A nanobody probe for identifying circulating tumor cells, a preparation method and application thereof

Abstract

The application discloses a nanobody probe for identifying circulating tumor cells, a preparation method and application thereof, and belongs to the technical field of biological detection. The probe takes EpCAM and Vimentin as double targets, is composed of a fluorescence reporter group coupled with a bispecific nanobody targeting two antigens, and the amino acid sequence of the bispecific nanobody is shown as SEQ ID NO. 5-SEQ ID NO. 8. The probe realizes synchronous capture of epithelial and mesenchymal CTCs, has the advantages of small molecular weight and high affinity, significantly improves the sensitivity and accuracy of CTC detection in an ex vivo peripheral blood sample, is simple to prepare, controllable in cost, and is suitable for in vitro qualitative and quantitative analysis of CTCs.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to a nanoantibody probe for identifying circulating tumor cells, its preparation method, and its application. Background Technology

[0002] Circulating tumor cells (CTCs) are tumor cells that detach from the primary lesion of a solid tumor and invade the peripheral blood circulation. Quantitative and subtyping detection of CTCs in peripheral blood can provide crucial reference for research on basic tumor mechanisms, clinical sample tracing, in vitro efficacy evaluation of antitumor drugs, and investigation of tumor metastasis mechanisms. However, the natural abundance of CTCs in peripheral blood is extremely low, typically only a small percentage (approximately 10 per 100 cells). 6 ~10 7 Only a few to dozens of CTCs exist in a single peripheral blood nucleated cell, and a large number of blood cells will form a strong background interference. How to achieve high specificity and high sensitivity in the identification and quantification of CTCs in peripheral ex vivo samples has become a key technical bottleneck that urgently needs to be overcome in the field of in vitro biological probe research and development.

[0003] Epithelial cell adhesion molecule (EpCAM / CD326) is a classic surface marker of epithelial tumors, highly expressed on the surface of epithelial tumor cells such as breast cancer, non-small cell lung cancer, colorectal cancer, and gastric cancer, while peripheral blood cells from healthy individuals express almost no of this molecule. Currently, most commercially available CTC enrichment detection reagents are based on full-length EpCAM monoclonal antibodies and are used for initial screening of CTCs in ex vivo blood samples. However, epithelial-mesenchymal transition (EMT) is a core biological process by which tumor cells acquire the ability to invade and migrate; mesenchymal CTCs that undergo EMT show downregulated or even complete absence of EpCAM expression. Relying solely on EpCAM single-target recognition systems is prone to missing such CTCs, failing to capture all CTC subtypes in the sample, significantly reducing the accuracy of CTC detection in peripheral blood samples, and severely limiting the clinical application scenarios of single-target probes.

[0004] Vimentin is a classic biomarker of mesenchymal phenotype. Its expression is specifically upregulated during tumor EMT, making it a characteristic marker of EpCAM-negative mesenchymal CTCs. Furthermore, healthy peripheral mature blood cells express almost no Vimentin. Therefore, a dual-target synergistic recognition strategy combining EpCAM and Vimentin can simultaneously capture epithelial CTCs (EpCAM-positive CTCs) in ex vivo samples. + Vim - ) and interstitial CTC (EpCAM) - Vim + This effectively compensates for the deficiency of single-target probes in missing EMT subtype CTCs and is also the mainstream research and development approach to improve the in vitro detection rate of all CTC subtypes.

[0005] Immunofluorescence staining, relying on fluorescently labeled antibodies and combined with fluorescence microscopy, enables the localization and quantitative analysis of antigens in biological samples. It is a mature technology in the field of bioassay, and its detection performance is highly dependent on the signal stability and targeted binding activity of the fluorescent probe. Currently, most in vitro CTC immunofluorescence probes are prepared using full-length IgG antibodies conjugated with fluorescent dyes. However, full-length IgG suffers from drawbacks such as high cost of large-scale production, large batch-to-batch activity fluctuations, and significant non-specific adsorption after fluorescence conjugation, which can significantly increase background fluorescence in blood samples and reduce the signal-to-noise ratio. Nanobodies (Nb) are derived from the VHH fragment of the heavy chain variable region of camel carnivora, with a molecular weight of only 12-15 kDa, approximately 1 / 10 that of traditional IgG. They possess advantages such as stable physicochemical properties, ease of genetic engineering modification, low-cost prokaryotic mass production, and low non-specific binding levels. They can also recognize occult antigenic epitopes that are difficult for traditional antibodies to bind, making them ideal targeting elements for optimizing the sensitivity of immunofluorescence detection.

