270] wherein,

[0271] Figure 17 (a) shows the pEF-CD276-VHH1-CAR plasmid sequencing alignment map;

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

[0273] The results show that the plasmid with correct sequence is successfully constructed.

[0274] Figure 18 The CAR lentivirus titer determination results are shown;

[0275] wherein,

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

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

[0278] The results show that the titer of lentivirus CD276-VHH1-CAR is 2.6 x 10 5 IU / mL; the titer of CD276-VHH2-CAR is 1.07 x 10 6 IU / mL; which can be used for subsequent infection of T cells.

[0279] Figure 19 shows the proportion of lymphocyte nuclei CD3 + T cells in PBMC;

[0280] wherein,

[0281] Figure 19 (a) shows the proportion of CD3 + T cells in PBMC before sorting;

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

[0283] The results show that the proportion of CD3 + T cells before sorting is 76.2%, the proportion of CD3 + T cells after magnetic bead sorting is 99.9%, and CD3 + T cells are successfully sorted.

[0284] Figure 20 shows the positive rate of CAR-T cells after lentivirus infection;

[0285] wherein,

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

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

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

[0289] Figure 21 shows the killing effect of CD276-VHH1-CAR-T cells and CD276-VHH2-CAR-T cells on five kinds of tumor cells under different effector-target ratios;

[0290] wherein,

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

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

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

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

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

[0296] The results show that the CAR-T cells constructed by the two nanobody sequences screened by the application have the killing ability on CD276 positive tumor cells.

[0297] Figure 22 shows the secretion of IFN-γ in the killing process of CAR-T cells;

[0298] wherein,

[0299] Figure 22 (a) shows the secretion of IFN-γ in the killing process of CAR-T on Raji cells;

[0300] Figure 22 (b) shows the secretion of IFN-γ in the killing process of CAR-T on HepG2 cells;

[0301] Figure 22 (c) shows the secretion of IFN-γ in the killing process of CAR-T on KYSE30 cells;

[0302] Figure 22 (d) shows the secretion of IFN-γ in the killing process of CAR-T on Hela cells;

[0303] Figure 22 (e) shows the secretion of IFN-γ in the killing process of CAR-T on KYSE270 cells.

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

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

[0306] From the test results, the two nanobodies CD276-VHH1 and CD276-VHH2 can be used as antigen recognition domains of CAR to kill tumor cells.

[0307] Test Example 3 (Nanobodies can be endocytosed by CD276 positive cells and can be used for the preparation of ADC drugs)

[0308] Test method:

[0309] Cell preparation: CD276 positive cells: Hela, HCT116; CD276 negative cells: Raji cells. 1-5x10 5 cells of each were collected in 1.5ml EP tubes, and 6 tubes were collected for each cell.

[0310] Antibody preparation: VHH-mIgGFc protein about 500ul for each.

[0311] Antibody incubation scheme: collect cells into EP tubes, centrifuge to remove supernatant. Add 25ul of corresponding protein, incubate at 4°C for 30min. Terminate incubation with PBS, wash away unbound antibodies, add 500ul of 4°C pre-cooled or 37°C pre-heated complete medium to resuspend the cells, incubate at 4°C and 37°C for 4h / 6h respectively, and detect once each time. After washing with PBS, add a fluorescent secondary antibody against mouse, incubate at 4°C for 30min. Terminate incubation and wash with PBS, and then detect by flow cytometry. The results of protein endocytosis of Raji cells, Hela cells and HCT116 cells are shown in Figure 23-25 Figures 6 shows the internalization ratio of CD276-VHH1 and CD276-VHH2 in different cells.

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

[0313]

[0314] Figure 23 Raji cell CD276-VHH1 / VHH2 endocytosis (50ug) results are shown;

[0315] wherein,

[0316] Figure 23 (a) shows the results of Raji cell CD276-VHH1 endocytosis (50ug);

[0317] Figure 23 (b) shows the results of Raji cell CD276-VHH2 endocytosis (50ug).

[0318] The results show that Raji cells not expressing CD276 have less endocytosis of CD276-VHH1 and CD276-VHH2 antibodies.

[0319] Figure 24 Results of Hela cell CD276-VHH1 / VHH2 endocytosis (50 μg) are shown;

[0320] wherein,

[0321] Figure 24 (a) Results of Hela cell CD276-VHH1 endocytosis (50 μg) are shown;

[0322] Figure 24 (b) Results of Hela cell CD276-VHH2 endocytosis (50 μg) are shown.

[0323] The results show that Hela cells expressing CD276 have more endocytosis of CD276-VHH1 and CD276-VHH2 antibodies.

[0324] Figure 25 Results of HCT116 cell CD276-VHH1 / VHH2 endocytosis (50 μg) are shown;

[0325] wherein,

[0326] Figure 25 (a) Results of HCT116 cell CD276-VHH1 endocytosis (50 μg) are shown;

[0327] Figure 25 (b) Results of HCT116 cell CD276-VHH2 endocytosis (50 μg) are shown.

[0328] The results show that HCT116 cells expressing CD276 have more endocytosis of CD276-VHH1 and CD276-VHH2 antibodies.

