An antibody against colorectal cancer and its application

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a colorectal cancer antibody and its applications. The antibody is composed of a camel-derived VHH-3 single-domain antibody and a membrane-penetrating peptide TAT fused together via a flexible linker peptide (GGGGS)3, and its amino acid sequence is shown in SEQ ID NO:9. This invention uses phage display technology to screen for high-affinity VHH-3 (KD=0.9524 nM) and further constructs the membrane-penetrating nanobody TAT-VHH-3, achieving soluble and efficient preparation using a prokaryotic expression system. Experiments confirm that TAT-VHH-3 can significantly inhibit the proliferation (CCK-8 assay) and migration (scratch assay) of colorectal cancer Caco-2 cells in a concentration-dependent manner. The nanobody of this invention has advantages such as small molecular weight (approximately 16.1 kDa), strong penetrability, and high stability, providing a novel tool for tumor therapy targeting CaMKⅡα.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a colorectal cancer antibody and its application. Background Technology

[0002] Colorectal cancer (CRC) is the third most common malignant tumor worldwide, with over 1.9 million new cases annually and the second leading cause of cancer-related death. Although surgical resection combined with radiotherapy and chemotherapy can improve the prognosis of early-stage patients, late-stage patients are prone to metastasis and recurrence, and traditional chemotherapy drugs (such as 5-fluorouracil and oxaliplatin) have drawbacks such as strong drug resistance and high systemic toxicity. Recent studies have found that calcium / calmodulin-dependent protein kinase IIα (CaMKIIα) is abnormally highly expressed in colorectal cancer. It activates signaling pathways such as Wnt / β-catenin and JAK / STAT by phosphorylating downstream targets (such as β-catenin and STAT3), driving tumor cell proliferation, migration, and epithelial-mesenchymal transition (EMT), and is significantly associated with poor patient prognosis.

[0003] Currently, inhibitors targeting CaMKIIα mainly include small molecule compounds (such as KN-93) and monoclonal antibodies, but they have the following limitations: Small molecule inhibitors: poor selectivity, easy off-target inhibition of other kinases (such as CaMKIIβ / γ), leading to cardiovascular toxicity; Traditional monoclonal antibodies: too large molecular weight (~150 kDa), difficult to penetrate solid tumor tissue, and unable to target intracellular CaMKIIα; Existing nanobodies: although they have the advantage of small molecular weight (~15 kDa), they lack efficient membrane penetration ability, require a carrier delivery system, and increase the complexity of preparation.

[0004] Cell-penetrating peptides (CPPs) offer novel approaches to improving the intracellular delivery of nanobodies. For example, the HIV-derived TAT cell-penetrating peptide (YGRKKRRQRRR) can directly cross the membrane via charge interactions, efficiently delivering fusion proteins to intracellular targets. However, existing cell-penetrating peptide-antibody conjugation technologies often employ chemical cross-linking or single linker peptides, which can easily lead to structural instability or loss of activity. Furthermore, cell-penetrating nanobodies targeting CaMKⅡα have not yet been reported, and their potential application in colorectal cancer treatment remains to be explored.

[0005] In conclusion, developing a nanobody that targets CaMKⅡα and possesses both high affinity and efficient membrane penetration is of great significance for overcoming the bottleneck in colorectal cancer treatment. Summary of the Invention

[0006] To address the shortcomings of existing CaMKⅡα inhibitors, such as poor penetration, low selectivity, and high preparation costs, this invention provides a colorectal cancer antibody and its application. This antibody combines small molecular weight, high affinity, and efficient membrane penetration, and can specifically inhibit the proliferation, migration, and invasion of colorectal cancer cells, breaking through the technical bottleneck of traditional antibodies being unable to target intracellular proteins.

[0007] This invention provides a transmembrane nanobody TAT-VHH-3 that targets CaMKⅡα;

[0008] In some embodiments, the amino acid sequence of the transmembrane nanobody TAT-VHH-3 is shown in SEQ ID NO:9, and it is composed of the transmembrane peptide TAT (YGRKKRRQRRR), the flexible linker peptide (GGGGS)3, and the camel-derived VHH-3 single-domain antibody linked sequentially.