[0006] Currently, no paired dual-target nanobody fluorescent probes targeting both EpCAM and Vimentin have been constructed. Traditional single-target IgG fluorescent probes and ordinary fluorescently modified probes cannot simultaneously identify two CTC subtypes, and they generally suffer from unstable fluorescence signals and easy decay of targeting activity, making them unsuitable for the research and clinical needs of comprehensive and highly sensitive CTC detection in small amounts of peripheral blood samples. Therefore, this invention proposes the development of an EpCAM / Vimentin dual-target nanobody fluorescent probe and the establishment of a corresponding in vitro CTC-specific detection system for in vitro samples. This is expected to solve the problems of missed detections and insufficient stability of existing probes, improve the overall technology system for in vitro CTC detection, and has significant theoretical and clinical application value. Summary of the Invention

[0007] The purpose of this invention is to provide a nanobody probe for identifying circulating tumor cells, its preparation method, and its application. Based on the EpCAM+Vimentin dual-target synergistic targeting strategy, and taking advantage of the small molecular weight, high affinity, strong stability, and ease of modification of nanobodies, a bispecific nanobody is constructed that can simultaneously identify epithelial, mixed, and stromal tumor cells, significantly reducing the false negative rate and improving detection specificity and sensitivity.

[0008] To achieve the above objectives, this application provides the following technical solution:

[0009] In a first aspect, this application provides a bispecific nanobody comprising a first nanobody domain targeting epithelial cell adhesion molecules, a second nanobody domain targeting vimentin, and a flexible linker peptide connecting the first and second nanobody domains; the first nanobody domain, the flexible linker peptide, and the second nanobody domain are tandemly linked.

[0010] According to some embodiments of the present application, the amino acid sequence of the first nanobody domain is as shown in SEQ ID NO.1 or SEQ ID NO.2, or has at least 90% homology with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2; the amino acid sequence of the second nanobody domain is as shown in SEQ ID NO.3 or SEQ ID NO.4, or has at least 90% homology with the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4.

[0011] The bispecific nanobody according to some embodiments of this application is any one of the following combinations:

[0012] The amino acid sequence of the first nanobody domain is shown in SEQ ID NO.1, and the amino acid sequence of the second nanobody domain is shown in SEQ ID NO.3;

[0013] The amino acid sequence of the first nanobody domain is shown in SEQ ID NO.1, and the amino acid sequence of the second nanobody domain is shown in SEQ ID NO.4;

[0014] The amino acid sequence of the first nanobody domain is shown in SEQ ID NO.2, and the amino acid sequence of the second nanobody domain is shown in SEQ ID NO.3;

[0015] The amino acid sequence of the first nanobody domain is shown in SEQ ID NO.2, and the amino acid sequence of the second nanobody domain is shown in SEQ ID NO.4.

[0016] According to some embodiments of the present application, the bispecific nanobody has the amino acid sequence of the flexible linker peptide as shown in SEQ ID NO.9.

[0017] In a second aspect, this application provides a nanobody probe comprising a fluorescent reporter group and a bispecific nanobody as described in any of the above schemes; the bispecific nanobody is coupled to the fluorescent reporter group.

[0018] According to the nanobody probes in some embodiments of this application, the fluorescent reporter group includes at least one of fluorescein isothiocyanate, phycoerythrin, allophycocyanin, anthocyanin dyes, or Alexa Fluor series dyes.

[0019] On a third-party level, this application provides a method for preparing the bispecific nanobody and the nanobody probe described in any of the above solutions, comprising the following steps:

[0020] Constructing a nanobody phage display library: Alpaca were immunized with epithelial cell adhesion molecules and vimentin, respectively, peripheral blood lymphocytes were isolated and total RNA was extracted. cDNA was obtained by reverse transcription and then amplified by PCR to obtain the VHH gene. The VHH gene was ligated to a phage vector, packaged and amplified to obtain a phage display library.

[0021] Screening for specific nanobodies: The phage display library was screened multiple times using epithelial cell adhesion molecules and vimentin as targets, respectively. Positive clones were screened by ELISA, and nanobody genes were obtained by sequencing.

[0022] Construction of genetically engineered bacteria: Based on the nanobody gene, a recombinant plasmid was constructed according to the tandem structure of the first nanobody domain-flexible linker peptide-second nanobody domain. The recombinant plasmid was transformed into competent Escherichia coli cells, and genetically engineered bacteria expressing bispecific nanobodies were screened to obtain the bacteria.

[0023] Induction of expression and purification: The genetically engineered bacteria were induced to express the bacteria, and the supernatant was collected and purified by affinity chromatography to obtain bispecific nanobodies.

[0024] Fluorescent conjugation: The bispecific nanobody is chemically conjugated with a fluorescent reporter group to obtain a nanobody probe.

[0025] In a fourth aspect, this application provides a nucleic acid molecule encoding a bispecific nanobody as described in any of the above schemes.

