CN116178556B - CEACAM 5-targeted nano antibody and preparation method and application thereof - Google Patents
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- CN116178556B CN116178556B CN202310028399.3A CN202310028399A CN116178556B CN 116178556 B CN116178556 B CN 116178556B CN 202310028399 A CN202310028399 A CN 202310028399A CN 116178556 B CN116178556 B CN 116178556B
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Classifications
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- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a CEACAM 5-targeted nano antibody, a preparation method and application thereof. Specifically provided is a nano-antibody targeting a neuroendocrine prostate cancer specific membrane marker CEACAM5, the nano-antibody comprises a nano-antibody B12, wherein the amino acid sequence of the nano-antibody B12 is shown as seq.id No. 1. The provided nano antibody B12 has higher affinity with CEACAM5, can target the CEACAM5 serving as a specific membrane marker of the neuroendocrine prostate cancer, is favorable for developing medicaments related to the nano antibody of the CEACAM5 serving as the specific membrane marker of the neuroendocrine prostate cancer, realizes the targeted treatment of the castration resistant neuroendocrine stage prostate cancer, and has great clinical application value. Meanwhile, the nano antibody is favorable for wide application due to small molecular weight.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a CEACAM 5-targeted nano antibody, and a preparation method and application thereof.
Background
Prostate cancer refers to an epithelial malignancy that occurs in the prostate. In general, prostate cancer (PCa) patients are mostly in the disease progression stage at the time of diagnosis, with distant metastasis of the tumor or the presence of high risk factors (Gleason score greater than 7, serum Prostate specific antigen greater than 20ng/ml or tumor stage greater than T2 b). Endocrine therapy, typified by androgen deprivation and androgen receptor antagonism, is the first choice for such patients (patients with metastatic lesions) or an important post-operative adjuvant therapy (patients with localized high risk prostate cancer). However, unlike localized low-risk prostate cancer patients, which are common in the western population, such patients inevitably develop treatment resistance with prolonged endocrine treatment time. Resulting in disease progression to castration-resistant prostate cancer stage (Castration resistant prostate cancer, CRPC).
Androgen receptor signaling, such as androgen receptor gene and promoter amplification, androgen receptor shear variant AR-V7 formation, is an important factor in the development of castration-resistant prostate cancer. In this regard, two-line endocrine therapy, which effectively blocks endogenous androgen synthesis (abiraterone) or androgen receptor nuclear entry (enzalutamide), is currently the primary therapeutic modality. While the above treatment can extend the survival period of CRPC patients, still 20% of patients will progress to the castration resistant prostate cancer neuroendocrine stage (Castration resistance prostate cancer-neuroendocrine, CRPC-NE) characterized by a high degree of invasion. Currently, chemotherapy, immunotherapy, etc. have limited efficacy on CRPC-NE. Meanwhile, CRPC-NE does not express a prostate specific antigen and is therefore difficult to diagnose by serological means. Imaging is the primary means of current diagnosis of CRPC-NE. However, the lack of membrane expression of the prostate specific membrane protein (Prostate specific membrane antigen) in CRPC-NE results in lack of sensitivity and specificity in imaging diagnostics. Thus, there is a need to develop effective therapeutic and diagnostic measures for CRPC-NE.
Lineage plasticity (Lineage plasticity) refers to the regulation of cellular development and differentiation processes by cells through epigenetic remodeling to acquire the retrodifferentiation or transdifferentiation process of a survival dominant phenotype. And is also an important mechanism for causing tumor drug resistance. Current studies reveal that prostate cancer acquires a neuroendocrine phenotype through a lineage plastic pathway and proceeds to CRPC-NE stage. Epigenetic remodeling is a central factor driving the process described above. Within prostate cancer, there is significant remodeling of the various epigenetic factors associated with androgen receptor signaling. Resulting in loss of androgen receptor signaling and aberrant activation of stem cell and neuroendocrine cell related genes. And its appearance is manifested by highly heterogeneous expression of CRPC-NE membrane proteins. Therefore, the effective recognition of CRPC-NE specific membrane proteins and the development of related antibody-coupled drugs (Antibody conjugated drug, ADC) are of great potential significance for the diagnosis and treatment of such diseases.
Disclosure of Invention
The application aims to provide a CEACAM 5-targeted nano antibody, a preparation method and application thereof, and aims to solve the technical problem that the prior art lacks a neuroendocrine prostate cancer marker-targeted nano antibody, so that the treatment of prostate cancer is affected.
In order to achieve the above application, the technical scheme adopted in the application is as follows:
in a first aspect, the present application provides a nanobody targeting a neuroendocrine prostate cancer specific membrane marker CEACAM5, the nanobody comprising nanobody B12, wherein the amino acid sequence of nanobody B12 is as shown in seq.id No. 1.