[0329] From the above test results, it can be seen that the two nanobodies CD276-VHH1 and CD276-VHH2 of the present application can be endocytosed by CD276 positive cells, and can be used for producing ADC and other drugs that need to enter cells to play a role.

[0330] Test Example 4 (Recognition and binding of nanobodies with esophageal cancer tissue)

[0331] Test method:

[0332] Paraffin section deparaffinization to water: put the section into the following reagents in turn for 10 min each time: environmental deparaffinization solution I, environmental deparaffinization solution II, environmental deparaffinization solution III, anhydrous ethanol I, anhydrous ethanol II, anhydrous ethanol III, and distilled water.

[0333] Antigen repair: the repair conditions are shown in Table 7. During the repair process, the buffer should be prevented from excessive evaporation, and the slice should not be dried. After the repair is completed, the slice is naturally cooled. Place the slice in PBS (pH 7.4) and shake for washing 3 times for 5 min each time on a decolorizing shaker.

[0334] Circle serum blocking: after the slice is slightly dried, use a histological pen to draw a circle around the tissue, and drop BSA (10% donkey serum is used for blocking when the primary antibody is goat-derived, and 3% BSA is used for blocking when the primary antibody is derived from other sources), and block for 30 min.

[0335] Addition of a primary antibody: drop the prepared primary antibody, and place the slice flat in a wet box for incubation at 4°C overnight.

[0336] Addition of a secondary antibody: place the slice in PBS (pH 7.4) and shake for washing 3 times for 5 min each time on a decolorizing shaker. Add the corresponding secondary antibody, and incubate at room temperature for 50 min in the dark.

[0337] DAPI re-staining of cell nuclei: place the slice in PBS (pH 7.4) and shake for washing 3 times for 5 min each time on a decolorizing shaker. Add DAPI staining solution, and incubate at room temperature for 10 min in the dark.

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

[0339] Mounting: mount with an anti-fluorescence quenching mounting agent.

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

[0341] Result interpretation: the cell nuclei in the DAPI channel are blue, and the positive in the 488 channel is green, and the results are as shown in Figure 26-29 .

[0342] Table 7 Antigen repair conditions

[0343]

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

[0345] Figure 27 The binding of CD276-VHH1 to esophageal cancer is shown.

[0346] Figure 28 The binding of CD276-VHH2 to esophageal cancer is shown.

[0347] Figure 29 The negative control of the binding of nanobody to esophageal cancer tissue is shown.

[0348] The above results show that the CD276-VHH1 and CD276-VHH2 nanobodies screened by the application can be used for immunohistochemical detection of the expression of CD276 in human esophageal cancer tissue.

[0349] In summary, the results of Test Examples 1-4 show that the anti-human CD276 nanobody provided by the application can bind to CD276 antigen, can sensitively detect CD276 antigen molecules; can be used as an antigen recognition domain of CAR and then construct CAR-T cells, has a significant killing effect on a variety of tumor cell lines expressing CD276 antigen; and can be used for preparing ADC and other drugs that need to enter CD276 positive cells to play a role. Therefore, it can be applied to the diagnosis and treatment of malignant tumors, and has excellent market prospects.

[0350] It is obvious to those skilled in the art that the application is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or essential characteristics of the application. Therefore, the embodiments should be regarded as exemplary and non-limiting, and the scope of the application is defined by the appended claims rather than the above description, and all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the application.

[0351] In addition, it should be understood that although the present specification is described in terms of embodiments, not every embodiment contains only one independent technical solution, and the description manner of the specification is only for the sake of clarity, those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be properly combined to form other embodiments that those skilled in the art can understand.

Claims

1. An anti-CD276 Nanobody, characterized in that, The anti-CD276 nanobody comprises complementarity determining regions CDR and framework regions FR; wherein, The complementarity determining regions CDR comprise complementarity determining regions CDR1-CDR3; The amino acid sequences of the complementarity determining regions CDR1-CDR3 are shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, respectively.

2. The anti-CD276 Nanobody of claim 1, wherein The amino acid sequences of the framework regions FR are shown in SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, respectively; or The amino acid sequences of the framework regions FR are shown in SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, respectively.

3. The anti-CD276 Nanobody of claim 1, wherein The anti-CD276 nanobody is CD276-VHH1 or CD276-VHH2; The amino acid sequences of the CD276-VHH1 and CD276-VHH2 are shown in SEQ ID No. 9-SEQ ID No.

10.

4. The anti-CD276 Nanobody of claim 1, wherein The anti-CD276 nanobody is selected from the group consisting of an immunized llama antibody, a chimeric antibody, a humanized antibody.

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 of claim 5, wherein The nucleotide sequence of the nucleic acid molecule is SEQ ID No. 11 or SEQ ID No.

12.

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

8. The carrier of claim 7, wherein, The vector is selected from the group consisting of a DNA molecule, an mRNA molecule or a cell.

9. Use of the vector according to any one of claims 7-8 for the manufacture of a CD276 antigen detection reagent or a CD276 adsorbing antigen reagent.

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