[0026] In a fifth aspect, this application provides a kit comprising a bispecific nanobody as described in any of the above-described schemes; or a nanobody probe as described in the above-described schemes; or a nucleic acid molecule as described in the above-described schemes; and an adjuvant acceptable for detection.

[0027] In a sixth aspect, this application provides the use of the bispecific nanobody described in any of the above-described schemes; or the nanobody probe described in the above-described schemes; or the nucleic acid molecule described in the above-described schemes; or the kit described in the above-described schemes in the preparation of preparations for the specific capture, adsorption and / or detection of circulating tumor cells.

[0028] The beneficial effects of this invention are:

[0029] This invention employs a dual-target synergistic targeting strategy using EpCAM and Vimentin to prepare a bispecific nanobody that can identify circulating tumor cells with different phenotypes, avoiding the problem of missed detection by single targets and comprehensively covering all stages of tumor development. The peptide exhibits nanomolar-level high affinity for both antigens, and combined with the fact that their expression in normal tissues does not overlap, it demonstrates excellent targeting specificity. Furthermore, the peptide is prepared based on nanobody technology, resulting in a small molecular weight, good penetration and stability, and easy modification and conjugation. It can meet diverse applications such as tumor detection, pathological analysis, and targeted therapy. Moreover, relying on phage display technology and the E. coli prokaryotic expression system, the process is mature, the cost is controllable, and it is suitable for large-scale production. Attached Figure Description

[0030] Figure 1 These are positive monoclonal identification images from Example 2 of the present invention, wherein A is an identification image of EpCAM-specific nanobody and B is an identification image of Vimentin-specific nanobody;

[0031] Figure 2 This is the kinetic curve of the monoclonal nanobody in Example 5 of the present invention;

[0032] Figure 3 This is the kinetic curve of the bispecific nanobody nanobody in Example 5 of the present invention;

[0033] Figure 4 This is the result of fluorescent staining in Example 7 of the present invention. Detailed Implementation

[0034] The above-described solutions and other embodiments of the present invention will be described in more detail below. The definitions of relevant terms and general rules are as follows:

[0035] (1) Unless otherwise stated, the term “sequence” as used herein (e.g., “antibody sequence”, “variable region sequence”, “VHH sequence”, “protein sequence”, etc.) shall, without contextual limitation, encompass both the corresponding amino acid sequence and the nucleic acid / nucleotide sequence encoding that amino acid sequence.

[0036] (2) Unless otherwise stated, all methods, procedures, processes and experimental techniques not described in detail herein are conventional and mature methods in the field and are known to those skilled in the art.

[0037] (3) The term "specificity" as used herein refers to the ability of antigen-binding molecules (such as the nanobodies and peptides of this invention) to distinguish and bind to different antigens and different antigenic epitopes. The specificity of antigen-binding molecules can be determined by their binding affinity and binding activity: among which the dissociation equilibrium constant (K... D K is used to characterize the binding strength between an antigen and an antigen-binding molecule. DThe smaller the value, the stronger the binding force between the two, and vice versa; K a As the associative constant, K a A larger value indicates a faster binding rate, while a smaller value indicates a slower binding rate; K d K is the dissociation constant. d A larger value indicates a faster dissociation rate of the complex, while a smaller value indicates a slower dissociation rate; the conversion relationship is satisfied: K D = K d / K a .

[0038] This invention provides a nanobody probe for recognizing circulating tumor cells (CTCs), its preparation method, and its application, to solve the problems of traditional probes being unable to simultaneously recognize two CTC subtypes, as well as unstable fluorescence signals and easy attenuation of targeting activity. Specifically, in a first aspect, this invention provides a bispecific nanobody comprising a first nanobody domain targeting epithelial cell adhesion molecules, a second nanobody domain targeting vimentin, and a flexible linker peptide connecting the first and second nanobody domains; the first nanobody domain, the flexible linker peptide, and the second nanobody domain are sequentially connected in series.

[0039] Preferably, the amino acid sequence of the first nanobody domain is as shown in SEQ ID NO.1 or SEQ ID NO.2, or has at least 90% homology with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2; the amino acid sequence of the second nanobody domain is as shown in SEQ ID NO.3 or SEQ ID NO.4, or has at least 90% homology with the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4.

[0040] Preferably, the amino acid sequence of the bispecific nanobody includes SEQ ID NO.5 to SEQ ID NO.8.

[0041] In a second aspect, the present invention provides a nanobody probe comprising a fluorescent reporter group and the above-mentioned bispecific nanobody; wherein the bispecific nanobody is coupled to the fluorescent reporter group; wherein the fluorescent reporter group comprises at least one of fluorescein isothiocyanate, phycoerythrin, allophycocyanin, anthocyanin dye or Alexa Fluor series dyes.