Further, the nanobody includes 4 framework regions FR1, FR2, FR3, FR4, and 3 complementarity determining regions CDR1, CDR2, CDR3; in the nano antibody B12, the amino acid sequence of FR1 is shown as SEQ ID NO.2, the amino acid sequence of FR2 is shown as SEQ ID NO.3, the amino acid sequence of FR3 is shown as SEQ ID NO.4, the amino acid sequence of FR4 is shown as SEQ ID NO.5, the amino acid sequence of CDR1 is shown as SEQ ID NO.6, the amino acid sequence of CDR2 is shown as SEQ ID NO.7, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 8.
Further, the base sequence of the nanobody B12 is shown in seq. ID No. 9.
In a second aspect, the present application provides the use of a nanobody targeting the neuroendocrine prostate cancer specific membrane marker CEACAM5 in the manufacture of a medicament for treating castration-resistant neuroendocrine stage prostate cancer.
Further, the drug comprises at least one of a CAR T-related drug, a CAR NK-related drug, a bispecific antibody-related drug, a nano-drug.
In a third aspect, the present application provides the use of a nanobody targeting the neuroendocrine prostate cancer specific membrane marker CEACAM5 in the manufacture of a medicament for identifying castration resistant prostate cancer neuroendocrine stage imaging.
In a fourth aspect, the present application provides a method for preparing a nanobody targeting a neuroendocrine prostate cancer specific membrane marker CEACAM5, comprising the steps of:
providing a neuroendocrine prostate cancer specific membrane marker CEACAM5-FC protein;
coating the neuroendocrine prostate cancer specific membrane marker CEACAM5-FC protein on an immune tube for enrichment screening to obtain a phage library;
performing PCR amplification and ELISA verification on the eluent of the phage library, performing second-generation sequencing, and synthesizing a gene sequence of the nano antibody according to a sequencing result;
cloning the gene sequence of the nano antibody into an expression vector to obtain a recombinant plasmid, transferring the recombinant plasmid into a host cell to induce expression and purifying to obtain the nano antibody of the targeted neuroendocrine prostate cancer specific membrane marker CEACAM 5.
Further, the concentration of the target protein is 50 to 80. Mu.g/mL.
Further, the expression vector is a PET-22B vector.
Further, in the step of enrichment screening, 2-3 rounds of enrichment screening are performed.
The application provides a nano antibody of a targeted neuroendocrine prostate cancer specific membrane marker CEACAM5, which comprises a nano antibody B12, wherein the amino acid sequence of the nano antibody B12 is shown as seq. ID No. 1. Since CEACAM5 can be used as a downstream target gene of a neural development related transcription factor ASCL1, it is expressed on the surface of CRPC-NE membrane. Meanwhile, the related study also confirms that CEA, an expression product of CEACAM5 gene, is elevated in serum level, and is related to visceral metastasis of CRPC-NE. Therefore, CEACAM5 can be used as a potential therapeutic target for diagnosis and treatment of CRPC-NE; the provided nano antibody B12 has higher affinity with CEACAM5, can target the CEACAM5 serving as a specific membrane marker of the neuroendocrine prostate cancer, is favorable for developing medicaments related to the nano antibody of the CEACAM5 serving as the specific membrane marker of the neuroendocrine prostate cancer, realizes the targeted treatment of the castration resistant neuroendocrine stage prostate cancer, and has great clinical application value. Meanwhile, the nano antibody is favorable for wide application due to small molecular weight.
The application of the nano-antibody of the specific membrane marker CEACAM5 of the targeted neuroendocrine prostate cancer provided in the second aspect of the application in preparing a medicament for treating castration-resistant neuroendocrine stage prostate cancer. Because CEACAM5 can be used as a potential treatment target of the neuroendocrine prostate cancer, and the provided nano antibody B12 of the targeted neuroendocrine prostate cancer specific membrane marker CEACAM5 has high-strength binding capacity to the CEACAM5, the application of the nano antibody B12 in preparing the medicament for treating the castration-resistant neuroendocrine stage prostate cancer can be beneficial to carrying out targeted treatment on the castration-resistant neuroendocrine stage prostate cancer, and improves the treatment targeting and treatment effects.
The application of the nano antibody of the targeted neuroendocrine prostate cancer specific membrane marker CEACAM5 in preparing the medicine for identifying the castration resistant prostate cancer neuroendocrine stage imaging is beneficial to the targeted neuroendocrine prostate cancer specific membrane marker CEACAM5, and the nano antibody B12 has high-strength binding capacity to CEACAM5, so that the application of the nano antibody B12 in preparing the medicine for identifying the castration resistant prostate cancer neuroendocrine stage imaging can be beneficial to the targeted imaging of castration resistant prostate cancer and the analysis and treatment of castration resistant prostate cancer.
According to the preparation method of the nano-antibody targeting the neuroendocrine prostate cancer specific membrane marker CEACAM5, which is provided by the fourth aspect of the application, the preparation method is used for carrying out antibody screening based on a phage natural nano-antibody library to obtain a nano-antibody B12, the nano-antibody B12 has higher binding force with the neuroendocrine prostate cancer specific membrane marker CEACAM5, and the preparation method is quick, simple and convenient, is beneficial to large-scale screening, and improves screening efficiency.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Wherein:
FIG. 1 is a graph showing the characterization of the expression of CEACAM5 in prostate cancer and normal tissues according to an embodiment.
