Colorectal cancer antibody and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a colorectal cancer antibody and application thereof. The antibody is formed by fusing a camel source VHH-3 single-domain antibody and a cell-penetrating peptide TAT through a flexible connecting peptide (GGGGS) 3, and the amino acid sequence of the antibody is as shown in SEQ ID NO: 9. According to the invention, high-affinity VHH-3 (KD = 0.9524 nM) is obtained by screening through a phage display technology, a transmembrane nano antibody TAT-VHH-3 is further constructed, and soluble efficient preparation is realized by using a prokaryotic expression system. Experiments prove that the TAT-VHH-3 can significantly inhibit proliferation (CCK-8 experiment) and migration (scratch experiment) of colorectal cancer Caco-2 cells, and the TAT-VHH-3 is concentration-dependent. The nano antibody has the advantages of being small in molecular weight (about 16.1 kDa), high in penetrability, high in stability and the like, and a novel tool is provided for CaMKII alpha targeting tumor treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a colorectal cancer antibody and applications thereof. Background Art

[0002] Colorectal cancer (CRC) is the third most common malignancy worldwide, with over 1.9 million new cases annually and the second highest mortality rate among cancer-related deaths. Although surgical resection combined with chemoradiotherapy can improve prognosis in early-stage patients, advanced-stage patients are prone to metastasis and recurrence, and traditional chemotherapeutic agents (such as 5-fluorouracil and oxaliplatin) have drawbacks such as strong drug resistance and systemic toxicity. Recent studies have revealed that calcium / calmodulin-dependent protein kinase IIα (CaMKIIα) is abnormally overexpressed in colorectal cancer. CaMKIIα activates Wnt / β-catenin and JAK / STAT signaling pathways 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 a molecular weight (~150 kDa), difficult to penetrate solid tumor tissue, and unable to target intracellular CaMKⅡα; existing nanoantibodies: although they have the advantage of a small molecular weight (~15 kDa), they lack efficient membrane penetration ability and need to rely on carrier delivery systems, which increases the complexity of preparation.

[0004] Cell-penetrating peptides (CPPs) offer new avenues for improving the intracellular delivery of nanobodies. For example, the HIV-derived TAT cell-penetrating peptide (YGRKKRRQRRR) can directly cross the membrane through 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 lead to structural instability or loss of activity. Furthermore, cell-penetrating nanobodies targeting CaMKIIα have yet to be reported, and their potential for colorectal cancer treatment urgently needs to be explored.

[0005] In summary, the development of a nanoantibody targeting CaMKⅡα with both high affinity and efficient membrane penetration ability is of great significance for breaking through the bottleneck of colorectal cancer treatment. Summary of the Invention

[0006] In response to the defects of existing CaMKⅡα inhibitors such as poor penetrance, low selectivity, and high preparation cost, the present invention provides a colorectal cancer antibody and its application. The antibody has a small molecular weight, high affinity and efficient membrane penetration ability, and can specifically inhibit the proliferation, migration and invasion of colorectal cancer cells, breaking through the technical bottleneck that traditional antibodies cannot target intracellular proteins.

[0007] The present invention provides a transmembrane nanoantibody TAT-VHH-3 targeting CaMKⅡα; In certain embodiments, the amino acid sequence of the membrane-penetrating nanobody TAT-VHH-3 is shown in SEQ ID NO: 9, which is composed of the membrane-penetrating peptide TAT (YGRKKRRQRRR), the flexible connecting peptide (GGGGS) 3 and the camel-derived VHH-3 single domain antibody connected in sequence.

[0008] The present invention provides a method for preparing the membrane-penetrating nanoantibody TAT-VHH-3; In some embodiments, the steps include: a) cloning the nucleotide sequence encoding SEQ ID NO: 9 into the prokaryotic expression vector pET-21b; b) Transform the recombinant plasmid into BL21(DE3) host bacteria and induce expression with IPTG; c) After sonication, the cells were lysed and the soluble TAT-VHH-3 protein was purified by Ni-NTA affinity chromatography.

[0009] The present invention provides a pharmaceutical composition comprising a transmembrane nanobody TAT-VHH-3 and a pharmaceutically acceptable carrier.

[0010] The present invention provides use of a transmembrane nanobody TAT-VHH-3 in preparing a drug for inhibiting the proliferation, migration or invasion of colorectal cancer cells.

[0011] In certain embodiments, the drug achieves anti-tumor effects by blocking the signaling pathway through targeted inhibition of CaMKⅡα activity.