[0042] Thirdly, the present invention provides a method for preparing the above-mentioned bispecific nanobodies and nanobodies probes, comprising the following steps:

[0043] S1. Constructing a nanobody phage display library: Alpaca were immunized with epithelial cell adhesion molecules and vimentin, respectively, peripheral blood lymphocytes were isolated and total RNA was extracted. cDNA was obtained by reverse transcription and then amplified by PCR to obtain the VHH gene. The VHH gene was ligated with a phage vector, packaged and amplified to obtain a phage display library.

[0044] S2. Screening for specific nanobodies: The phage display library was screened multiple times using epithelial cell adhesion molecules and vimentin as targets, respectively. Positive clones were screened by ELISA, and nanobody genes were obtained by sequencing.

[0045] S3. Constructing genetically engineered bacteria: Based on the nanobody gene, a recombinant plasmid is constructed according to the tandem structure of the first nanobody domain - flexible linker peptide - second nanobody domain. The recombinant plasmid is transformed into competent Escherichia coli cells, and genetically engineered bacteria expressing bispecific nanobodies are screened to obtain the bacteria.

[0046] S4. Induction of expression and purification: The genetically engineered bacteria were induced to express the bacteria, and the supernatant was collected and purified by affinity chromatography after cell lysis to obtain bispecific nanobodies;

[0047] S5. Fluorescence coupling: The bispecific nanobody is chemically coupled with a fluorescent reporter group to obtain a nanobody probe.

[0048] Fourthly, the present invention provides a nucleic acid molecule that encodes the above-mentioned bispecific nanobody.

[0049] Fifthly, the present invention provides the application of the bispecific nanobody in immunoassay, enrichment and / or purification.

[0050] Preferably, the bispecific nanobody is used in the preparation of CTC purification kits and CTC detection kits.

[0051] Preferably, the application of the bispecific nanobody in capturing and detecting CTC cells.

[0052] Example

[0053] The embodiments of the present invention are described in detail below with reference to examples. However, the present invention is not limited to these embodiments. Any modifications and substitutions made within the scope of the present invention without impairing its technical effects are within the scope of protection of the present invention.

[0054] Example 1: Construction of nanobody library.

[0055] The phage display library used in this invention is an immune library based on T7 phage, and the establishment steps are as follows:

[0056] (1) Two alpacas numbered E01 and V01 were immunized with EpCAM antigen and Vimentin antigen respectively, for a total of 4 immunizations. After immunization, jugular vein blood was collected from the two alpacas, peripheral blood lymphocytes were separated, and total RNA was extracted from the cells using PureLink™ RNA Mini Kit (catalog number: 12183018A, Life Technologies).

[0057] (2) The total RNA obtained was reverse transcribed to obtain cDNA, and then the VHH gene was amplified by two rounds of nested PCR.

[0058] The first round of PCR used cDNA as a template, and amplification was performed using upstream primer UP primer1 and downstream primer DOWN primer1, respectively. The target band of 650-750 bp was recovered and used as the template for the second round of PCR. The upstream and downstream primers for the second round of PCR were UP primer2 and DOWN primer2, respectively. The PCR product of 450-500 bp was recovered after amplification.

[0059] The primer sequences are as follows:

[0060] UP primer1: AGGTGGTCCTGGCTGCTCT

[0061] DOWN primer1:GGTACGTGCTGTTGAACTGTA

[0062] UP primer2: CTAGTC GAATTC CGCCCAGGTGCAGCTC

[0063] DOWN primer2: AGCGACTAAGCTTTGAGGAGACGGT

[0064] (3) The PCR products were double-digested with EcoRI and HindIII restriction endonucleases. The digested products were separated by agarose gel electrophoresis, and the nucleic acid band of 350-500 bp was recovered to obtain the VHH gene fragment.

[0065] (4) Use T4 DNA ligase to ligate the above VHH gene fragment with the T7 vector. The vector kit used is T7Select® 10-3 Cloning Kit (Merck Millipore Novagen®, catalog number: 70550-3).

[0066] (5) Mix the ligation product with the phage packaging protein and assemble to obtain complete T7 phage particles. Amplify the phage mixture system and construct the original phage library.

[0067] (6) The titer of the original phage library obtained was 7.63 × 10⁻⁶ after library quality testing. 9 pfu / mL, library diversity reached 6.2 × 10⁻⁶. 6 .

[0068] Example 2: Screening of nanobodies.

[0069] A. EpCAM-specific nanobody screening

[0070] Phage panning experiments were conducted using 96-well plates. The specific procedures were as follows: EpCAM antigen was diluted with TBS buffer to a concentration of 10 μg / mL, and 100 μL of antigen dilution was added to each well. The plates were incubated at 4°C for 12 h. The liquid in the wells was aspirated, and the plates were washed three times with TBS buffer and then blotted dry. Subsequently, 300 μL of 1% protein-free blocking buffer (Sangon Biotech Co., Ltd.) was added to each well, and the plates were blocked and incubated at room temperature for 2 h.

