Anti-CEACAM6 nanobodies, their preparation methods, nucleic acids, vectors, host cells and applications, detection reagents or kits

Abstract

This application relates to anti-CEACAM6 nanobodies, their preparation methods, nucleic acids, vectors, host cells, applications, detection reagents, or kits. The anti-CEACAM6 nanobodies include heavy chain variable regions, specifically CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID No:12, the amino acid sequence of CDR2 is shown in SEQ ID No:14, and the amino acid sequence of CDR3 is shown in SEQ ID No:16. This antibody exhibits higher affinity, better stability, and better specificity.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to anti-CEACAM6 nanobodies and their preparation methods, nucleic acids, vectors, host cells and applications, detection reagents or kits. Background Technology

[0002] Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a glycosylphosphatidylinositol-anchored cell surface glycoprotein belonging to the CEA family. In normal adult tissues, CEACAM6 expression levels are extremely low. However, it is significantly overexpressed in various epithelial-derived malignancies, particularly pancreatic ductal adenocarcinoma, gastric cancer, colon cancer, breast cancer, and non-small cell lung cancer. Studies have shown that CEACAM6 overexpression is closely related to tumor cell proliferation, apoptosis resistance, invasion, metastasis, and chemotherapy resistance. Its expression rate in pancreatic cancer is as high as 80%, and it is often upregulated in precancerous lesions and early cancer stages, making it a highly attractive tumor diagnostic biomarker and therapeutic target.

[0003] Tumor screening mainly relies on imaging technology and serum biomarkers. However, imaging technology is not sensitive enough for early or small lesions, making it difficult to detect tumors smaller than 1 cm in diameter, and it cannot effectively distinguish between inflammation and malignant lesions. Furthermore, the sensitivity and specificity of serum biomarkers cannot meet the increasing demand for "early screening, early diagnosis and early treatment," especially for occult tumors such as pancreatic cancer, which are often discovered at an advanced stage, missing the best treatment opportunity.

[0004] Furthermore, while studies have reported the use of anti-CEACAM6 monoclonal antibodies for immunohistochemistry or in vitro detection, traditional antibodies have drawbacks when used in in vivo molecular imaging, including slow tissue penetration, slow blood clearance, and high background signal, affecting imaging sensitivity and timeliness. Additionally, their complex structure leads to high production costs, making them unsuitable for population screening.

[0005] Currently, there are a few academic reports on anti-CEACAM6 antibodies, which have preliminarily confirmed their binding activity and some in vitro application potential. However, existing anti-CEACAM6 antibodies still suffer from many problems, such as insufficient affinity. Therefore, there is an urgent need to develop an antibody targeting CEACAM6 with higher affinity and better stability. Summary of the Invention

[0006] Based on this, this application provides an anti-CEACAM6 nanobody, which has higher affinity, better stability and better specificity.

[0007] The technical solution to the above-mentioned technical problems in this application is as follows:

[0008] The first aspect of this application provides an anti-CEACAM6 nanobody, the anti-CEACAM6 nanobody comprising a heavy chain variable region, the heavy chain variable region comprising CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 being shown in SEQ ID No:12, the amino acid sequence of CDR2 being shown in SEQ ID No:14, and the amino acid sequence of CDR3 being shown in SEQ ID No:16.

[0009] In some embodiments, at least one of the following features is included:

[0010] (1) The amino acid sequence of the heavy chain variable region is shown in SEQ ID No:18;

[0011] (2) Compared with (1), the sequence identity is at least 80% of the amino acid sequence.

[0012] In some embodiments, the anti-CEACAM6 nanobody further includes a heavy chain constant region, the amino acid sequence of which is shown in SEQ ID No:20.

[0013] A second aspect of this application provides a nucleic acid encoding the aforementioned anti-CEACAM6 nanobody.

[0014] A third aspect of this application provides a vector whose nucleotide sequence includes the nucleotide sequence of the aforementioned nucleic acid.

[0015] A third aspect of this application provides a host cell that expresses the aforementioned anti-CEACAM6 nanobody, or that includes the aforementioned nucleic acid or the aforementioned vector.

[0016] The fourth aspect of this application provides a method for preparing an anti-CEACAM6 nanobody, comprising:

[0017] The host cells described above were cultured to prepare the anti-CEACAM6 nanobody.

[0018] The fifth aspect of this application provides the use of the aforementioned anti-CEACAM6 nanobody, the aforementioned nucleic acid, the aforementioned carrier, or the aforementioned host cell in the preparation of products for diagnosing and / or treating diseases targeting CEACAM6.

[0019] In some embodiments, the diseases targeting CEACAM6 include one or more of colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, medullary thyroid carcinoma, liver cancer, lung cancer, ovarian cancer, and urinary tract tumors.

[0020] The sixth aspect of this application provides a detection reagent or detection kit, comprising the aforementioned anti-CEACAM6 nanobody.