FIG. 2 is a graph of membrane expression profiling of CEACAM5 of a specific example in a CRPC-NE cell line.
FIG. 3 is a schematic diagram of phage library screening and analysis of the sequence of nanobody B12 obtained in accordance with the specific examples.
FIG. 4 is a diagram showing the analysis of nanobody B12 identification obtained by purification in the specific example.
FIG. 5 is an in vitro experimental study analysis chart of the nanobody B12 targeting CEACAM5 antigen of the specific embodiment.
FIG. 6 is an in vitro study analysis of nanobody B12 targeting CEACAM5 positive cell lines of specific examples.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In a first aspect, the embodiment provides a nano-antibody targeting a neuroendocrine prostate cancer specific membrane marker CEACAM5, the nano-antibody comprises a nano-antibody B12, wherein the amino acid sequence of the nano-antibody B12 is shown as seq.id No. 1.
The embodiment provides a nano antibody of a targeted neuroendocrine prostate cancer specific membrane marker CEACAM5 in the first aspect, wherein the nano antibody comprises a nano antibody B12, and the amino acid sequence of the nano antibody B12 is shown as seq. ID No. 1. Since CEACAM5 can be used as a downstream target gene of a neural development related transcription factor ASCL1, it is expressed on the surface of CRPC-NE membrane. Meanwhile, the related study also confirms that CEA, an expression product of CEACAM5 gene, is elevated in serum level, and is related to visceral metastasis of CRPC-NE. Therefore, CEACAM5 can be used as a potential therapeutic target for diagnosis and treatment of CRPC-NE; the provided nano antibody B12 has higher affinity with CEACAM5, can target the CEACAM5 serving as a specific membrane marker of the neuroendocrine prostate cancer, is favorable for developing medicaments related to the nano antibody of the CEACAM5 serving as the specific membrane marker of the neuroendocrine prostate cancer, realizes the targeted treatment of the castration resistant neuroendocrine stage prostate cancer, and has great clinical application value. Meanwhile, the nano antibody is favorable for wide application due to small molecular weight.
In some embodiments, a nanobody targeting the neuroendocrine prostate cancer specific membrane marker CEACAM5, the nanobody comprising nanobody B12, wherein the amino acid sequence of nanobody B12 is as shown in seq.id No.1, wherein seq.id No.1 is: MAVQLVESGGGLVQPGESLRLSCAASGVTFSTYGMGWARQVPGKGLEWVCG TYSDGSTYCADSVKGRFTISRDNAKNTVYLQMTSLKPEDTAVYYCTAPKHEY GTNWYERTIYSNELDYWGQGTQVTVSS.
In some embodiments, the nanobody comprises 4 framework regions FR1, FR2, FR3, FR4, and 3 complementarity determining regions CDR1, CDR2, CDR3.
In the nano antibody B12, the amino acid sequence of FR1 is shown as SEQ ID NO.2, and SEQ ID NO.2 is: MAVQLVESGGGLVQPGESLRLSCAAS.
The amino acid sequence of FR2 is shown as SEQ ID NO.3, and SEQ ID NO.3 is WARQVPGKGLEWVC.
The amino acid sequence of FR3 is shown as SEQ ID NO.4, and SEQ ID NO.4 is YCADSVKGRFTISRDNAKNTVYLQMTSLKPEDTAVYYCT.
The amino acid sequence of FR4 is shown as SEQ ID NO.5, and SEQ ID NO.5 is WGQGTQVTVSS.
The amino acid sequence of CDR1 is shown as SEQ ID NO.6, and SEQ ID NO.6 is GVTFSTYGMG.
The amino acid sequence of CDR2 is shown as SEQ ID NO.7, and SEQ ID NO.7 is GTYSDGST.
The amino acid sequence of CDR3 is shown as SEQ ID NO.8, and SEQ ID NO.8 is APKHEYGTNWYERTIYSNELDY.
In some embodiments, the nanobody B12 has a base sequence as shown in seq.id No.9, seq.id No. 9:
ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGTTTGGTGCAGCCTGGGGAGTCTCTGAGACTCTCCTGTGCAGCTTCTGGTGTCACCTTCAGTACATATGGGATGGGCTGGGCCCGCCAGGTTCCAGGGAAGGGACTCGAGTGGGTGTGCGGAACTTATAGTGATGGTAGCACATATTGTGCAGACTCCGTGAAGGGCCGCTTCACCATCTCCAGAGACAACGCCAAGAATACGGTGTATTTGCAAATGACTAGCCTGAAACCTGAAGATACGGCCGTTTATTACTGTACAGCCCCCAAACATGAATACGGGACTAACTGGTACGAGAGGACAATTTACTCGAATGAACTTGATTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。
in a second aspect, embodiments of the present application provide the use of nanobodies targeting the neuroendocrine prostate cancer specific membrane marker CEACAM5 in the manufacture of a medicament for treating castration-resistant neuroendocrine stage prostate cancer.