[0071] After blocking, the blocking solution was aspirated, the plate was washed 6 times with TBST buffer and blotted dry. 100 μL of the amplified phage library was added to each well and incubated at room temperature for 30 min. After incubation, the plate was washed 10 times with TBST buffer to remove unbound phages. Then, T7 elution buffer containing 1% SDS was added for elution, and the plate was incubated at room temperature for 30 min. The eluent was collected and amplified for the next round of panning.

[0072] After three rounds of screening, the elution buffer was used for solid plate amplification. Twelve single phage plaques were randomly selected and inoculated sequentially into deep-well plates containing 1 mL of host bacterial culture for phage amplification. Each clone was numbered according to its inoculation location. After amplification, the plates were centrifuged, and the supernatant containing the phages was collected.

[0073] Antigen coating was performed using ELISA plates: EpCAM antigen at a concentration of 1 μg / mL was hydrophobically coated, with a blank negative control (no antigen coating) included. Blocking was performed using 3% BSA. 100 μL of the phage amplification supernatant was added to each well, followed by incubation and washing 10 times with TBST buffer. Horseradish peroxidase-labeled anti-VHH HRP IgG nanobody was then added, followed by further incubation and washing 5 times with TBS buffer. ELISA chromogenic solution was then added for colorimetric reaction, and the absorbance (OD) of each well was measured at 450 nm. 450 ).

[0074] The absorbance ratio of the test wells to the negative control wells was calculated. A ratio greater than 10 was considered a positive clone, indicating that the phage clone significantly bound to the corresponding antigen. A higher absorbance ratio indicates stronger binding ability and affinity between the phage and the antigen. For detailed screening results, please refer to [link to relevant documentation]. Figure 1 A.

[0075] B. Vimentin-specific nanobody screening

[0076] Vimentin antigen was diluted to a concentration of 10 μg / mL using TBS buffer. 100 μL of the antigen dilution was added to each well of a 96-well plate and incubated at 4°C for 12 h. Subsequent panning, amplification, and positive clone identification procedures were the same as in Part A of this embodiment. Screening results are detailed in [link to sample plate]. Figure 1 B.

[0077] Positive clones obtained from screening for two types of antigens, with an absorbance ratio greater than 10 and ranking among the top two in binding ability, were selected for gene sequencing identification. PCR amplification was performed using the corresponding plaque amplification solution as a template, employing upstream primer UP primer3 and downstream primer DOWN primer3.

[0078] The primer sequences are as follows:

[0079] UP primer 3: TTCCTTAA CATATG GCCCAGGTGCAGCTA

[0080] DOWN primer3: AAGGAA CTCGAG CACGGTGACCAGGGTC

[0081] A portion of the PCR amplification products were sent to a professional institution for sequencing to obtain the nanobody gene sequences corresponding to each positive clone. The names and sequence numbers of each monoclonal nanobody are detailed in Table 1.

[0082] Table 1. Names and Serial Numbers of Monoclonal Nanobodies

[0083]

[0084] SEQ ID No. 1:

[0085] AQVQLQESGGGLVQAGGSLRLSCAASGAGRTDFHIMGWFRQAPGKEREFVAGIAGTLSATRYTDSVKGRFTISRDNAQNTVYLQMNSLRPEDTAVYYCETGRLFPMQASVDNKWGQGTQVTVSS

[0086] SEQ ID No. 2:

[0087] AQVQLQESGGGLVQPGGSLRLSCAASGAGLSDPHIMGWFRQAPGKEREFVARIGTLASARTYTDSVKGRFTISGDNAQNTVYLQMNSLKPEDTAVYYCETGRHFFMQASVDNKWGQGTQVTVSS

[0088] SEQ ID No. 3:

[0089] AQVQLQESGGGLVQPGGSLRLSCAVSGSHGADLPEMHWFRQAPGKQRELVATIATATSLAYADSVKGRFTISRENTKNTVYLQMSSLKPEDTAVYYCDFLATMVGSQNKPEWGQGTQVTVSS

[0090] SEQ ID No.4:

[0091] AQVQLQESGGGLVQAGGSLRLSCVASGSLSADLFTMHWFRQAPGKQRDLVATITAAGTLAYAGSAKGRFTISRDNGMNTAFLQMNSLKPEDTAVYYCDPLASMVGSQNLPEWGQGTQVTVSS.