[0009] This invention provides a method for preparing transmembrane nanobody TAT-VHH-3;

[0010] In some embodiments, the following steps are included:

[0011] a) The nucleotide sequence encoding SEQ ID NO:9 was cloned into the prokaryotic expression vector pET-21b;

[0012] b) Transform the recombinant plasmid into the BL21(DE3) host bacterium and induce expression with IPTG;

[0013] c) After ultrasonic lysis of the bacterial cells, soluble TAT-VHH-3 protein was obtained by Ni-NTA affinity chromatography.

[0014] The present invention provides a pharmaceutical composition comprising a transmembrane nanobody TAT-VHH-3 and a pharmaceutically acceptable carrier.

[0015] This invention provides the application of transmembrane nanobody TAT-VHH-3 in the preparation of drugs that inhibit the proliferation, migration or invasion of colorectal cancer cells.

[0016] In some embodiments, the drug exerts its antitumor effect by targeting and inhibiting CaMKⅡα activity to block the signaling pathway.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects:

[0018] 1. High affinity: TAT-VHH-3 has a KD value of 0.9524 nM for CaMKⅡα, indicating high specificity;

[0019] 2. Excellent penetration: The transmembrane peptide TAT endows nanobodies with efficient transmembrane capabilities, enabling them to directly reach intracellular targets;

[0020] 3. Significant anti-tumor effect: It achieves low-toxicity and high-efficiency tumor inhibition by blocking the downstream signaling pathway of CaMKⅡα;

[0021] 4. Low preparation cost: The prokaryotic expression system simplifies the production process and is suitable for large-scale preparation. Attached Figure Description

[0022] Figure 1 Protein purification map of CaMKⅡα recombinant antigen.

[0023] Figure 2 ELISA was used to identify and screen bacteriophages based on affinity.

[0024] Figure 3 Schematic diagram of the structure of the pET-21b-TAT-VHH-3 expression vector.

[0025] Figure 4 SDS-PAGE analysis of TAT-VHH-3 protein purification results.

[0026] Figure 5 The proliferative effect of TAT-VHH-3 transmembrane nanobody on colorectal cancer cells.

[0027] Figure 6 The effect of TAT-VHH-3 transmembrane nanobody on the migration of colorectal cancer cells. Detailed Implementation

[0028] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0029] Example 1: Preparation of CaMKⅡα recombinant antigen

[0030] The nucleotide sequence corresponding to CaMKⅡα was downloaded from the NCBI database (NCBI Reference Sequence: NP_001390241.1). Subclones were constructed by Sangon Biotech (Shanghai) Co., Ltd., and then ligated into the HIS-tagged pET-21a(+) vector after double digestion with XhoⅠ and XbaⅠ. Randomly selected clones were sequenced. The correctly sequenced CaMKⅡα recombinant plasmid was transformed into BL21(DE3) strain. Randomly selected positive single colonies were inoculated into 5 mL of LB medium containing ampicillin and cultured overnight. The next day, the overnight culture was transferred to 1 L of fresh LB medium at a 1:100 ratio and incubated at 37℃ and 200 r·min. -1 After shaking and incubating until OD600 nm = 0.8, add [a specific ingredient] to a final concentration of 0.2 mmol·L⁻¹. −1 IPTG, 15℃, 120 r·min-1 Overnight induction. Collect 7000g of the overnight induced bacterial culture, centrifuge for 10 min to obtain bacterial cells; resuspend and wash twice with PBS, then sonicate (300 W, 30 min); centrifuge at 20000g, 4℃ for 1 h. Collect the supernatant after centrifugation, filter the obtained bacterial culture supernatant through a 0.22 μm filter, and purify the target protein using a Ni-NTA Agarose (Qiagen) nickel column, see [link to relevant documentation]. Figure 1 .

[0031] Figure 1 The results showed that the protein purification spectrum of the CaMKⅡα recombinant antigen exhibited a single peak, indicating high purity, and it could be used for subsequent immunization.