The application of the nano-antibody of the targeted neuroendocrine prostate cancer specific membrane marker CEACAM5 in preparing the medicine for treating castration-resistant neuroendocrine stage prostate cancer is provided in the second aspect of the embodiment. Because CEACAM5 can be used as a potential treatment target of the neuroendocrine prostate cancer, and the provided nano antibody B12 of the targeted neuroendocrine prostate cancer specific membrane marker CEACAM5 has high-strength binding capacity to the CEACAM5, the application of the nano antibody B12 in preparing the medicament for treating the castration-resistant neuroendocrine stage prostate cancer can be beneficial to carrying out targeted treatment on the castration-resistant neuroendocrine stage prostate cancer, and improves the treatment targeting and treatment effects.
In some embodiments, the drug comprises at least one of a CAR T-related drug, a CAR NK-related drug, a bispecific antibody-related drug, a nano-drug.
A third aspect of embodiments of the present application provides the use of nanobodies targeting the neuroendocrine prostate cancer specific membrane marker CEACAM5 in the preparation of a medicament for identifying castration-resistant prostate cancer neuroendocrine stage imaging.
The application of the nano antibody of the targeted neuroendocrine prostate cancer specific membrane marker CEACAM5 in preparing the medicament for identifying the castration resistant prostate cancer neuroendocrine stage imaging provided by the third aspect of the embodiment of the application is beneficial to carrying out targeted imaging on castration resistant prostate cancer and analyzing and treating castration resistant prostate cancer because the nano antibody B12 of the targeted neuroendocrine prostate cancer specific membrane marker CEACAM5 has high-strength binding capacity on CEACAM5, and the application of the nano antibody B12 in preparing the medicament for identifying the castration resistant prostate cancer neuroendocrine stage imaging is beneficial to carrying out targeted imaging on castration resistant prostate cancer.
In a fourth aspect, the present embodiment provides a method for preparing a nano-antibody targeting a neuroendocrine prostate cancer specific membrane marker CEACAM5, including the following steps:
s01, providing a CEACAM5-FC protein which is a neuroendocrine prostate cancer specific membrane marker;
s02, coating the CEACAM5-FC protein of the neuroendocrine prostate cancer specific membrane marker on an immune tube for enrichment screening to obtain a phage library;
s03, performing PCR amplification and ELISA verification on the eluent of the phage library, performing second-generation sequencing, and synthesizing a gene sequence of the nano antibody according to a sequencing result;
s04, cloning the gene sequence of the nano antibody into an expression vector to obtain a recombinant plasmid, transferring the recombinant plasmid into a host cell to induce expression and purifying to obtain the nano antibody of the targeted neuroendocrine prostate cancer specific membrane marker CEACAM 5.
According to the preparation method of the nano-antibody of the targeted neuroendocrine prostate cancer specific membrane marker CEACAM5, which is provided by the fourth aspect of the application, the preparation method is based on the phage natural nano-antibody library for antibody screening to obtain the nano-antibody B12, the nano-antibody B12 has higher binding force with the neuroendocrine prostate cancer specific membrane marker CEACAM5, and the preparation method is rapid, simple and convenient, is beneficial to large-scale screening, and improves screening efficiency.
In step S01, a neuroendocrine prostate cancer specific membrane marker CEACAM5-FC protein is provided. In some embodiments, the CEACAM5-FC protein is purchased from ACRObiosystems.
In step S02, the neuroendocrine prostate cancer specific membrane marker CEACAM5-FC protein is coated on an immune tube for enrichment screening to obtain a phage library.
In some embodiments, the natural alpaca-derived phage display nanobody library is screened using an immune tube method, with a selected phage display library capacity of 2x10 9 . The screening steps are as follows: coating target protein on an immune tube according to the concentration of 50 mug/mL, and carrying out enrichment screening for 3 rounds; third round phage eluate was used for plating.
In step S03, the eluent of the phage library is subjected to PCR amplification and ELISA verification, then second generation sequencing is carried out, and the gene sequence of the nanobody is synthesized according to the sequencing result.
In some embodiments, 192 of the monoclonal antibodies are randomly picked for ELISA validation. Monoclonal was picked as ELISA using a third round of phage eluate plating, with CEACAM5 ELISA readings 3 times greater than the corresponding BSA readings, and FC antigen ELISA readings less than 2 times positive for BSA readings. Sequencing and determining sequence information by 2 times of positive monoclonal sent company identified by phage ELISA; and designing and synthesizing the screened nano antibody according to the sequencing information.
S04, cloning the gene sequence of the nano antibody into an expression vector to obtain a recombinant plasmid, transferring the recombinant plasmid into a host cell to induce expression and purifying to obtain the nano antibody of the targeted neuroendocrine prostate cancer specific membrane marker CEACAM 5.