[0092] Example 3: Construction of genetically engineered bacteria

[0093] Bispecific nanobody sequence design: Using monoclonal nanobodies E1 and E2 as the N-terminus of the peptides, (G4S)3 as the flexible linker (amino acid sequence as shown in SEQ ID No. 9), and monoclonal nanobodies V1 and V2 as the C-terminus of the peptides, bispecific nanobodies E1-V1, E1-V2, E2-V1, and E2-V2 were constructed. The amino acid sequences corresponding to the above four bispecific nanobodies are shown in SEQ ID No. 5 to SEQ ID No. 8, respectively.

[0094] SEQ ID No. 5:

[0095] AQVQLQESGGGLVQAGGSLRLSCAASGAGRTDFHIMGWFRQAPGKEREFVAGIAGTLSATRYTDSVKGRFTISRDNAQNTVYLQMNSLRPEDTAVYYCETGRLFPMQASVDNKWGQGTQVTVSSGGGGSG GGGSGGGGSAQVQLQESGGGLVQPGGSLRLSCAVSGSHGADLPEMHWFRQAPGKQRELVATIATATSLAYADSVKGRFTISRENTKNTVYLQMSSLKPEDTAVYCDFLATMVGSQNKPEWGQGTQVTVSS

[0096] SEQ ID No.6:

[0097] AQVQLQESGGGLVQAGGSLRLSCAASGAGRTDFHIMGWFRQAPGKEREFVAGIAGTLSATRYTDSVKGRFTISRDNAQNTVYLQMNSLRPEDTAVYYCETGRLFPQASVDNKWGQGTQVTVSSGGGGSGGGGSGGGGSAQVQLQESGGGLVQAGGSLRLSCVASGSLSADLFTMHWFRQAPGKQRDLVATITAAGTLAYAGSAKGRFTISRDNGMNTAFLQMNSLKPEDTAVYYCDPLASMVGSQNLPEWGQGTQVTVSS

[0098] SEQ ID No.7:

[0099] AQVQLQESGGGLVQPGGSLRLSCAASGAGLSDPHIMGWFRQAPGKEREFVARIGTLASARTYTDSVKGRFTISGDNAQNTVYLQMNSLKPEDTAVYYCETGRHFFMQASVDNKWGQGTQVTVSSGGGGSGGGGSGGGGSAQVQLQESGGGLVQPGGSLRLSCAVSGSHGADLPEMHWFRQAPGKQRELVATIATATSLAYADSVKGRFTISRENTKNTVYLQMSSLKPEDTAVYYCDFLATMVGSQNKPEWGQGTQVTVSS

[0100] SEQ ID No.8:

[0101] AQVQLQESGGGLVQPGGSLRLSCAASGAGLSDPHIMGWFRQAPGKEREFVARIGTLASARTYTDSVKGRFTISGDNAQNTVYLQMNSLKPEDTAVYYCETGRHFFMQASVDNKWGQGTQVTVSSGGGGSG GGGSGGGGSAQVQLQESGGGLVQAGGSLRLSCVASGSLSADLFTMHWFRQAPGKQRDLVATITAAGTLAYAGSAKGRFTISRDNGMNTAFLQMNSLKPEDTAVYYCDPLASMVGSQNLPEWGQGTQVTVSS

[0102] SEQ ID No. 9:

[0103] GGGGSGGGGSGGGGS.

[0104] Based on the designed bispecific nanobody sequence, a professional institution was commissioned to synthesize the full gene sequence, and recombinant plasmids were constructed together with nanobodies E1, E2, V1, and V2. The resulting recombinant plasmids were introduced into competent E. coli cells, and after screening and identification, genetically engineered bacteria capable of stably expressing the corresponding monoclonal nanobodies and bispecific nanobodies were obtained.

[0105] Example 4: Preparation of monoclonal nanobodies and bispecific nanobodies

[0106] (1) Using TB medium as the culture system, the eight genetically engineered bacteria constructed in Example 3 were inoculated at a volume percentage of 5% and cultured at 37°C for 3-5 h. Then, the inducer isopropyl-β-D-thiogalactoside (IPTG) was added and its final concentration was adjusted to 0.25 mM. The mixture was left to stand overnight to induce protein expression.

[0107] (2) After the induction reaction is completed, the bacterial solution is centrifuged at 4000 rpm for 20 min, and the wet precipitate of bacterial cells is collected to obtain wet bacterial cells enriched with the target protein.

[0108] (3) Add lysis buffer to the wet bacterial cells at a mass-to-volume ratio of 1:10; the lysis buffer consists of 10 mM imidazole, 500 mM sodium chloride, and 0.02 M phosphate buffer, with a pH of 7.4. The bacterial cells are then homogenized using a high-pressure homogenizer at a pressure of 700 bar.

[0109] (4) After the bacterial cells are broken, the mixture is centrifuged at 4°C and 10,000 rpm for 20 min and the supernatant is collected.