[0021] The anti-CEACAM6 nanobody obtained through screening in this application specifically binds to the CEACAM6 antigen with high affinity. In vitro experiments confirmed that this antibody not only exhibits high specificity for CEACAM6-positive cell lines, but its short half-life in mice also perfectly matches the ideal pharmacokinetic characteristics of "fast in, fast out" for therapeutic nanobodies, enabling rapid and specific enrichment in positive tumor tissues after injection, while maintaining extremely low background signal in normal organs. Therefore, the nanobody of this application successfully overcomes the inherent defects of traditional monoclonal antibodies, such as slow tissue penetration, slow blood clearance, and high background signal, providing a high-performance core ligand for the development of next-generation diagnostic and therapeutic tools for early non-invasive tumor screening, high-resolution molecular imaging (such as PET / CT), and targeted radionuclide therapy (RDC). Attached Figure Description

[0022] Figure 1 The image shows the screening of CEACAM6 antibody and the expression of Pat30 nanobody in Example 1. Figure 1 To screen for CEACAM6 antibodies, the affinity of designed antibodies at different concentrations (200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM) was detected by SPR (left side), with the x-axis representing time and the y-axis representing protein detection signal. Flow cytometry (right side) was used to further verify the binding level of the antibodies to CEACAM6-positive cells; the x-axis represents different groups and the y-axis represents the relative median fluorescence intensity.

[0023] Figure 2 This is a schematic diagram of the modification of the Pat30 nanobody in Example 1 and an SDS-PAGE. The SDS-PAGE shows that the relative molecular mass of the nanobody is about 16kD under both reducing and non-reducing conditions.

[0024] Figure 3 The non-competitive ELISA assay for Pat30 nanobody affinity in Example 1 shows a logarithmic-absorbance line graph of Pat30 nanobody concentration under different CEACAM6 coating concentrations (0, 0.125, 0.25, 0.5 μg / mL); the final calculated KD is 1.644 nM; (ns, not significant, p < 0.05, p < 0.01, p < 0.001, error bars represent mean ± SD), the x-axis is the concentration of Pat30 nanobody, and the y-axis is OD. 450 value;

[0025] Figure 4 This is a diagram showing the results of Pat30 nanobody specifically binding to CEACAM6 positive cell lines in Example 1; Figure 4 In Figure A, the flow cytometry histogram of average fluorescence intensity and the corresponding bar graph of relative average fluorescence intensity are shown. The Pat30 nanobody has good binding ability to CEACAM6 positive cell lines H2122, KM-12, and H1395, but almost no binding to the negative cell line H157. Figure 4 B represents the results of immunofluorescence analysis of Pat30 nanobody. p < 0.01, p < 0.001, p <0.0001, error bars represent mean ± SD); Pat30 Nanobody binding indicates the binding effect of Pat30 nanobodies; Pat30 Nanobody immunofluorescence analysis indicates the immunofluorescence analysis of Pat30 nanobodies.

[0026] Figure 5 This is a confocal microscope image of Pat30 nanobody immunofluorescence staining in Example 1. Figure 5 The green fluorescence represents the Pat30 nanobody, and the blue fluorescence represents the cell nucleus;

[0027] Figure 6 This is a graph showing the in vivo property verification results of the Pat30 nanobody in Example 1; Figure 6 A and B in the diagram represent the in vivo half-life verification of the Pat30 nanobody. Figure 6 Figure A shows the flowchart for the half-life experiment in mice. Whole blood was collected at 0 min, 10 min, 30 min, 1 h, 2 h, and 4 h after tail vein injection, and plasma was obtained by centrifugation. The plasma drug concentration at each time point was detected by ELISA. Figure 6 Figure B shows the fitted half-life curve, with a calculated half-life of approximately 69 min. The horizontal axis represents time, and the vertical axis represents the concentration of nanobody in serum.

[0028] Figure 7Figures A, B, and C demonstrate the in vivo binding ability of the Pat30 nanobody. Figure 7 In the diagram, A is a schematic of the experiment. Nude mice were subcutaneously induced to form tumors using CEACAM6 positive cells H2122 and negative cells H157. After tumor formation, Pat30 nanobody was injected via the tail vein. The mice were sacrificed 1 hour later, and the tumors and important organs were collected for tissue immunofluorescence staining. Figure 7 Image B is a confocal microscope image of Pat30 nanobody tissue immunofluorescence staining, where green fluorescence represents Pat30 nanobody and blue fluorescence represents cell nuclei. Figure 7 C represents the quantitative analysis of the average fluorescence intensity of Pat30 nanobody tissue staining. p<0.0001, error bars represent mean±SD), the x-axis represents different groups, and the y-axis represents the average fluorescence intensity. Detailed Implementation

[0029] To facilitate understanding of this application, a more complete description will be provided below. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of this application will be more thorough and complete.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0031] Antibodies are immunoglobulins that specifically bind to antigens. Nanobodies are variable region fragments derived from camel heavy chain antibodies. They possess a nanoscale size (2.5nm × 4nm × 3nm) and a molecular weight only about one-tenth that of traditional antibodies (approximately 15kDa), yet retain all four conserved framework regions and three complementarity-determining regions (CDRs) that determine binding ability, making them the smallest functional antigen-binding fragments. Nanobodies exhibit significant advantages such as high solubility, high stability, strong tissue penetration, low immunogenicity, and ease of genetic engineering and large-scale production. As the smallest natural antibodies, nanobodies possess unique diagnostic and therapeutic characteristics. They can penetrate tissues relatively easily and efficiently cross the blood-brain barrier, thus demonstrating good targeting ability for solid tumors and metastases throughout the body. These characteristics give them great potential in in vitro diagnostics, molecular imaging, and targeted therapy, especially in the early detection and precise intervention of solid tumors.

