CN114044823B

CN114044823B - Nano antibody targeting cadherin 17 and application thereof

Info

Publication number: CN114044823B
Application number: CN202111305763.3A
Authority: CN
Inventors: 王继刚; 李志杰; 马静博; 徐小龙; 戴凌云; 陈昆
Original assignee: Shenzhen Peoples Hospital
Current assignee: Shenzhen Peoples Hospital
Priority date: 2021-11-05
Filing date: 2021-11-05
Publication date: 2022-07-05
Anticipated expiration: 2041-11-05
Also published as: CN114044823A

Abstract

The invention provides a nano antibody targeting cadherin 17 and application thereof, wherein the complementary determining region of the amino acid sequence of the nano antibody targeting cadherin 17 is CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 4 and CDR3 shown in SEQ ID NO. 6; or CDR1 shown in SEQ ID NO. 9, CDR2 shown in SEQ ID NO. 11, and CDR3 shown in SEQ ID NO. 13. The nanometer antibody targeting cadherin 17 has good binding activity with CDH17 protein, has nanomolar affinity with CDH17 protein, has high affinity, and has specific recognition capability on CDH17 protein on the surface of cell membrane.

Description

Nano antibody targeting cadherin 17 and application thereof

Technical Field

The invention belongs to the technical field of immunology or molecular biology, and particularly relates to a cadherin 17-targeting nano antibody and application thereof.

Background

With the deep knowledge of molecular mechanisms and signal transduction of tumor genomics, proteomics, cancer cell-related proliferation, apoptosis, tumor angiogenesis, etc., some key molecules and signal pathways of various cancers in the digestive system are gradually revealed. A variety of targeted therapeutic drugs that are able to target these critical molecules or signaling pathways have emerged and have extended the patient's survival to some extent. For the treatment of digestive system tumors, targeting drugs that have been widely used clinically today are Avastin targeting VEGF, Erbitux and Vectibx targeting EGFR, and Herceptin targeting HER2, among others. In addition, there are a number of inhibitors of small molecule kinases that target key signaling pathways, such as Tarceva, Sorafenib, Gleevec, and Stivarga, among others. However, the targeted drugs only reduce the mortality rate of patients with a very small fraction of digestive system tumors. There is a great deal of evidence that the vast majority of patients do not benefit from these drugs, and some suffer from serious and even fatal side effects. Patients who are initially effective also develop resistance to the targeted drug, thereby losing their response to the drug. Taking an EGFR antibody as an example, 25-77% of colorectal cancer patients highly express the EGFR, and most of the patients initially have a good effect on the EGFR antibody, but then experience a rapid disease progression period due to drug resistance; the expression rate of HER2 in colorectal cancer is only 2-6%, so the HER2 antibody can only benefit a very small part of colorectal cancer patients. The other targeted drugs mentioned above have similar conditions in different tumors of the digestive system, and many fail in clinical trials. Therefore, for digestive system tumors, there is an urgent need to find new and better molecular targets and develop more effective molecular targeted therapeutic strategies.

Cadherin 17 (Cadherin 17, CDH 17), also known as Liver-gut Cadherin (LI-Cadherin), is a member of a Cadherin superfamily that has unique structures and functions. CDH17 consists of seven cadherin repeats constituting the extracellular segment, and the intracellular segment consists of only 24 amino acids. In recent years, various research institutes around the world have demonstrated that CDH17 exhibits high expression levels in a significant proportion of the above-mentioned cancer tissues in tumors of the digestive system such as gastric cancer, colorectal cancer, pancreatic cancer and partial liver cancer; especially in adenocarcinoma. Moreover, after the primary cancer highly expressing CDH17 molecule is transferred, the tumor tissue in the metastatic focus still keeps high expression CDH 17. Based on the analysis of TCGA data, the CDH17 gene in the tissues of stomach cancer, colorectal cancer, pancreatic cancer and partial liver cancer is shown to be expressed higher than that in corresponding normal tissues, and the expression levels of other normal organs are extremely low. Based on the current research situation, CDH17 has great potential as a new therapeutic target for digestive system malignant tumor and metastatic tumor thereof. However, no targeting antibody against CDH17 is currently available.

Disclosure of Invention

Aiming at the technical problems, the invention discloses a nano antibody targeting cadherin 17 and application thereof, wherein the nano antibody has good binding activity and high affinity with cadherin 17 and can be used in imaging and treatment products of CDH17 protein in various diseases.