[0110] (5) The supernatant obtained was filtered through a 0.45 μm filter membrane and then purified by affinity chromatography column (GE Healthcare, US) for separation of the target protein. The packing material of the affinity chromatography column was Ni Sepharose High Performance.

[0111] (6) Take the purified target protein sample for SDS-PAGE electrophoresis to identify the protein purity; select high-purity protein samples and determine the protein solution concentration using the BCA method.

[0112] Example 5: Affinity Analysis

[0113] Surface plasmon resonance (SPR) technology was used to determine the binding activity of monoclonal nanobodies and bispecific nanobodies to the target antigens EpCAM and Vimentin, respectively. EpCAM and Vimentin were immobilized on the surface of independent CM5 sensor chips via amino coupling, with antigen immobilization response values ​​controlled between 500 and 800 RU. Seven concentration gradients of test sample solutions ranging from 1 nM to 100 nM were prepared and sequentially injected for analysis, with the experimental mobile phase flow rate set at 45 μL / min. After each round of detection, the sensor chip was regenerated using a pH 1.5 glycine-hydrochloric acid buffer.

[0114] Based on real-time binding response curves collected at different sample concentrations, kinetic parameters such as the binding rate constant Ka, dissociation rate constant Kd, and equilibrium dissociation constant KD were fitted and solved. The detection results are as follows: Figure 2 and Figure 3 As shown, the response curves in each figure correspond to the sample concentrations from top to bottom as follows: 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.5625 nM. The kinetic parameters obtained from the fitting of each group are summarized in Tables 2 and 3.

[0115] Experimental results show that the monoclonal nanobodies E1 and E2 of this invention have high binding affinity for EpCAM, and their equilibrium dissociation constant KD is in the nanomolar range (10⁻⁶). -9 M); V1 and V2 have high binding affinity for Vimentin, and their equilibrium dissociation constant KD is also in the nanomolar range (10). -9 M).

[0116] Compared to monoclonal nanobodies, the bispecific nanobodies of this invention exhibit significantly enhanced binding affinity for EpCAM and Vimentin, with their equilibrium dissociation constant KD reaching the sub-nanomolar level (10⁻⁶). -10 M).

[0117] Table 2. Affinity of monoclonal nanobodies to EpCAM and Vimentin

[0118]

[0119] Table 3. Affinity of bispecific nanobodies to EpCAM and Vimentin

[0120]

[0121] Example 6: Antibody Labeling / Probe Preparation

[0122] In this embodiment, fluorescein isothiocyanate (FITC) was used as a fluorescent reporter group to complete the fluorescent conjugation preparation of monoclonal nanobody probes and bispecific nanobody probes, respectively. The detailed labeling process is as follows:

[0123] Take 1 mg / mL solutions of E1 nanobody, V1 nanobody, and E1-V1 bispecific nanobody protein in phosphate-buffered saline (PBS) as the solvent; add 10 molar equivalents of FITC powder to each protein system, mix thoroughly by pipetting, and incubate overnight at 4°C in the dark; after incubation, replace the buffer by ultrafiltration centrifugation to remove unbound free FITC impurities and purify to obtain three fluorescently labeled probes, named E1-FITC, V1-FITC, and E1-V1-FITC respectively.

[0124] Example 7: Immunofluorescence Analysis

[0125] MDA-MB-231 cells (EpCAM) were seeded and cultured in 24-well cell culture plates. - Vimentin + ) and MCF-7 cells (EpCAM) + Vimentin - After the cells adhered and grew to a suitable density, the cells were fixed and blocked according to the standard cell immunofluorescence assay procedure.

[0126] First, cell nuclei were specifically stained and labeled using DAPI staining solution. Then, cells were incubated in groups using single-specific fluorescent probes E1-FITC, V1-FITC, and dual-specific fluorescent probes E1-V1-FITC, respectively. After staining, the cells were mounted, and fluorescence imaging of each group was acquired using a laser confocal microscope. The imaging results are shown below. Figure 4 As shown.

[0127] Fluorescence imaging results showed that clear DAPI nuclear fluorescence signals were visible in all cell groups. The single-specific probe E1-FITC only produced fluorescence signals on the surface of MCF-7 cell membranes and showed no specific fluorescence in MDA-MB-231 cells; the single-specific probe V1-FITC only produced fluorescence signals on the surface of MDA-MB-231 cell membranes and showed no specific fluorescence in MCF-7 cells. In stark contrast, the dual-specific probe E1-V1-FITC showed significant fluorescence signals on the cell membranes of both MCF-7 and MDA-MB-231 cells.