[0032] A "vector" is a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40).

[0033] "Host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0034] One embodiment of this application provides an anti-CEACAM6 nanobody, which includes a heavy chain variable region, comprising CDR1, CDR2 and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID No:12, the amino acid sequence of CDR2 is shown in SEQ ID No:14, and the amino acid sequence of CDR3 is shown in SEQ ID No:16.

[0035] Specifically, the amino acid sequence shown in SEQ ID No:12 is VYTMG, the amino acid sequence shown in SEQ ID No:14 is AQIMWGAGTNTHYADSVKG, and the amino acid sequence shown in SEQ ID No:16 is ANRGIPIAGRQYDY.

[0036] The anti-CEACAM6 nanobody obtained through screening in this application specifically binds to the CEACAM6 antigen with high affinity. In vitro experiments confirmed that this antibody not only exhibits high specificity for CEACAM6-positive cell lines, but its short half-life in mice also perfectly matches the ideal pharmacokinetic characteristics of "fast in, fast out" for therapeutic nanobodies, enabling rapid and specific enrichment in positive tumor tissues after injection, while maintaining extremely low background signal in normal organs. Therefore, the nanobody of this application successfully overcomes the inherent defects of traditional monoclonal antibodies, such as slow tissue penetration, slow blood clearance, and high background signal, providing a high-performance core ligand for the development of next-generation diagnostic and therapeutic tools for early non-invasive tumor screening, high-resolution molecular imaging (such as PET / CT), and targeted radionuclide therapy (RDC).

[0037] In some of these embodiments, at least one of the following features is included: (1) the amino acid sequence of the above-mentioned heavy chain variable region is as shown in SEQ ID No:18;

[0038] (2) Compared with (1), the sequence identity is at least 80% of the amino acid sequence.

[0039] Specifically, the amino acid sequence shown in SEQ ID No:18 is QVQLEESGGGLVQAGGSLRLSCRTSGRTNSVYTMGWFRQAPGKEREFVAQIMWGAGTNTHYADSVKGRFTISRDSAESTVYLQMNSLKPEDTAVYYCAANRGIPIAGRQYDYWGQGTQVTVSS.

[0040] It is understood that in other embodiments, the heavy chain variable region is not limited to the polypeptide with an amino acid sequence as shown in SEQ ID No:18, but may also be other polypeptides containing the above-mentioned heavy chain complementarity-determining region (amino acid sequences as shown in SEQ ID No:12, 14 and 16).

[0041] Optionally, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No:17, and the nucleotide sequence shown in SEQ ID No:17 is: 5'-CAGGTCCAGCTGGAGGAGTCCGGTGGCGGTCTGGTGCAGGCAGGCAGGCGGATCACTGAGACTGAGCTGTCGCACCTCCGGTAGAACCAACTCCGTGTACACAATGGGCTGGTTTAGACAGGCTCCAGGCAAAGAAAGGGAGTTCGTGGCACAGATCATGTGGGGAGCAGGTACAAACACACATTACGCTGACTCTGTGAAAGGCCGCTTTACCATCTCCAGAGACAGTGCCGAAAGCACTGTGTACCTGCAGATGAACAGCCTTAAACCTGAGGATACAGCCGTGTACTACTGTGCCGCTAACAGAGGTATTCCAATCGCTGGTAGACAGTACGATTATTGGGGCCAGGGCACACAGGTGACCGTCTCTTCC-3'.

[0042] It is understood that, based on the degeneracy of codons, in other embodiments, the nucleic acid encoding the heavy chain variable region is not limited to the above, and may also be other nucleic acids capable of encoding the heavy chain variable region of the amino acid sequence as shown in SEQ ID No:17.

[0043] In some embodiments, the above-mentioned anti-CEACAM6 nanobody further includes a heavy chain constant region, the amino acid sequence of which is shown in SEQ ID No:20.

[0044] Specifically, the amino acid sequence shown in SEQ ID No:20 is: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. It is understood that in other embodiments, the amino acid sequence of the heavy chain constant region is not limited to the above, and may also be other polypeptide fragments that can serve as the constant region.

[0045] Optionally, the nucleotide sequence of the nucleic acid encoding the heavy chain constant region is as shown in SEQ ID No:19, and the nucleotide sequence shown in SEQ ID No:19 is: 5'--3'.

[0046] It is understood that, based on the degeneracy of codons, in other embodiments, the nucleic acid encoding the heavy chain constant region is not limited to the above, but may also be other nucleic acids capable of encoding the heavy chain constant region of the amino acid sequence as shown in SEQ ID No:19.

[0047] This application also provides a nucleic acid encoding the aforementioned anti-CEACAM6 nanobody according to an embodiment.

[0048] In some embodiments, the nucleic acid includes a nucleic acid fragment encoding a heavy chain variable region of an anti-CEACAM6 nanobody with an amino acid sequence as shown in SEQ ID No. 17. Further, the nucleic acid also includes a nucleic acid fragment encoding a heavy chain constant region with an amino acid sequence as shown in SEQ ID No. 19.