In contrast, the technical scheme adopted by the invention is as follows:

a nanometer antibody targeting cadherin 17 is characterized in that the complementarity determining regions of the amino acid sequence of the nanometer antibody targeting cadherin 17 are CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 4 and CDR3 shown in SEQ ID NO. 6; specifically, the sequence of CDR1 shown in SEQ ID NO. 2 is GRTFSSNG, the sequence of CDR2 shown in SEQ ID NO. 4 is SWNAGTTL, and the sequence of CDR3 shown in SEQ ID NO. 6 is REYYGGSYYPSREYEYG;

or CDR1 shown in SEQ ID NO. 9, CDR2 shown in SEQ ID NO. 11, and CDR3 shown in SEQ ID NO. 13. Specifically, the sequence of CDR1 shown in SEQ ID NO. 9 is GSILSFNS, the sequence of CDR2 shown in SEQ ID NO. 11 is SIHKTSSN, and the sequence of CDR3 shown in SEQ ID NO. 13 is VQRGQSRIS.

As a further improvement of the invention, the framework region of the amino acid sequence of the nano antibody targeting cadherin 17 comprises FR1 shown in SEQ ID NO. 1, FR2 shown in SEQ ID NO. 3, FR3 shown in SEQ ID NO. 5 and FR4 shown in SEQ ID NO. 7.

As a further improvement of the invention, the amino acid sequence of the nano antibody targeting cadherin 17 is shown in SEQ ID NO. 15.

As a further improvement of the invention, the framework region of the amino acid sequence of the nano antibody targeting cadherin 17 comprises FR1 shown in SEQ ID NO. 8, FR2 shown in SEQ ID NO. 10, FR3 shown in SEQ ID NO. 12 and FR4 shown in SEQ ID NO. 14.

As a further improvement of the invention, the amino acid sequence of the nano antibody targeting cadherin 17 is shown in SEQ ID NO. 16.

The nano antibody of the technical scheme of the invention is an antibody of a specific targeting cadherin 17, can be combined with the targeting cadherin 17, and has high antigen binding property, high affinity, low immunogenicity and stronger tissue penetrating power.

The invention also discloses a nucleic acid, which is: nucleic acid encoding a cadherin 17-targeting nanobody as described in any of the above or a complementary sequence thereof.

The invention also discloses an expression vector which comprises the nucleic acid.

The invention also discloses a host cell which comprises the expression vector.

The invention also discloses application of the cadherin 17-targeting nano antibody, which is used as a detection reagent targeting cadherin 17, a living body imaging probe, a chimeric immune cell or a therapeutic antibody.

Compared with the prior art, the invention has the beneficial effects that:

the nanometer antibody targeting cadherin 17 has good binding activity with CDH17 protein, has nanomolar affinity with CDH17 protein, has high affinity, and has specific recognition capability on CDH17 protein on the surface of cell membrane. And the nano antibody has smaller molecular weight and better tissue penetrability, can be expressed and purified by using an escherichia coli system, and has low preparation cost.

Drawings

FIG. 1 is an immunofluorescence assay of the expression of the CDH17 protein on the membrane of IM95 in an example of the invention.

FIG. 2 is a diagram of the expression analysis of CDH17 protein in the immunohistochemical detection of gastric cancer, liver cancer, colon cancer and pancreatic cancer in the examples of the present invention; wherein (a) is gastric cancer, (b) is colon cancer, (c) is liver cancer, and (d) is pancreatic cancer.

FIG. 3 is a schematic representation of the CDH17 protein domain in an example of the invention.

FIG. 4 is an electrophoresis staining diagram of CDH17 protein after the extracellular domain 1-3 is expressed and purified.

FIG. 5 is an electrophoretogram of His and HA tag fused Nanobody proteins A1 and E8 according to an embodiment of the present invention.

Fig. 6 is WB detection results of a1 and E8 using His and HA tag antibodies according to examples of the present invention.

FIG. 7 shows the results of ELISA detection of the binding of A1 and E8 nanobodies to CDH17 protein.

FIG. 8 is a graph of an analysis of the affinity of the A1, E8 nanobody clone to CDH17 protein; wherein (a) is an affinity analysis chart of the A1 clone and the CDH17 protein, and (b) is an affinity calculation curve of the A1 clone and the CDH17 protein; (c) is an affinity analysis chart of the E8 clone and the CDH17 protein, and (d) is an affinity calculation curve of the E8 clone and the CDH17 protein;

FIG. 9 shows the CDH17 binding capacity assay of various concentrations of nanobodies of the present examples to MKN45, TMK1, IM95, and AGS cell surfaces; wherein (a) is on MKN45 cells, (b) is on TMK1 cells, (c) is on IM95 cells, and (d) is on AGS cells.