[0032] Example 2: Preparation and Identification of Anti-CaMKⅡα Recombinant Antigen Nanobodies

[0033] The CaMKⅡα recombinant antigen and adjuvant were mixed at a 1:1 volume ratio and administered via immunization to the neck lymph nodes of camels for a total of 7 times, once a week. After immunization, 120 mL of peripheral blood was collected from the camel neck and diluted with an equal volume of physiological saline. 10 mL of lymphocyte separation medium was added to every 15 mL of peripheral blood dilution, and peripheral blood lymphocytes were separated by density gradient centrifugation. Total RNA was extracted from the lymphocytes using Trizol and reverse transcribed into cDNA. Using this cDNA as a template, the VHH fragment was obtained through two rounds of PCR. The VHH fragment amplified in the second round was inserted into the pMECS phage vector to construct the plasmid VHH-pMECS. After electroporation into TG1 competent cells, the plasmid was added to 1 mL of SOC medium and cultured at 37°C for 1 h. 10 µL of the bacterial culture was serially diluted and plated onto 60 mm diameter LB-AMP-GLU plates and incubated overnight at 37°C. Colonies were collected and added to culture medium to construct a phage display antibody library. M13K07 helper phage was used to infect TG1 electrocompetent cells to amplify the M13K07 helper phage. Phage particles were then added to complete the large-scale amplification of the phage particles, so that CaMKⅡα protein was expressed on the surface of the phage.

[0034] Immunotubes were coated with 30 μg·mL⁻¹ CaMKⅡα and incubated overnight at 4 °C. They were then blocked with MPBS (PBS + 2.5% skim milk powder) at room temperature for 2 h. The overnight recovered CaMKⅡα nanobody library was precipitated with PEG-NaCl, and the phages were incubated with MPBS for 1 h. The blocked phages were added to the blocked immunotubes and incubated at 37 °C for 2 h. The tubes were then washed with PBS. 2 mL of 100 mmol·L⁻¹ ammonium chloride solution was used. -1The phages were eluted with triethanolamine solution and then neutralized with 2 mL of Tris-HCl. The eluted phages were transferred to 16 mL of TG1 bacterial suspension (OD400 nm = 0.4) and incubated at 37 °C for 30 min. After infecting the TG1 bacterial suspension, the phages were centrifuged, resuspended in 2YT medium, and plated onto 2YTAG solid culture plates. The plates were then incubated overnight at 30 °C to obtain the primary library. After the above three rounds of screening, 20 single clones were picked from the quaternary library culture plates. Phage supernatant treated with blocking buffer was added to CaMKⅡα recombinant protein-coated ELISA plates to allow for specific binding. The plates were then incubated with HRP-conjugated anti-M13 phage antibody for affinity identification and screening. The absorbance was measured at 450 nm using a microplate reader. The ELISA results were analyzed. Figure 2 .

[0035] Figure 2 The results showed that VHH3 had the strongest positive reaction among the 20 sample strains, indicating the best affinity.

[0036] The positive clone strain VHH3 was amplified and plasmids were extracted. Based on the flanking sequences of the multiple cloning site of the pMECS phage vector, universal sequencing primers were designed for PCR amplification of the VHH gene and partial vector sequence. The purified PCR product was then sent to a professional sequencing company (BGI Genomics) for bidirectional sequencing using an ABI 3730xl sequencer. The raw sequencing data were subjected to base identification and quality assessment using the Phred algorithm. The vector sequence (pMECS backbone) was removed using Vector NTI Advance 11.5 software. The forward (PMF) and reverse (PMR) sequencing results were spliced ​​to obtain the complete VHH gene sequence. This sequence (named VHH-3) was obtained and is shown in Table 1.

[0037] Table 1 shows the amino acid sequence results of VHH-3.