[0049] Optionally, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region of the anti-CEACAM6 nanobody, as shown in SEQ ID No. 18, is as shown in SEQ ID No. 17. It is understood that, due to codon degeneracy, in other embodiments, the nucleotide sequence of the nucleic acid fragment encoding the heavy chain variable region, as shown in SEQ ID No. 17, is not limited to the above and may be other sequences.

[0050] Optionally, the nucleotide sequence of the nucleic acid encoding the heavy chain constant region of the anti-CEACAM6 nanobody, as shown in SEQ ID No. 20, is as shown in SEQ ID No. 19. It is understood that, based on codon degeneracy, in other embodiments, the nucleotide sequence of the nucleic acid fragment encoding the heavy chain constant region, as shown in SEQ ID No. 20, is not limited to the above and may be other sequences.

[0051] In some embodiments, the nucleic acid of any of the above embodiments further includes transcription elements, such as promoters and terminators. It is understood that in some embodiments, the nucleic acid of any of the above embodiments may not include transcription elements. In this case, during use, the nucleic acid can be inserted into a vector having the corresponding transcription elements for expression.

[0052] Another embodiment of this application provides a vector whose nucleotide sequence includes the nucleotide sequence of the aforementioned nucleic acid. The nucleic acid fragment encoding the heavy chain variable region as shown in SEQ ID No. 18 and the nucleic acid fragment encoding the heavy chain constant region as shown in SEQ ID No. 20.

[0053] In some embodiments, the vector is pcDNA. TM 3.1(+) carrier.

[0054] Another embodiment of this application provides a host cell that expresses the above-mentioned anti-CEACAM6 nanobody, or that includes the above-mentioned nucleic acid or the above-mentioned vector.

[0055] In one embodiment, the host cell is a CHO. Of course, in other embodiments, the host cell is not limited to CHO and can be other cells.

[0056] Another embodiment of this application provides a method for preparing anti-CEACAM6 nanobodies, comprising:

[0057] The host cells described above were cultured to prepare the anti-CEACAM6 nanobody.

[0058] After culturing the transformed host cells for a period of time, the host cells or the culture medium in which the host cells were cultured were collected to prepare a primary product containing anti-CEACAM6 nanobodies; then the primary product was purified to prepare purified anti-CEACAM6 nanobodies.

[0059] This application also provides the use of the above-mentioned anti-CEACAM6 nanobody, the above-mentioned nucleic acid, the above-mentioned carrier or the above-mentioned host cell in the preparation of products for diagnosing and / or treating diseases targeting CEACAM6.

[0060] In some embodiments, the diseases targeting CEACAM6 include one or more of colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, medullary thyroid carcinoma, liver cancer, lung cancer, ovarian cancer, and urinary tract tumors.

[0061] Another embodiment of this application also provides a detection reagent or detection kit, including the above-described anti-CEACAM6 nanobody.

[0062] In some embodiments, the above-mentioned detection may be for the purpose of diagnosing or treating a disease or for the purpose of diagnosing or treating a non-disease.

[0063] The following detailed description is provided with reference to specific embodiments. Unless otherwise specified, the embodiments do not include components other than unavoidable impurities. Unless otherwise specified, the reagents and instruments used in the embodiments are conventionally selected in the art. Experimental methods not specifying specific conditions in the embodiments are implemented according to conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

[0064] Example 1

[0065] 1. Screening for anti-CEACAM6 antibodies

[0066] To improve the binding properties and exploitability of anti-CEACAM6 antibodies, this application uses computational methods to identify evolutionarily feasible mutations from large-scale evolutionary information embedded in millions of natural protein sequences—mutations most likely to maintain structure and function while enhancing binding affinity or stability.

[0067] Nanobodies were designed by modifying the sequence of the single-domain antibody AFAI. This antibody was initially isolated from a phage display library derived from llamas and screened against non-small cell lung cancer (A549) cells; its target antigen was later identified as a variant of carcinoembryonic antigen-associated cell adhesion molecule 6 (CEACAM6). The probability of each residue in the AFAI variable domain sequence was calculated using ESM-1b and ESM-1v protein language models. These models, trained on the UniRef50 and UniRef90 protein databases, capture general patterns of amino acid covariance reflecting evolutionary constraints on protein folding and stability. Substitutes that predicted a higher model conditional probability relative to wild-type residues were considered favorable. Subsequently, a set of high-confidence restricted mutations was selected for experimental evaluation after consensus was reached among multiple models.

[0068] Following computational predictions, a small group of AFAI variants were generated using site-directed mutagenesis, incorporating single or combined amino acid substitutions. This strategy enabled the optimization of the original nanobody framework with minimal experimental screening—fewer than twenty variants per design cycle—while maintaining complete antigen specificity. The resulting variants exhibited enhanced immunoreactivity against tumors expressing CEACAM6 and maintained selectivity in normal tissues. The modified antibody sequences are shown in Table 1 below.