[0012] Compared with the prior art, the present invention has at least the following beneficial effects: 1. High affinity: TAT-VHH-3 has a KD value of 0.9524 nM for CaMKⅡα, indicating strong specificity; 2. Excellent penetrability: The membrane-penetrating peptide TAT gives the nanoantibody efficient transmembrane ability, allowing it to directly reach intracellular targets; 3. Significant anti-tumor effect: By blocking the CaMKⅡα downstream signaling pathway, it achieves low toxicity and high efficiency tumor suppression; 4. Low preparation cost: The prokaryotic expression system simplifies the production process and is suitable for large-scale preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Protein purification map of CaMKⅡα recombinant antigen.

[0014] Figure 2 ELISA was used to identify and screen the phage affinity.

[0015] Figure 3 Schematic diagram of the structure of the pET-21b-TAT-VHH-3 expression vector.

[0016] Figure 4 SDS-PAGE analysis of TAT-VHH-3 protein purification results.

[0017] Figure 5 The proliferation effect of TAT-VHH-3 transmembrane nanoantibody on colorectal cancer cells.

[0018] Figure 6 The migration effect of TAT-VHH-3 transmembrane nanoantibody on colorectal cancer cells. DETAILED DESCRIPTION

[0019] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0020] Example 1 Preparation of CaMKⅡα recombinant antigen 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 using XhoⅠ and XbaⅠ enzymes. Random clones were selected for sequencing. The correctly sequenced CaMKⅡα recombinant plasmid was transformed into the BL21(DE3) strain, and positive single clones were randomly selected and inoculated into 5 mL of LB medium containing ampicillin for overnight culture. The next day, the overnight culture was transferred to 1 L of fresh LB medium at a ratio of 1:100 and incubated at 37°C at 200 rpm. -1 The culture was shaken until OD600 nm = 0.8, and a final concentration of 0.2 mmol·L −1 IPTG, 15℃, 120 r·min -1Overnight induction. Collect the overnight induced bacterial suspension and centrifuge at 7000g for 10 min to obtain the cells. Resuspend and wash twice with PBS, then disrupt using an ultrasonic disruptor (300 W, 30 min). Centrifuge at 20000g at 4°C for 1 h. Collect the supernatant after centrifugation, filter the obtained bacterial suspension through a 0.22 μm filter, and purify the target protein using a nickel column containing Ni-NTA Agarose (Qiagen). Figure 1 .

[0021] Figure 1 The results showed that the protein purification profile of CaMKⅡα recombinant antigen showed a single peak with high purity and could be used for subsequent immunization.

[0022] Example 2 Preparation and identification of anti-CaMKⅡα recombinant antigen nanobody Camels were immunized with CaMKⅡα recombinant antigen and adjuvant at a 1:1 volume ratio via the cervical lymph nodes for a total of seven rounds, once weekly. Following immunization, 120 mL of peripheral blood was collected from the camel's neck and diluted with an equal volume of saline. Peripheral blood lymphocytes were isolated by density gradient centrifugation. Total RNA from lymphocytes was extracted with Trizol and reverse-transcribed into cDNA, which served as a template for two PCR reactions to generate VHH fragments. The VHH fragments amplified from these two rounds were inserted into the pMECS phagemid vector to construct the plasmid VHH⁃pMECS. After electroporation into TG1 competent cells, the plasmid was added to 1 mL of SOC medium and incubated at 37°C for 1 hour. Ten microliters of the bacterial suspension was then plated onto 60-mm-diameter LB-AMP-GLU plates, which were then incubated inverted overnight at 37°C. Colonies were collected and transferred to culture medium to construct a phage display antibody library. M13K07 helper phage was used to infect TG1 electrocompetent cells for M13K07 helper phage amplification, and then phagemids were added thereto to complete large-scale amplification of phagemids, so that CaMKⅡα protein was expressed on the phage surface.

[0023] Immunotubes were coated with 30 μg·mL-1 CaMKⅡα and blocked with MPBS (PBS + 2.5% skim milk powder) for 2 h at room temperature after overnight incubation at 4°C. Phages from the overnight revived CaMKⅡα nanobody library were precipitated with PEG-NaCl and 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 -1The phages were eluted with triethanolamine solution and then neutralized by adding 2 mL of Tris-HCl. The eluted phages were transferred to 16 mL of TG1 bacterial solution (OD400 nm = 0.4) and incubated in a 37°C water bath for 30 min. After the phages infected the TG1 bacterial solution, they were centrifuged, resuspended with 2YT medium and spread on 2YTAG solid culture plates, and cultured overnight at 30°C to obtain the primary library bacteria. After the above three rounds of screening, 20 monoclonal clones were picked from the fourth-level library bacterial culture plate, and the phage supernatant treated with blocking solution was added to the ELISA enzyme-labeled plate coated with CaMKⅡα recombinant protein to allow specific binding, and then incubated with HRP-coupled anti-M13 phage antibodies for affinity identification and screening. The absorbance value was measured at a wavelength of 450 nm using an enzyme reader, and the ELISA results were analyzed, see. Figure 2 .