FIG. 10 is a photograph of the nano-antibody localized on the surface of MKN45 and AGS cell membrane observed by confocal laser microscopy according to the embodiment of the present invention.

Fig. 11 is an image of control nanobodies and CDH 17-targeted E8 nanobodies of the present invention in MKN45 gastric cancer mice, where (a) is the control nanobody and (b) is the E8 nanobody.

FIG. 12 is a graph of the fluorescence signal analysis of the control nanobody and the E8 nanobody at the tumor site according to the embodiment of the present invention.

Detailed Description

Preferred embodiments of the present invention are described in further detail below.

1. In vitro and in vivo verification of CDH17 expression

1) In vitro validation (immunofluorescence)

AGS and IM95 cells were revived and after 3 passages the cells were seeded in 24-well plates for subsequent experiments. The method comprises the following specific steps:

a) cells were placed on ice, washed 3 times with PBS and fixed in 4% paraformaldehyde for 10 minutes;

b) washed 3 times with PBS and blocked with 4% donkey serum for 1 hour;

c) and (3) sealing the mixture by using sealing liquid according to the proportion of 1: CDH17 antibody was diluted 100 and incubated with cells for 1 hour;

d) precooled PBS was washed 3 times with blocking solution at 1: the Alexa 488-conjugated secondary antibody was diluted 1000 and incubated with the cells for 1 hour;

e) and washing with precooled PBS 3 times, mounting by using a mounting medium containing DAPI, and detecting the expression condition of the CDH17 protein by using a fluorescence inverted microscope.

The cell surface condition of the gastric cancer cells detected by the immunofluorescence IM95 is shown in FIG. 1, and the result shows that CDH17 is highly expressed.

2) In vivo validation (immunohistochemistry)

Paraffin tissue chips of purchased stomach cancer, colon cancer, liver cancer and pancreatic cancer are dewaxed and hydrated for antigen retrieval, and then endogenous peroxidase activity is blocked by a 3% hydrogen peroxide solution. After washing with TBS 2-3 times, 4% donkey serum was blocked for 1 hour. Then, sealing liquid is used for sealing according to the proportion of 1: CDH17 antibody was diluted at 200 and incubated with the chip for 1 hour. And cleaning with TBS for 2-3 times, and sealing with sealing liquid according to the weight ratio of 1: biotin-labeled secondary antibodies were diluted 200 times and incubated with the chips for 1 hour. And (3) washing with TBS for 2-3 times, dropwise adding a proper amount of horse radish peroxidase-labeled avidin working solution, and incubating for 30 minutes at room temperature. TBS washing is performed for 2-3 times. And (3) fully washing by tap water after the DAB color developing agent develops color for 2 minutes, dehydrating, and sealing.

The expression of CDH17 in gastric cancer, colon cancer, liver cancer and pancreatic cancer detected by immunohistochemistry is shown in FIG. 2, and the results show that all of them are in high expression state.

2. Screening and verification of CDH17 protein extracellular domain nano antibody

1) CDH17 domain expression purification

According to the extracellular structure of CDH17, 1-3 domain genes of CDH17 protein are designed and synthesized, and purified protein is expressed and used for screening nano antibodies. The expression purification steps are as follows:

a) in order to prevent the formation of inclusion bodies and the degradation of protein, the induction conditions are simulated to be searched at the low temperature of 16 ℃ by IPTG with different concentrations;

b) carrying out a large amount of induction expression according to the induction conditions of the preliminary experiment, and carrying out bacterium breaking under the working condition of a high-pressure bacterium breaking instrument of 1000W;

c) 17000 g, centrifuging at 4 ℃ for 30 min, taking the supernatant and incubating with Ni filler at 4 ℃ for 1 hour;

d) after the Ni column is purified, molecular sieve separation is carried out, the AKTA parameter is set to be 0.5 mL flow rate/min, and the flow rate is collected once every 1 mL;

e) and determining the purity of the target protein according to the electrophoresis result, and determining the protein concentration by using a BCA method.

The schematic diagram of the CDH17 protein domain is shown in FIG. 3, and it can be seen that the CDH 171-3 domain protein is about 40 kD in size and is suitable for being used as a target protein for screening nanobodies. After codon optimization, CDH 171-3D gene is synthesized for escherichia coli induced expression and purification, in order to avoid protein degradation and generation of inclusion bodies, 0.2mM IPTG is adopted for induction at low temperature of 16 ℃ overnight, finally, the high-purity target protein is obtained after purification through a Ni column and a molecular sieve, an electrophoresis staining graph is shown in figure 4, electrophoresis Coomassie brilliant blue staining is carried out after the CDH17 protein extracellular section 1-3 structural domain expression and purification, and the method can be used for subsequent nano antibody screening.