[0069] Table 1

[0070]

[0071]

[0072]

[0073] 2. Preparation of anti-CEACAM6 protein-specific nanobodies

[0074] Following the conventional DNA recombination procedure described in Molecular Cloning, the above base sequence was cloned into the corresponding site in pcDNA3.1 to construct a complete eukaryotic expression vector. The plasmid containing the above antibody nucleotide sequence was then transferred to CHO cells for stable antibody expression. The specific antibody expression process is as follows: (1) CHO cells were cultured in CHO CD medium at 2×10⁻⁶ m³ / h. 5 Inoculate cells / mL with 4 mM glutamine. Build up cell density to 4 × 10⁶ cells / mL. 6 Cells were collected at a density of 1 × 10⁶ cells / mL with a viability greater than 95%. Cells were collected by centrifugation, resuspended in electroporation buffer, and adjusted to a density of 1 × 10⁶ cells / mL. 7(2) Take a sterile EP tube, add 0.8 mL of cell suspension, add 5 μg of plasmid, mix gently, incubate for 15 min, and then incubate on ice for 5 min. Set the parameters of the electroporator to 280 V, 3 pulses, 5 ms pulse time, 0.784 s pulse interval, and 4 mm inner diameter of the electroporation cup. Add 0.5 mL of cell and plasmid mixture to each electroporation cup, place it in the electroporator and start the electroporation program. After completion, quickly aspirate the cells from the electroporation cup and transfer them to a 125 mL conical flask containing 20 mL of CHO CD medium. Repeat the above steps to electroporate the second electroporation cup. Gently mix the cells in the shake flask, then tighten the lid and place it in a shaker at 37 °C. (3) After 48 h, transfer the cells to a centrifuge tube and centrifuge at 1000 rpm for 5 min. Remove the supernatant and resuspend the cells in 20 mL of CHO CD medium containing 2 μM, 5 μM, and 20 μM MSX. Calculate the cell density and viability. Cell density at 1.2 × 10⁻⁶ 6 (4) Transfer the cells to a new shake flask. Starting from day 7, observe cell density and viability every two days. Cell density started at 1.2 × 10⁻⁶ cells / mL. 6 The cell density decreases from 10 to 15 days, reaching its lowest point between days 5 and 7. The cell density then begins to increase. When the cell density reaches 1 × 10⁻⁶, the density rises. 6 After reaching a cell density of 3 × 10⁶ cells / mL, the cells were passaged to a cell density of 3 × 10⁶ cells / mL using CD OPM medium containing 50 μM MSX. 5 (5) When the cell density increases to 4 × 10⁶ cells / mL, continue culturing. 6 After reaching a cell / mL level, cells were expanded or passaged using CD OPM medium containing 50 μM MSX, with a minimum seeding density of 3 × 10⁻⁶ cells / mL. 5 (6) Antibody collection method: Antibody purification was performed using protein A affinity chromatography. Antibodies Pat30, Pat32, Pat35, Pat36, and Pat42 were obtained.

[0075] 3. Affinity assay of anti-CEACAM6 nanobody

[0076] Further experimental screening was conducted to identify antibodies with better affinity, which were then used to ultimately modify them into nanobodies. Kinetic analysis of the five antibodies was performed using the SPR assay. Figure 1(As shown on the left). The specific experimental protocol is as follows: SPR was performed using the Cytiva Biacore 8 K surface plasmon resonance molecular interaction system. 0.4 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.1 M N-hydroxysuccinimide (NHS) were used as coupling reagents to immobilize CEACAM6-his (HY-P78397, MCE) on the surface of a CM5 chip (BR100399, Cytiva). DPBS at a flow rate of 30 μL / min was used as the mobile phase, sodium acetate at pH 2.5 as the elution buffer, and 10 mM glycine-hydrochloride buffer at pH 3 as the regeneration buffer. The affinity of all antibodies was tested sequentially, and the binding constant (Kb) was calculated using the Biacore 8 K system. a ), dissociation rate constant (K) d Finally, the dissociation equilibrium constant (KD) is calculated.

[0077] Flow cytometry was also used to detect the binding of antibodies to CEACAM6 positive and negative cells in vitro. Figure 1 (As shown on the right). Specifically, one day in advance, at 1×10 4Cell number / well density: Positive cells KM12 and LoVo, and negative cells MDA-MB-231 were seeded in 24-well plates. Cells were digested using TrypLE until they became round. An equal volume of complete culture medium was added to stop digestion, and the cell suspension was transferred to a 1.5 mL EP tube and centrifuged at 2000 rpm for 5 min. After centrifugation, the supernatant was discarded, and the cells were washed 1-3 times with PBS buffer, centrifuged at 2000 rpm for 5 min after each wash, and the supernatant was discarded. Pat30, Pat32, Pat35, Pat36, and Pat42 were added at 5 μg / mL and incubated for 60 min. After incubation, the cells were centrifuged at 2000 rpm for 5 min, and the supernatant was discarded. The cells were washed 1-3 times with PBS buffer, centrifuged at 2000 rpm for 5 min after each wash, and the supernatant was discarded. After incubating with 1:200 diluted Fluorescein (FITC)–conjugated Goat Anti-Human IgG (H+L) (SA00003-12, Protentech) for 60 min, wash cells 1-3 times with PBS buffer, centrifuging at 2000 rpm for 5 min after each wash and discarding the supernatant. Resuspend cells in 200 μL of PBS buffer in each tube before flow cytometry. (The flow cytometer used was a Thermo Attune NxT. Before loading the cells, blank cells were used to adjust the voltage and bring the initial fluorescence intensity of each group to the same baseline. FlowJo analysis was performed after flow cytometry.)