In some embodiments, nanobody gene sequences are cloned into a PET-22B vector while fusion expressing a hemagglutinin TAG (hemagglutinin HA TAG) with the HIS TAG for subsequent detection. The expression purification steps are as follows: a) Due to the periplasmic expression system, induction was carried out overnight at 30℃and 250rpm using ITPG at a concentration of 1.0 mM; b) The cells were collected by centrifugation, washed with PBS and resuspended in phosphate solution containing 10nM imidazole; c) Adding polymyxin according to the proportion of 50 ten thousand units per 50ml of bacterial liquid, and shaking table schizomycete for 1 hour at 37 ℃; d) Centrifuging 17000g at 4deg.C for 30min, and incubating supernatant with Ni filler at 4deg.C for 1 hr; e) The mycoproteins were removed by washing with gradient imidazole/phosphate solutions (10 mM and 20 mM). 15% SDS-PAGE gel electrophoresis separation, coomassie brilliant blue staining and western blot analysis identification.
The following description is made with reference to specific embodiments.
Example 1
In vitro verification of expression characteristics (tissue/cell level) of CEACAM5 in CRPC-NE
The test process comprises the following steps:
1) Tissue immunohistochemical staining
CRPC-NE prostate cancer tissue was derived from the third affiliated hospital of the university of medical science, south, for a total of 4 cases. 3 cases of PDX model CRPC-NE tissue were collected. Normal human tissue, prostate cancer, benign prostatic hyperplasia and normal prostate tissue are derived from tissue chips (midget light bloom). Paraffin-embedded tissues dewaxed and hydrated for antigen retrieval. OCT embedded tissue, after slicing OCT was cleared with PBS and fixed with acetone/methanol solution (4:1) for 10 min. All tissues were blocked with 3% hydrogen peroxide solution for endogenous peroxidase activity. After 2-3 times of TBS washing, 4% donkey serum was blocked for 1 hour. Then the blocking liquid is used for blocking the mixture according to the following formula 1: the CEACAM5 antibody was diluted 100 and incubated with the chip for 1 hour. Washing for 2-3 times by TBS again, and sealing by using a sealing liquid according to the proportion of 1:200 dilution of biotin labelled secondary antibody, incubated with the chip for 1 hour. And (3) after TBS is cleaned for 2-3 times, a proper amount of horseradish peroxidase-labeled avidin working solution is dripped, and the mixture is incubated for 30 minutes at room temperature. TBS is washed 2-3 times. After the DAB color developing agent develops color for 2 minutes, tap water is fully washed, dehydrated, transparent and sealed.
2) Western blotting experiment of cell extraction
The prostate cancer cell lines LNCAP, 22RV1, VCAP, PC3, and NCI-H660 were taken at the logarithmic phase of growth. Pre-cooling on ice for 10 min, washing with PBS for 3 times and air-drying. The cells were lysed thoroughly using RIPA-potent lysates with pre-addition of PMSF, protease and phosphatase inhibitors. After centrifugation at 12000rpm for 5min, the supernatant was collected and loading buffer was added at 1:4 and the deformed protein was boiled at 95℃for 5 min. And (3) taking the prepared 10% SDS-PAGE gel, and transferring the cell lysate to a PVDF membrane after electrophoresis separation until protein markers are fully separated. After blocking with 10% nonfat dry milk for 1 hour, CEACAM5 antibodies were diluted 1:100 and incubated overnight in a refrigerator at 4 ℃. TBST was washed three times, secondary antibodies were incubated and washed 3 times again with TBST solution. ECL development exposure.
3) Cellular immunofluorescence assay
22RV1, VCAP (CEACAM 5-) and NCI-H660, HT29 (CEACAM 5+) cell lines were implanted into a polylysine coated Perkin Elmer ultra cell well plate. The method comprises the following specific steps: a) Cells were placed on ice, washed 3 times with PBS, and fixed with 0.25% paraformaldehyde for 10 min; b) Washing 3 times with PBS, blocking with 4% bsa for 1 hour; c) The sealing liquid is used for sealing according to the proportion of 1:100 dilution of CEACAM5 antibody, co-incubation with cells for 1 hour at room temperature; d) Pre-cooling PBS was used for washing 3 times, and blocking solution was used at a ratio of 1: diluting Alexa 594 conjugated secondary antibody by 1000, and incubating with cells for 1 hour; e) Washing 3 times by precooling PBS, nuclear dyeing by Hoechst33342, and detecting the expression condition of CEACAM5 protein by a fluorescence inverted microscope.
4) Flow cytometry membrane surface antigen detection
The CEACAM5 positive cell line NCI-H660 and HT29 were taken. Cells were collected after pancreatin digestion and washed with PBS. After fixation of 4% pfa for 10 min, PBS wash again. CEACAM5 antibodies were diluted with 4% BSA solution at a ratio of 1:100 and incubated for 1 hour at room temperature. After 3 washes in PBS, a 1:1000 dilution of Alexa 488-conjugated secondary antibody was added again and incubated for 1 hour at room temperature. After the complete PBS cleaning, the cell line membrane surface antigen characteristics are detected by FITC channels.