[0024] Figure 2 The results showed that VHH3 had the strongest positive reaction among the 20 sample strains, that is, it had the best affinity effect.

[0025] The positive clone strain VHH3 was amplified and plasmid extracted. Universal sequencing primers were designed based on the sequences flanking the multiple cloning site of the pMECS phagemid vector to amplify the VHH gene and partial vector sequence using PCR. The purified PCR product was then sent to a professional sequencing company (BGI) for bidirectional sequencing using an ABI 3730xl sequencer. The raw sequencing data were subjected to base calling 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 then spliced ​​to obtain the complete VHH gene sequence. This sequence (designated VHH-3) was sequenced and is shown in Table 1.

[0026] Table 1 The amino acid sequence of VHH-3

[0027] Example 3 Preparation and activity identification of transmembrane nanobodies targeting CaMKⅡα A transmembrane nanobody TAT-VHH-3 targeting CaMKⅡα was designed. The transmembrane peptide TAT sequence (YGRKKRRQRRR) was flexibly linked to the N-terminus of the nanobody VHH-3 via (GGGGS)3. Its amino acid sequence is as follows: YGRKKRRQRRRGGGGSGGGGSGGGGSEVQLWSCIFLVATATGVHSKAQSYWMGWFRQAPGKEREFVSFSIYDMNWYRLAPGKRDNSKNTVYLQMQMSSLKADDTAVYYDYWGQGTQVTVSSADSVKGRFTISRISSGDTSYVTVSS (SEQ ID NO: 9).

[0028] The prokaryotic expression vector pET-21b-TAT-VHH-3 was delivered by Sangon Biotech (Shanghai) Co., Ltd., and its structural formula is as follows Figure 3 As shown, the pET-21b-TAT-VHH-3 recombinant plasmid was transformed into BL21 (DE3) expression competent cells, IPTG induced expression, ultrasonic lysed the bacteria, centrifuged and separated the supernatant and precipitate, and subjected to SDS-PAGE analysis respectively. The protein expression form of the membrane-penetrating nanoantibody was soluble; the chromatography column was prepared and soaked with 75% ethanol for 30 minutes, and then washed with 2 column volumes of deionized water; 3 mL of Ni-NTA Beads 6FF resin was added to the chromatography column, allowed to stand for 10 minutes, and the storage buffer was washed out with 3 to 5 column volumes of deionized water; the chromatography column was equilibrated with 5 column volumes of Lysis buffer; the supernatant after ultrasonic lysis was added to the chromatography column, incubated at 4°C overnight, and the effluent was collected; the column was washed with different gradients of Wash Buffer to wash away impurities until the OD280nm value of the washing liquid was close to the baseline; a linear gradient elution was used, using an elution containing imidazole concentrations from low to high. Buffer was sequentially eluted from the column, and each gradient eluate was collected until the OD280nm value of the eluate was close to the baseline. The results of TAT-VHH-3 protein purification were analyzed by SDS-PAGE. Figure 4 .

[0029] Figure 4 The results showed that the molecular weight of TAT-VHH-3 protein was approximately 16.10 kDa, which was in line with expectations.

[0030] The affinity constant (KD) of TAT-VHH-3 for CaMKⅡα was determined by ELISA. CaMKⅡα protein (1 μg / mL, 100 μL / well) was coated onto ELISA plates and incubated at 4°C overnight (16-18 h). The plates were washed three times with PBST for 5 min each. 200 μL / well of blocking buffer (5% skim milk powder-PBS) was added and blocked at 37°C for 2 h, followed by washing three times. 50 μL of TAT-VHH-3 (a dilution ladder of TAT-VHH-3 was prepared, covering 0.001-1000 nM) was added to each well and incubated at 37°C for 1 h, followed by washing five times. 50 μL of HRP-anti-HIS (1:5000) (Anti-6His tag antibody [HRP], Catalog No.: ab1187, Abcam) was then added to each well and incubated at 37°C for 1 h, followed by washing five times. 100 μL of TMB colorimetric solution was added to each well and the mixture was incubated in the dark for 15 min. 50 μL of 2M H2SO4 was added to terminate the reaction, and the OD450 nm value was immediately measured using a microplate reader. A four-parameter logistic regression model (4PL) was used to fit the competition curve (GraphPad Prism 9.0), with log[TAT-VHH-3 concentration] as the abscissa and OD450 nm as the ordinate. Figure 5 , KD value was converted according to Cheng-Prusoff equation.