[0078] like Figure 1 As shown, except for Pat35, the other four antibodies all exhibited extremely strong affinity for CEACAM6, reaching the 0.1 nM level, and showed strong binding ability to CEACAM6-positive cell lines KM12 and LoVo, but no binding to the negative cell line MDA-MB-231. Pat30 was the antibody with the strongest affinity among them. Pat30 was further modified into a nanobody, and the specific expression procedure was consistent with the VHH-Fc antibody expression procedure described above. The sample was obtained by mixing 10 μg of Pat30 nanobody with loading buffer (antibody to loading buffer volume ratio of 4:1), incubating at 100℃ for 30 min, and centrifuging at 10000 rpm for 1 min at room temperature. The pre-cast gel was then installed in the electrophoresis tank, electrophoresis buffer was injected, the comb was adjusted, and the sample was loaded. The procedure involved loading 10 μL of protein marker and 10 μg of the sample to be tested. Electrophoresis settings were: 80 V, 90 min, and electrophoresis was stopped when the bromophenol blue indicator band reached the bottom. The gel was then stained with Coomassie Brilliant Blue for 30 min, followed by destaining in destaining solution for 1 h. This process was repeated 5 times before photographing. The final determined molecular weight of the expressed protein was approximately 16 kDa, consistent with expectations. Figure 2 (As shown).

[0079] Previous ELISA studies have verified the excellent affinity of the Pat30 nanobody. The specific protocol is as follows: First, CEACAM6-Hfc (TMPY-06760, TargetMol) was coated overnight using antigen coating buffer at concentration gradients of 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, and 0 μg / mL. After washing three times with washing buffer, the nanobody was blocked with 5% skim milk for 2 hours. Then, 100 μL of diluted Pat30 nanobody was added at concentration gradients of 1 ng / mL, 2 ng / mL, 4 ng / mL, 8 ng / mL, 16 ng / mL, 32 ng / mL, 64 ng / mL, 128 ng / mL, 256 ng / mL, and 512 ng / mL, and incubated for 2 hours. The nanobody was then eluted three times with washing buffer. Then, 100 μL of 1:2000 diluted anti-His tag Antibody-HRP was added as a secondary antibody and incubated for 2 h. After washing three times, 100 μL of TMB chromogenic solution was added, and stop solution was added after 15 min. The 450 nm reading was recorded using an ELISA reader. Plotting absorbance OD450 on the ordinate and Lg (Pat30 nanobody concentration) on the abscissa, the affinity of the Pat30 nanobody was calculated to be 1.644 using the EC50 of the ELISA fitted curve. 10 -9 The M value, reaching the nanomolar level, indicates that the Pat30 nanobody has extremely high affinity for CEACAM6. Figure 3 (As shown).

[0080] The antibody sequence was sequenced, and the sequenced results were analyzed to obtain the variable region sequence of the antibody. The sequencing results of the nucleic acid fragments and variable regions of the anti-CEACAM6 nanobody are shown in Table 2.

[0081] Table 2

[0082]

[0083] 4. Pat30 nanobodies exhibit excellent CEACAM6 cell binding ability.

[0084] To verify that the Pat30 nanobody can bind to cell lines expressing CEACAM6 positively on their cell surface in vitro, thus ensuring that it will not cause non-specific attack on normal tissues in vivo, a batch of CEACAM6 positive cell lines were first screened and validated. Since CEACAM6 is a cell surface antigen, flow cytometry was used to preliminarily identify lung cancer cells H1395 and H2122, and colorectal cancer cells KM12-SM, which showed high levels of CEACAM6 expression, while lung cancer cell line H157 was a CEACAM6 negative cell line.

[0085] To verify the ability of the Pat30 nanobody to specifically bind to CEACAM6-positive cell lines, 1×10⁻⁶ cells were injected one day in advance. 4 Cell number / well density: H2122, H1395, KM-12-SM, and H2122 cells were seeded in 24-well plates. Cells were digested using TrypLE until they became round. An equal volume of complete culture medium was added to stop digestion, and the cell suspension was transferred to a 1.5 mL EP tube and centrifuged at 2000 rpm for 5 min. After centrifugation, the supernatant was discarded, and the cells were washed 1-3 times with PBS buffer, centrifuged at 2000 rpm for 5 min after each wash, and the supernatant was discarded. Cells were then incubated at room temperature for 60 min with PBS buffer containing 5 μg / mL Pat30 nanobody at a 1:200 dilution. After incubation, the cells were centrifuged at 2000 rpm for 5 min, and the supernatant was discarded. Cells were then washed 1-3 times with PBS buffer, centrifuged at 2000 rpm for 5 min after each wash, and the supernatant was discarded. A 1:200 dilution of CoraLite® Plus 488-conjugated 6... After incubation with His, His-Tag Monoclonal antibody (CL488-66005, Protentech) for 60 min, cells were washed 1-3 times with PBS buffer, centrifuged at 2000 rpm for 5 min after each wash, and the supernatant was discarded. Cells were resuspended in 200 μL of PBS buffer in each tube before flow cytometry analysis. (The flow cytometer used was a Thermo Attune NxT. Before loading the fluorescent antibody-incubated cells, blank cells were used to adjust the voltage and ensure the initial fluorescence intensity of each group was at the same baseline. FlowJo analysis was performed after flow cytometry analysis.) The results showed that the Pat30 nanobody bound well to the three positive cell lines H2122, H1395, and KM-12-SM, but almost noly to the negative cell line H157. These results indicate that the Pat30 nanobody has excellent binding ability to CEACAM6 positive cells in vitro. Figure 4 (As shown in A).