[0038]

[0039] Example 3: Preparation and activity identification of transmembrane nanobodies targeting CaMKⅡα

[0040] The transmembrane nanobody TAT-VHH-3 targeting CaMKⅡα was designed by flexibly linking the transmembrane peptide TAT sequence (YGRKKRRQRRR) to the N-terminus of the nanobody VHH-3 via (GGGGS)3. Its amino acid sequence is as follows:

[0041] YGRKKRRQRRRGGGGSGGGGSGGGGSEVQLWSCIFLVATATGVHSKAQSYWMGWFRQAPGKEREFVSFSIYDMNWYRLAPGKRDNSKNTVYLQMQMSSLKADDTAVYYDYWGQGTQVTVSSADSVKGRFTISRISSGDTSYVTVSS (SEQ ID NO: 9).

[0042] The prokaryotic expression vector pET-21b-TAT-VHH-3, delivered by Sangon Biotech (Shanghai) Co., Ltd., has the following structural formula: Figure 3 As shown, the pET-21b-TAT-VHH-3 recombinant plasmid was transformed into BL21(DE3) competent cells, and expression was induced by IPTG. The cells were lysed by sonication, and the supernatant and precipitate were separated by centrifugation. SDS-PAGE analysis showed that the protein expression form of the transmembrane nanobody was soluble. The chromatography column was prepared by soaking in 75% ethanol for 30 min, followed by washing with 2 column volumes of deionized water. 3 mL of Ni-NTA Beads 6FF resin was added to the column, allowed to stand for 10 min, and then washed with 3-5 column volumes of deionized water to remove the storage buffer. The column was equilibrated with 5 column volumes of Lysis buffer. The supernatant obtained after sonication was added to the column and incubated overnight at 4°C. The eluent was collected. The column was washed with different gradients of wash buffer to remove contaminating proteins until the OD280nm value of the wash liquid was close to the baseline. A linear gradient elution was used, using elution solutions containing increasing concentrations of imidazole. The column was eluted sequentially with buffer, and the eluents from each gradient were collected until the OD280nm value of the eluent was close to the baseline. SDS-PAGE analysis of the TAT-VHH-3 protein purification results was performed. Figure 4 .

[0043] Figure 4 The results showed that the molecular weight of the TAT-VHH-3 protein was approximately 16.10 kDa, which was in line with expectations.

[0044] The affinity constant (KD) between TAT-VHH-3 and CaMKⅡα was determined by ELISA. CaMKⅡα protein (1 μg / mL, 100 μL / well) was coated onto an ELISA plate and incubated overnight (16-18 h) at 4°C. The plate was washed three times with PBST for 5 min each time. 200 μL of blocking buffer (5% skim milk powder-PBS) was added to each well, and the plate was blocked at 37°C for 2 h, followed by three washes. 50 μL of TAT-VHH-3 (using a dilution ladder to cover 0.001-1000 nM) was added to each well, and the plate was incubated at 37°C for 1 h, followed by five washes. Then, 50 μL of HRP-anti-HIS (1:5000) (Anti-6His tag antibody [HRP], catalog number: ab1187, Abcam) was added to each well, and the plate was incubated at 37°C for 1 h, followed by five washes. Add 100 μL of TMB chromogenic solution to each well and react in the dark for 15 min. Stop the reaction by adding 50 μL of 2M H2SO4, and immediately measure the OD450 nm value using a microplate reader. Plot log[TAT-VHH-3 concentration] on the x-axis and OD450 nm on the y-axis, and fit the competition curve using a four-parameter logistic regression model (4PL) (GraphPad Prism 9.0). See [link to graph]. Figure 5 The KD value is converted according to the Cheng-Prusoff equation.

[0045] Figure 5 The results showed that the fitted curve R²=0.9983, indicating a good linear relationship between the data; meanwhile, the KD value was 0.9524nM, indicating that TAT-VHH-3 has a high affinity for CaMKⅡα.

[0046] Example 4: Inhibitory effect of TAT-VHH-3 transmembrane nanobody on colorectal cancer cells

[0047] The Caco-2 cell line for colorectal cancer was cultured in RPMI 1640 containing 10% FBS and antibiotics at 37°C in a 5% CO2 incubator. Caco-2 cells were seeded into 6-well plates at 2.5 × 10⁶ cells per well. 5 Caco-2 cells were divided into low-, medium-, and high-dose groups and a control group.