[0031] Figure 5 The results showed that the fitting curve R²=0.9983, indicating that the data had a good linear relationship; at the same time, the KD value was 0.9524nM, indicating that TAT-VHH-3 had a high affinity for CaMKⅡα.

[0032] Example 4 Inhibitory effect of TAT-VHH-3 transmembrane nanobody on colorectal cancer cells The colorectal cancer Caco-2 cell line was placed in RPMI1640 containing 10% FBS and antibiotics and cultured in a 37°C, 5% CO2 incubator. Caco-2 cells were seeded into 6-well plates at 2.5×10 cells per well. 5 Caco-2 cells were divided into low-dose, medium-dose, high-dose groups and a control group: Control group: Caco-2 cells were incubated with an equal volume of PBS for 3 h, then cultured with conventional culture medium for a further 36 h; Low-dose group: Caco-2 cells were incubated with TAT-VHH-3 at a final concentration of 5 μg / mL for 3 h, then cultured in conventional medium for a further 36 h. Medium-dose group: Caco-2 cells were incubated with TAT-VHH-3 at a final concentration of 25 μg / mL for 3 h, then cultured in conventional medium for a further 36 h; High-dose group: Caco-2 cells were incubated with TAT-VHH-3 at a final concentration of 50 μg / mL for 3 h, then cultured in conventional medium for a further 36 h; CCK-8 assay for Caco-2 cell proliferation: 10 μL 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 with a microplate reader. The cell proliferation inhibition rate was calculated as follows: inhibition rate (%) = [1 - (OD dose group / OD control group)] × 100%, see Figure 5 .

[0033] Figure 5 The results showed that TAT-VHH-3 significantly inhibited the proliferation of Caco-2 cells in a concentration-dependent manner.

[0034] Scratch assay to detect cell migration ability: Caco-2 cells in logarithmic growth phase were collected and 5×10 5 / well density in 6-well plates, and 2 mL of complete culture medium was added to each well. Culture at 37°C until the cell confluence reached 90%-100% (about 24 h) and a uniform monolayer was formed. Use a 200 μL sterile pipette tip, vertically close to the bottom of the well plate, and evenly scratch three parallel scratches along the ruler (about 0.5 cm apart); gently wash with PBS three times to remove detached cell debris, and replace with low-serum culture medium containing 1% FBS; add PBS or different concentrations of TAT-VHH-3 (5, 25, 50 μg / mL) according to the group, with 3 replicates in each group. Continue to culture in a 37°C incubator, and observe the healing of the scratch at 0h and 24h respectively; use an inverted microscope (100× field of view) to capture images of the scratch area at a fixed position, and analyze the scratch area using Image J software: initial scratch area (S0): pixel area of ​​the scratch area at 0h. End scratch area (S t ): Pixel area of ​​the scratch area 24 h after treatment, calculate migration rate: healing rate (%) = [(initial scratch area - end point scratch area) / initial scratch area] × 100%; see Figure 6 .

[0035] Figure 6 The results showed that TAT-VHH-3 significantly inhibited Caco-2 cell migration in a concentration-dependent manner. The high-dose group (50 μg / mL) showed a significantly lower migration rate than the control group (p<0.001), indicating that the nanoantibody effectively blocks the migration of colorectal cancer cells by targeting CaMKⅡα.

[0036] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

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

2. The method for preparing an antibody according to claim 1, wherein: The following steps are involved: a) cloning the nucleotide sequence encoding SEQ ID NO: 9 into the prokaryotic expression vector pET-21b; b) Transform the recombinant plasmid into BL21(DE3) host bacteria and induce expression with IPTG; c) After ultrasonic lysis of the bacteria, soluble transmembrane nanobodies were obtained by purification via Ni-NTA affinity chromatography.

3. A pharmaceutical composition, characterized in that Comprising the antibody according to claim 1 and a pharmaceutically acceptable carrier.

4. Use of the antibody according to claim 1 in the preparation of a drug for inhibiting the proliferation, migration or invasion of colorectal cancer cells.

5. The use according to claim 4, characterized in that The drug achieves anti-tumor effect by blocking the signal pathway through targeted inhibition of CaMKⅡα activity.

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