Analysis of results:
immunohistochemical analysis of the expression profile of CEACAM5 in prostate adenocarcinoma (classified according to the international urological coordination prostate cancer pathological grading system, ISUP grading, group treatment), CRPC-NE and normal human tissues. The results are shown in FIG. 1, wherein A in FIG. 1 and B in FIG. 1 are the expression characteristics and the positive rate analysis of CEACAM5 protein in CRPC-NE tissue; c of FIG. 1 and D of FIG. 1 are analysis of expression characteristics and positive rate of CEACAM5 in prostate adenocarcinoma tissues; e of FIG. 1 and F of FIG. 1 are expression characteristics of CEACAM5 in normal human tissue. It can be seen that CRPC-NE tissue has higher CEACAM5 expression positive rate. The positive rate of the kit in CRPC-NE clinical specimens and PDX tissues can reach 50% (2/4) and 66% (2/3) respectively. However, in prostate adenocarcinoma, prostate hyperplasia and normal tissues, CEACAM5 immunohistochemical staining was almost negative. Meanwhile, immunohistochemistry of normal human tissues showed positive expression of CEACAM5 in only part of the digestive tract tissues (esophagus, colon), and negative expression in stomach, small intestine, liver, pancreas, lung, kidney, bladder, testis, spleen and heart tissues. From the above results, CEACAM5 is considered to be a membrane protein marker of high expression in CRPC-NE tissue.
Subsequently, the expression profile of CEACAM5 in prostate cancer cell lines was analyzed using western immunoblotting techniques. As a result, CEACAM5 was found to be positively expressed in the CRPC-NE-derived NCI-H660 cell line. Subsequent use of immunofluorescence analysis, as shown in fig. 2, of CEACAM5 membrane expression profile in CRPC-NE cell line, wherein a of fig. 2 is immunoblot analysis of CEACAM5 endogenous expression in prostate cancer cell line; FIGS. 2B and 2C are membrane expression profiles of CEACAM5 in the CRPC-NE cell line and CDX model; FIG. 2D is a measurement of the positive rate of membrane expression of CEACAM5 in a CRPC-NE derived NCI-H660 cell line. CEACAM5 localization to NCI-H660 cell line membranes can be found. NCI-H660CDX tissue samples were also immunohistochemical for the demonstration of membrane surface positive expression characteristics of CEACAM 5. Flow cytometry analysis showed that the positive rate of CEACAM5 in the CRPC-NE derived cell line NCI-H660 was 37.8%.
Taken together, CEACAM5 is suggested to be an important membrane protein marker for CRPC-NE and can be used as a potential target for antibody targeted therapy.
Example 2
Screening, purifying and identifying targeted CEACAM nano antibody phage library
The test process comprises the following steps:
1) Nanobody screening
The CEACAM5-FC protein is purchased from ACRObiosystems. Screening a natural alpaca-derived phage display nanobody library by adopting an immune tube method, wherein the selected phage display library capacity is 2x109. The screening steps are as follows: a) Coating target protein on an immune tube according to the concentration of 50 mug/mL, and carrying out enrichment screening for 3 rounds; b) 192 monoclonal were randomly picked for ELISA validation using a third round of phage eluate plating. Monoclonal was picked as ELISA using a third round of phage eluate plating, with CEACAM5 ELISA readings 3 times greater than the corresponding BSA readings, and FC antigen ELISA readings less than 2 times positive for BSA readings. c) Sequencing positive monoclonal identified by 2 times of phage ELISA (enzyme-linked immunosorbent assay) to determine sequence information; d) Designing and synthesizing the screened nano antibody according to the sequencing information, and carrying out expression purification by escherichia coli; e) The affinity of the nanobody is preliminarily identified by ELISA affinity experiments, the nanobody with better affinity is selected, and the affinity constant is determined by surface plasmon resonance (surface plasmon resonance, SPR) after expression and purification.
2) Purification expression of nanobodies
The nanobody gene sequence is cloned into a PET-22B carrier, and simultaneously, a hemagglutinin TAG (hemagglutinin HA TAG) and an HIS TAG are fused and expressed for subsequent detection. The expression purification steps are as follows: a) Due to the periplasmic expression system, induction was carried out overnight at 30℃and 250rpm using ITPG at a concentration of 1.0 mM; b) The cells were collected by centrifugation, washed with PBS and resuspended in phosphate solution containing 10nM imidazole; c) Adding polymyxin according to the proportion of 50 ten thousand units per 50ml of bacterial liquid, and shaking table schizomycete for 1 hour at 37 ℃; c) Centrifuging 17000g at 4deg.C for 30min, and incubating supernatant with Ni filler at 4deg.C for 1 hr; g) The mycoproteins were removed by washing with gradient imidazole/phosphate solutions (10 mM and 20 mM). 15% SDS-PAGE gel electrophoresis separation, coomassie brilliant blue staining and western blot analysis identification.