[0048] Control group: Caco-2 cells were co-incubated with an equal volume of PBS for 3 h, then cultured in conventional culture medium for another 36 h;

[0049] Low-dose group: Caco-2 cells were co-incubated with TAT-VHH-3 at a final concentration of 5 μg / mL for 3 h, then cultured in conventional medium for another 36 h.

[0050] Medium dose group: Caco-2 cells were co-incubated with TAT-VHH-3 at a final concentration of 25 μg / mL for 3 h, then cultured in conventional medium for another 36 h;

[0051] High-dose group: Caco-2 cells were co-incubated with TAT-VHH-3 at a final concentration of 50 μg / mL for 3 h, then cultured in conventional medium for another 36 h.

[0052] CCK-8 assay for Caco-2 cell proliferation: 10 μL of CCK-8 reagent was added to each well; the cells were incubated at 37°C in the dark for 2 h, and the absorbance (OD value) at 450 nm was measured using a microplate reader. The cell proliferation inhibition rate was calculated as follows: Inhibition rate (%) = [1 - (OD dose group / OD control group)] × 100%, see [link to relevant documentation]. Figure 5 .

[0053] Figure 5 The results showed that TAT-VHH-3 significantly inhibited the proliferation of Caco-2 cells in a concentration-dependent manner.

[0054] Scratch assay to detect cell migration ability:

[0055] Take Caco-2 cells in the logarithmic growth phase and use 5 × 10⁻⁶ cells. 5 Cells were seeded at a density of 2 mL of complete culture medium per well in 6-well plates. Cells were incubated at 37°C until 90%-100% confluence (approximately 24 h) was achieved, forming a uniform monolayer. Using a 200 μL sterile pipette tip, three parallel scratches (approximately 0.5 cm apart) were made vertically and evenly along a ruler against the bottom of the plate. Cells were gently washed three times with PBS to remove cell debris and then replaced with low-serum culture medium containing 1% FBS. PBS or different concentrations of TAT-VHH-3 (5, 25, 50 μg / mL) were added according to the grouping, with three replicates per group. Cells were incubated at 37°C, and scratch healing was observed at 0 h and 24 h. Images of the scratched area were taken at a fixed position using an inverted microscope (100× field of view), and the scratch area was analyzed using ImageJ software: Initial scratch area (S0): pixel area of ​​the scratched area at 0 h. Ending scratch area (S... t The following is a calculation of the migration rate based on the pixel area of ​​the scratch region 24 hours after treatment: Healing rate (%) = [(Initial scratch area - Ending scratch area) / Initial scratch area] × 100%; see Figure 6 .

[0056] Figure 6The results showed that TAT-VHH-3 significantly inhibited Caco-2 cell migration in a concentration-dependent manner. After intervention with the high-dose group (50 μg / mL), the migration rate was significantly lower than that of the control group (p<0.001), indicating that the nanobody effectively blocked the migration process of colorectal cancer cells by targeting CaMKⅡα.

[0057] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A colorectal cancer antibody, characterized in that, The amino acid sequence of the antibody is shown in SEQ ID NO:9, and it is composed of a membrane-penetrating peptide TAT, a flexible linker peptide (GGGGS)3, and a camel-derived VHH-3 single-domain antibody linked sequentially.

2. The method for preparing the antibody according to claim 1, characterized in that, Includes the following steps: a) The nucleotide sequence encoding SEQ ID NO:9 was cloned into the prokaryotic expression vector pET-21b; b) The recombinant plasmid was transformed into the BL21(DE3) host bacterium, and expression was induced by IPTG; c) After ultrasonic lysis of bacterial cells, soluble transmembrane nanobodies were obtained by Ni-NTA affinity chromatography purification.

3. A pharmaceutical composition, characterized in that, It comprises the antibody as described in claim 1 and a pharmaceutically acceptable carrier.

4. The use of the antibody according to claim 1 in the preparation of a medicament for treating colorectal cancer.

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