[0086] Furthermore, to more intuitively observe the binding ability of the Pat30 nanobody to positive cells, immunofluorescence technology was used to observe the binding of the Pat30 nanobody to cells. One day prior to observation, 1 × 10⁻⁶ cells were used. 5 Cell number / well density: Four cell types—H2122, H1395, KM-12-SM, and H157—were seeded in 3 cm confocal microplates. After 24 h of culture, the original culture medium was discarded, and the cells were washed 1-3 times with PBS buffer. Using the corresponding complete culture medium for each cell type, a medium containing 10 μg / mL Pat30 nanobody was prepared. 500 μL of the nanobody-containing medium was added to the observation area of ​​the slide in the center of each confocal microplate. After incubation at 37 ℃ for 1 h, the culture medium was discarded, and the cells were washed 1-3 times with PBS buffer to remove excess unbound nanobody. 500 μL of 4% paraformaldehyde was added to the central slide area of ​​the microplate, and the fixative was removed after 10 min. The fixative was then washed 3 times with PBS buffer. Next, a 1:200 dilution of secondary antibody solution (FITC-labeled goat anti-human IgG Fc antibody) was prepared using PBS buffer containing 5% BSA. 500 μL of the secondary antibody solution was added to the central slide area of ​​the dish, and the mixture was incubated at room temperature for 1 h. The solution was then washed three times with PBS buffer. Finally, commercially available DAPI + anti-fluorescence quenching mounting medium was added to the central slide area of ​​the dish. The results were observed and images were taken using a Nikon confocal microscope, and analyzed using ImageJ. The images show that the Pat30 nanobody exhibits green fluorescence, while DAPI-stained cell nuclei show blue fluorescence. Positive cells H2122, H1395, and KM-12-SM show very obvious green fluorescence, while negative cells H157 show almost no green fluorescence. Figure 5 As shown). Quantitative analysis of immunofluorescence also showed that the binding level of Pat30 nanobody to positive cells was significantly higher than that to negative cells H157 (as shown). Figure 4 (As shown in Figure B). This further demonstrates that the Pat30 nanobody exhibits excellent binding ability to CEACAM6-positive cells in vitro.

[0087] 5. Verification of the half-life of Pat30 nanobody in mice

[0088] To achieve the desired diagnostic effect, nanobodies possess both strong affinity and a relatively short half-life in vivo, enabling rapid accumulation at tumor sites for imaging and quick excretion or degradation within the body, thus minimizing the antibody's impact on the human body. Therefore, the half-life of Pat30 nanobodies was first determined in mice. Balb / c mice were administered 0.5 mg / kg Pat30 nanobodies via tail vein injection. Whole venous blood was collected by tail clipping at 0 min, 10 min, 30 min, 1 h, 2 h, and 4 h post-administration. The whole blood was centrifuged at 2000 rpm, and the supernatant serum was separated and temporarily stored at -80℃. After all collection was completed, ELISA experiments were performed to determine the blood concentration of Pat30 nanobodies at different time points. Figure 6 As shown in Figure A), the specific procedure is as follows: First, CEACAM6 antigen is coated overnight at a concentration of 0.5 μg / mL using antigen coating buffer. After washing three times with washing buffer, it is blocked for 2 hours with blocking buffer. Serum diluted 1:100 and Pat30 nanobody at a predetermined concentration (used for plotting the standard curve) are added and bound for 2 hours, then washed away with washing buffer. Next, 100 μL of 1:1000 diluted anti-His tag Antibody-HRP is added as a secondary antibody and incubated for 2 hours. After washing, 100 μL of TMB chromogenic solution is added, and after 15 minutes, stop solution is added. The 450 nm reading is recorded using an ELISA reader. Based on the concentration-absorbance ratio in the standard curve, a linear regression equation between absorbance OD and Pat30 nanobody concentration is obtained. The blood drug concentration at each time point can then be calculated, and a half-life curve is plotted. According to the computer calculation results, the half-life of Pat30 nanobody in mice is 69.43 min (…). Figure 6 (As shown in B).