Analysis of results:
as shown in FIG. 3, A of FIG. 3 is the library enrichment of more than 50-fold by 3 rounds of phage nanobody library screening. Subsequently, 192 clones from the library obtained in the third round were selected for secondary phage ELISA primary validation to obtain 1 positive clone, designated nanobody B12 (as shown in B of fig. 3), and specific amino acid sequences were:
MAVQLVESGGGLVQPGESLRLSCAASGVTFSTYGMGWARQVPGKGLEW VCGTYSDGSTYCADSVKGRFTISRDNAKNTVYLQMTSLKPEDTAVYYCTAPK HEYGTNWYERTIYSNELDYWGQGTQVTVSS。
further, after optimizing codons according to the B12 sequence, a pet.22b vector was introduced and induced to express in escherichia coli. After colistin is split, the Ni column captures the B12 nano antibody with His tag protein. The bacterial proteins were removed by washing with gradient imidazole/phosphate solutions (10 mM and 20 mM). As shown in fig. 4, a high purity nanobody B12 was obtained. Wherein, a in fig. 4 is a coomassie blue staining result of the purified nanobody B12, B in fig. 4 is an analysis result of the HIS and HA tag carried by the nanobody verified by western blotting, and it is determined that the purified nanobody B12 is obtained.
Example 3
In vitro experimental study of B12 nano antibody targeting CEACAM5 antigen
The test process comprises the following steps:
1) Dot blot experiments
CEACAM5 positive cells HT29 and NCI-H660 total protein were extracted with RIPA potent lysate containing PMSF, protease and phosphatase inhibitor, and BCA was quantified. Nitrocellulose membrane was taken and 1cm3 cells were drawn with a pencil. 5. Mu.L of the solution to be examined was prepared in a gradient of 0, 0.4, 0.8, 1.2, 1.6 and 2.0. Mu.g and was dropped into the cell. And (5) airing at room temperature. After blocking with 10% nonfat milk powder for 1 hour, 15ml of nanobody working solution was prepared at a concentration of 20. Mu.g/ml using 4% BSA. The nanobody working solution and nitrocellulose membrane were placed in a 10cm dish and incubated overnight at 4 ℃. PBST is washed 3 times, and anti-HA antibody coupled with horseradish peroxide is added according to the proportion of 1:1000 for incubation for 1 hour at room temperature. PBST washing was again performed. ECL development exposure.
2) ELISA assay for nanobodies
ELISA plates were coated with CEACAM5-FC protein. Meanwhile, the coated FC antigen and BSA served as negative references. Blocking with 4% BSA, adding various concentrations of nano-antibodies, incubating for 1 hour at room temperature, rinsing with PBS for 3 times, incubating for 1 hour at room temperature with anti-HA antibody, amplifying signal with horseradish peroxidase-labeled anti-HA antibody, and developing TMB.
3) Surface plasmon resonance experiments
This experiment was used to verify the direct interaction of the in vitro purified nanobody expressed in vitro with the in vitro purified antigen protein and to calculate the equilibrium constants of the two. Purified antigen proteins are immobilized on a chip, nanobodies with different concentrations are sequentially added to analyze the affinity with the antigen proteins, reaction signals within 360 seconds are recorded, a kinetic curve is made, and relevant parameters are calculated.
Analysis of results:
the result is shown in FIG. 5 through Dot blot experiments, wherein A in FIG. 5 is that the nano antibody can recognize CEACAM5 antigen in the lysate of CEACAM5 positive cell line (NCI-H660 and HT 29) by Dot blot experiments. The purified nano antibody B12 can be found to effectively recognize CEACAM5 antigen in CEACAM5 positive cell lysate. It appears to be a linear correlation between the spot signal intensity and the amount of cell lysate protein.
FIG. 5B shows the affinity and specificity of the antigen CEACAM5 recognized by nanobody B12 as determined by Elisa experiments. Elisa also demonstrates that nanobody B12 has high specificity and affinity in recognizing CEACAM5 antigen.
Fig. 5C shows that nanobody B12 recognizes CEACAM5 antigen titers at nanomolar level (kd=6.653×10 -8 M). SPR experiments confirm that B12 nanobody recognizes CEACAM5 antigen titers at nanomolar level (kd=6.653×10 -8 M). In conclusion, nanobody B12 can effectively recognize CEACAM5 antigen.
Example 4
In vitro binding studies of B12 nanobody-targeted CEACAM5 positive cell line
The test process comprises the following steps:
1) Immunofluorescence analysis of nanobody CEACAM5+ cell line
The CEACAM5 positive HT29 and NCI-H660 cells were resuscitated and after 3 passages the cells were seeded in 96-well plates for subsequent experiments. The method comprises the following specific steps: a) Washing 3 times with PBS, fixing 0.25% paraformaldehyde for 10 minutes; b) Washing 3 times with PBS, blocking with 4% bsa for 1 hour; c) Diluting the nanobody to 1 mu M working solution by using a blocking solution and incubating the nanobody and cells for 1 hour at room temperature; d) PBST was washed 3 times, and incubated with anti-HA antibody diluted 1:100 for 1 hour at room temperature; e) PBST was washed 3 times, and 1 was added with blocking solution: 1000 dilution of Alexa 594 conjugated secondary antibody, incubated with cells for 1 hour; f) PBST cleared residual antibody, hoechst33342 nuclear stain, and observed under an inverted fluorescence microscope.