[0128] The experimental results show that the bispecific probe of the present invention has dual target recognition capability, can simultaneously recognize tumor cells with different phenotypes, and can achieve specific labeling and immunofluorescence visualization analysis of EMT intermediate tumor cells.

[0129] Industrial availability

[0130] The bispecific nanobody involved in this invention relies on phage display technology and E. coli prokaryotic expression system. The production process is stable, efficient, and low-cost, making it easy to achieve large-scale industrial production. The bispecific nanobody probe can be applied to in vitro diagnostic fields such as clinical tumor screening, circulating tumor cell capture, and immunofluorescence, effectively solving the problem of high false negative rate of traditional single-target reagents. At the same time, the technical route of this invention is highly versatile and can be further extended to the development of other targeted molecules. Overall, it has a wide range of applications, good market prospects, and outstanding industrial utilization value and transformation potential.

[0131] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A bispecific nanobody, characterized in that, It includes a first nanobody domain that targets epithelial cell adhesion molecules, a second nanobody domain that targets vimentin, and a flexible linker peptide that connects the first and second nanobody domains; the first nanobody domain, the flexible linker peptide, and the second nanobody domain are connected in series.

2. The bispecific nanobody according to claim 1, characterized in that, The amino acid sequence of the first nanobody domain is as shown in SEQ ID NO.1 or SEQ ID NO.2, or has at least 90% homology with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2; the amino acid sequence of the second nanobody domain is as shown in SEQ ID NO.3 or SEQ ID NO.4, or has at least 90% homology with the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.

4.

3. The bispecific nanobody according to claim 2, characterized in that, The bispecific nanobody is any one of the following combinations: The amino acid sequence of the first nanobody domain is shown in SEQ ID NO.1, and the amino acid sequence of the second nanobody domain is shown in SEQ ID NO.3; The amino acid sequence of the first nanobody domain is shown in SEQ ID NO.1, and the amino acid sequence of the second nanobody domain is shown in SEQ ID NO.4; The amino acid sequence of the first nanobody domain is shown in SEQ ID NO.2, and the amino acid sequence of the second nanobody domain is shown in SEQ ID NO.3; The amino acid sequence of the first nanobody domain is shown in SEQ ID NO.2, and the amino acid sequence of the second nanobody domain is shown in SEQ ID NO.

4.

4. The bispecific nanobody according to claim 2, characterized in that, The amino acid sequence of the flexible linker peptide is shown in SEQ ID NO.

9.

5. A nanobody probe, characterized in that, It includes a fluorescent reporter group and a bispecific nanobody according to any one of claims 1 to 4; the bispecific nanobody is coupled to the fluorescent reporter group.

6. The nanobody probe according to claim 5, characterized in that, The fluorescent reporter group includes at least one of fluorescein isothiocyanate, phycoerythrin, allophycocyanin, anthocyanin dyes, or Alexa Fluor series dyes.

7. A method for preparing a bispecific nanobody as described in any one of claims 1 to 4 and a nanobody probe as described in claim 5, characterized in that, Includes the following steps: Constructing a nanobody phage display library: Alpaca were immunized with epithelial cell adhesion molecules and vimentin, respectively, peripheral blood lymphocytes were isolated and total RNA was extracted. cDNA was obtained by reverse transcription and then amplified by PCR to obtain the VHH gene. The VHH gene was ligated to a phage vector, packaged and amplified to obtain a phage display library. Screening for specific nanobodies: The phage display library was screened multiple times using epithelial cell adhesion molecules and vimentin as targets, respectively. Positive clones were screened by ELISA, and nanobody genes were obtained by sequencing. Construction of genetically engineered bacteria: Based on the nanobody gene, a recombinant plasmid was constructed according to the tandem structure of the first nanobody domain-flexible linker peptide-second nanobody domain. The recombinant plasmid was transformed into competent Escherichia coli cells, and genetically engineered bacteria expressing bispecific nanobodies were screened to obtain the bacteria. Induction of expression and purification: The genetically engineered bacteria were induced to express the bacteria, and the supernatant was collected and purified by affinity chromatography to obtain bispecific nanobodies. Fluorescent conjugation: The bispecific nanobody is chemically conjugated with a fluorescent reporter group to obtain a nanobody probe.

8. A nucleic acid molecule encoding the bispecific nanobody according to any one of claims 1 to 4.

9. A reagent kit, characterized in that, It includes bispecific nanobodies according to any one of claims 1 to 4; or nanobodies probes according to claims 5 or 6; or nucleic acid molecules according to claim 8, and adjuvants acceptable for detection.

10. The use of the bispecific nanobody of any one of claims 1 to 4, or the nanobody probe of claim 5 or 6; or the nucleic acid molecule of claim 8; or the kit of claim 9, in the preparation of a specific capture, adsorption and / or detection formulation for circulating tumor cells.

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