[0089] 6. Binding of Pat30 nanobodies in mice

[0090] After verifying that the nanobody has a suitable half-life in mice, further mouse experiments were conducted to confirm that the Pat30 nanobody can bind well to tumors and exhibit less non-specific binding to other tissues and organs. Since human tumor cell lines were used, 4-week-old BALB / c nude mice were employed in the experiments. CEACAM6-positive cell line H2122 and CEACAM6-negative cell line H157 were used at a concentration of 1×10⁻⁶. 6Cells were inoculated subcutaneously in the right axilla of nude mice at a density of 1 mg / kg. Experiments were conducted one week after tumor growth. Nude mice were administered the Pat30 nanobody via tail vein injection at a dosage of 1 mg / kg. Because previous experiments showed that the half-life of the Pat30 nanobody in mice is approximately 1 hour, mice were sacrificed one hour after tail vein injection, and tumor tissue and tissues from important organs (heart, liver, spleen, lung, kidney, colon) were collected. After fixation in 4% paraformaldehyde, the tissues were embedded in paraffin and sectioned. Figure 7 (As shown in Figure A). Before dewaxing, the slides were dried in a 60°C oven for 2 hours. Dewaxing and hydration were performed according to the following steps: 1) Immersion in xylene for 10 minutes, repeated once; 2) Immersion in anhydrous ethanol for 5 minutes; 3) Immersion in 95% ethanol for 5 minutes; 4) Immersion in 75% ethanol for 5 minutes; 5) Washing with distilled water for 1 minute, then placing in PBS buffer. The slides were then immersed in EDTA buffer (pH=8.0) and heated in a microwave oven on medium-high heat for 10 minutes to complete antigen retrieval. After cooling naturally to room temperature, the slides were washed three times with PBS buffer. The slides were then immersed in 3% hydrogen peroxide solution and treated at room temperature for 15 minutes. The specimens were blocked with goat serum for 1 hour, and the blocking solution was discarded without washing. Finally, a 1:100 dilution of secondary antibody solution (FITC-labeled goat anti-human IgG Fc antibody) was prepared in PBS buffer containing 5% BSA and incubated at room temperature for 1 hour. Commercially available DAPI + anti-fluorescence quenching mounting medium was added, and the results were observed and images were taken using a Nikon confocal microscope. The images were analyzed using ImageJ. Ultimately, it was observed that the Pat30 nanobody could bind well to the positive tumor H2122, but showed virtually no binding to the negative tumor H157 and other tissues and organs. Figure 7 (As shown in Figure B). Quantitative analysis of tissue immunofluorescence also showed that the binding level of Pat30 nanobody to positive cells was significantly higher than that to negative cells H157 and other tissues and organs (as shown in Figure B). Figure 7 (As shown in C). Furthermore, the Pat30 nanobody showed almost no binding to the colon and lungs, tissues in which CEACAM6 is relatively abundant in the human body.

[0091] In summary, this application provides a novel nanobody, Pat30, that targets CEACAM6 with high affinity. Compared to existing CEACAM6 nanobodies with insufficient affinity and lack of systematic validation, this application yields a nanobody with binding activity reaching the nanomolar level (KD=1.644 nM). This antibody not only exhibits high specificity for in vitro binding to CEACAM6-positive cell lines, but its short half-life of approximately 69 minutes in mice better matches the ideal pharmacokinetic characteristics of "fast in, fast out" for therapeutic nanobodies, enabling rapid and specific enrichment in positive tumor tissues one hour after injection, while maintaining extremely low background signal in normal organs. Therefore, the nanobody of this application successfully overcomes the inherent defects of traditional monoclonal antibodies, such as slow tissue penetration, slow blood clearance, and high background signal, providing a high-performance core ligand for the development of next-generation diagnostic and therapeutic tools for early non-invasive screening of tumors (especially malignant tumors that highly express CEACAM6, such as pancreatic cancer, gastric cancer, and colon cancer), high-resolution molecular imaging (such as PET / CT), and targeted radionuclide therapy (RDC).

[0092] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0093] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A nanobody against CEACAM6, characterized in that, The anti-CEACAM6 nanobody includes a heavy chain variable region, which includes CDR1, CDR2 and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID No:12, the amino acid sequence of CDR2 is shown in SEQ ID No:14 and the amino acid sequence of CDR3 is shown in SEQ ID No:

16.

2. The anti-CEACAM6 nanobody as described in claim 1, characterized in that, Includes at least one of the following features: (1) The amino acid sequence of the heavy chain variable region is shown in SEQ ID No:18; (2) Compared with (1), the sequence identity is at least 80% of the amino acid sequence.

3. The anti-CEACAM6 nanobody as described in claim 1, characterized in that, The anti-CEACAM6 nanobody further includes a heavy chain constant region, the amino acid sequence of which is shown in SEQ ID No:

20.

4. A nucleic acid, characterized in that, It encodes the anti-CEACAM6 nanobody as described in any one of claims 1 to 3.

5. A carrier, characterized in that, The nucleotide sequence of the vector includes the nucleotide sequence of the nucleic acid described in claim 4.

6. A host cell, characterized in that, It expresses the anti-CEACAM6 nanobody as described in any one of claims 1 to 3, or it includes the nucleic acid as described in claim 4 or the vector as described in claim 5.

7. A method for preparing the anti-CEACAM6 nanobody according to claim 1, characterized in that, include: The host cells described in claim 6 are cultured to prepare the anti-CEACAM6 nanobody.

8. The use of the anti-CEACAM6 nanobody according to any one of claims 1 to 3, the nucleic acid according to claim 4, the vector according to claim 5, or the host cell according to claim 6 in the preparation of products for diagnosing and / or treating diseases targeting CEACAM6.

9. The application as described in claim 8, characterized in that, The diseases targeting CEACAM6 include one or more of the following: colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, medullary thyroid carcinoma, liver cancer, lung cancer, ovarian cancer, and urinary tract tumors.

10. A detection reagent or detection kit, characterized in that, Including the anti-CEACAM6 nanobody as described in any one of claims 1 to 3.

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