2)Cell Elisa
Nanobody and IR800-Mal according to 5:1, and reacting overnight at 4 ℃. The purity was then analyzed by SDS-PAGE and the labeling effect of the protein was detected in the fluorescent mode. E.g.4% paraformaldehyde, was fixed in 96-well plates for EACAM5 positive HT29 and NCI-H660 cells. Blocking with 4% BSA for 1 hour. Nanobody was diluted with blocking solution to gradient working solution (up to 750 nM). And incubated with cells for 1 hour at room temperature. PBST was washed 3 times. And detecting the combination condition of the cell surface nano antibody by the full-automatic electrophoresis fluorescence immunoassay analyzer.
Analysis of results:
as shown in fig. 6, immunofluorescence showed that nanobody B12 was effectively localized on the NCI-NE cell line NCI-H660 cell membrane positive for CEACAM5, compared to negative control nanobody C9. The Cell Elisa experiment further shows that nanobody B12 has high recognition efficacy on both CEACAM5 positive Cell lines HT29 and NCI-H660.
In summary, the present application provides a nanobody targeting neuroendocrine prostate cancer specific membrane marker CEACAM5, the nanobody includes a nanobody B12, wherein the amino acid sequence of the nanobody B12 is shown in seq id No. 1. Since CEACAM5 can be used as a downstream target gene of a neural development related transcription factor ASCL1, it is expressed on the surface of CRPC-NE membrane. Meanwhile, the related study also confirms that CEA, an expression product of CEACAM5 gene, is elevated in serum level, and is related to visceral metastasis of CRPC-NE. Therefore, CEACAM5 can be used as a potential therapeutic target for diagnosis and treatment of CRPC-NE; the provided nano antibody B12 has higher affinity with CEACAM5, can target the CEACAM5 serving as a specific membrane marker of the neuroendocrine prostate cancer, is favorable for developing medicaments related to the nano antibody of the CEACAM5 serving as the specific membrane marker of the neuroendocrine prostate cancer, realizes the targeted treatment of the castration resistant neuroendocrine stage prostate cancer, and has great clinical application value. Meanwhile, the nano antibody is favorable for wide application due to small molecular weight.
The foregoing disclosure is illustrative of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims.
Claims (4)
1. A nano-antibody B12 targeting a neuroendocrine prostate cancer specific membrane marker CEACAM5, wherein the amino acid sequence of the nano-antibody B12 is shown as seq. ID No. 1.
2. Use of nanobody B12 of the targeted neuroendocrine prostate cancer specific membrane marker CEACAM5 according to claim 1 for the preparation of a medicament for the treatment of castration resistant neuroendocrine stage prostate cancer.
3. The use of claim 2, wherein the drug comprises at least one of a CAR T-related drug, a CAR NK-related drug, a bispecific antibody-related drug, a nano-drug.
4. Use of nanobody B12 of the targeted neuroendocrine prostate cancer specific membrane marker CEACAM5 as claimed in claim 1 for the preparation of a medicament for identifying castration resistant prostate cancer neuroendocrine stage imaging.
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| CN102056945A (en) * | 2008-04-07 | 2011-05-11 | 埃博灵克斯股份有限公司 | Amino acid sequences directed against the Notch pathways and uses thereof |
| CN112028997A (en) * | 2020-08-04 | 2020-12-04 | 中山大学附属第五医院 | anti-CEACAM 5 nano antibody |
| WO2022116079A1 (en) * | 2020-12-03 | 2022-06-09 | 上海吉倍生物技术有限公司 | Humanized anti-ceacam5 antibody, and preparation method therefor and use thereof |
| CN115505043A (en) * | 2021-06-23 | 2022-12-23 | 上海吉倍生物技术有限公司 | Antibodies specifically binding glycosylated CEACAM5 |
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| CN102056945A (en) * | 2008-04-07 | 2011-05-11 | 埃博灵克斯股份有限公司 | Amino acid sequences directed against the Notch pathways and uses thereof |
| CN112028997A (en) * | 2020-08-04 | 2020-12-04 | 中山大学附属第五医院 | anti-CEACAM 5 nano antibody |
| WO2022116079A1 (en) * | 2020-12-03 | 2022-06-09 | 上海吉倍生物技术有限公司 | Humanized anti-ceacam5 antibody, and preparation method therefor and use thereof |
| CN115505043A (en) * | 2021-06-23 | 2022-12-23 | 上海吉倍生物技术有限公司 | Antibodies specifically binding glycosylated CEACAM5 |
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