2) Nanobody screening

Screening natural alpaca phage display nano antibody library by using an immune tube method, wherein the capacity of the selected phage display library is 2x10⁹. A1 and E8 nanometer antibodies are obtained through selection. Wherein the framework region of the amino acid sequence of the A1 nano antibody is FR1 shown in SEQ ID NO. 1, FR2 shown in SEQ ID NO. 3, FR3 shown in SEQ ID NO. 5, and FR 387 shown in SEQ ID NO. 7FR 4; the complementarity determining region is CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 4, CDR3 shown in SEQ ID NO. 6; the amino acid sequence of the antibody is shown in SEQ ID NO. 15. That is, the sequence of the A1 nanobody is: MAVQLVESGGGLVQAGDSLRLSCAASGRTFSSNGMGWFRQAPGKGREFVAAISWNAGTTLYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAAREYYGGSYYPSREYEYGYWGQGTQVTVSS is added.

The framework region of the amino acid sequence of the E8 nano antibody is FR1 shown as SEQ ID NO. 8, FR2 shown as SEQ ID NO. 10, FR3 shown as SEQ ID NO. 12 and FR4 shown as SEQ ID NO. 14; the complementarity determining regions are CDR1 shown in SEQ ID NO. 9, CDR2 shown in SEQ ID NO. 11, and CDR3 shown in SEQ ID NO. 13; the amino acid sequence of the antibody is shown as SEQ ID NO. 16. That is, the sequence of the E8 nanobody is: MAVQLVESGGGLVQPGGSLRLSCTASGSILSFNSMGWHRQAPGRQRELVAGISIHKTSSNYANFVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAAVQRGQSRISYWGQGTQVTVSS are provided.

3) Purified expression of Nanobodies

A1 nano antibody and E8 nano antibody gene sequences are cloned into a PET-14B vector, and a hemagglutinin tag (hemagglutinin HA tag) is fused and expressed for subsequent detection. The expression purification steps are as follows:

a) to prevent the formation of inclusion bodies and protein degradation, induction was performed at low temperature of 16 ℃ using IPTG at a concentration of 0.2 mM;

d) and after the Ni column is purified, the molecular sieve separation is carried out, the AKTA parameter is set to be 0.5 mL flow rate/min, and the molecular sieve is collected every 1 mL.

The A1 nano antibody, the E8 nano antibody and the control nano antibody C9 are expressed and purified, anti-His and anti-HA WB detection is carried out to confirm the correctness of the protein, and the results are shown in FIG. 5 and FIG. 6. As can be seen from FIG. 5, the 2 His and HA tags fused nanobody proteins A1 and E8 are expressed and purified to obtain high-purity nanobodies with the size of about 16 kD. Direct ELISA verification was performed, and the results are shown in fig. 7, confirming that the 2 nanobodies have binding activity to CDH17 protein.

4) ELISA experiment of Nanobody

And (2) fusing an HA label into the gene coding sequence of the nano antibody to express the nano antibody with the HA label, coating CDH 171-3 domain protein on an ELISA plate, sealing, adding the nano antibodies with various concentrations, incubating at room temperature for 1 hour, rinsing with PBS for 3 times, incubating at room temperature for 1 hour with anti-HA antibody, amplifying signals by using horseradish peroxidase-labeled anti-HA antibody, developing TMB, and simultaneously performing control of irrelevant nano antibodies and blank control of irrelevant protein antigens.

5) Surface plasmon resonance experiment

This experiment was used to verify that the in vitro expression of purified nanobodies interacts directly with the in vitro purified antigenic proteins and to calculate the equilibrium constants for the two. Purified antigen protein is fixed on a chip, nano antibodies with different concentrations are sequentially added to analyze the affinity with the antigen protein, reaction signals within 360 seconds are recorded, a kinetic curve is made, and each relevant parameter is calculated.

The results of the affinity constant test of clone A1 and clone E8 on CDH17 protein are shown in FIG. 8, and the affinities of A1 and E8 on CDH17 protein are 377nM and 70.3nM, respectively.

6) Targeting detection of cells by nano-antibody

Cells positive for CDH17, MKN45, TMK1, AGS, and IM95 were revived and seeded in 96-well plates after 3 passages for subsequent experiments. The method comprises the following specific steps:

a) cells were placed on ice, washed 3 times with PBS and fixed in 4% paraformaldehyde for 10 min;

b) washed 3 times with PBS and blocked with 4% donkey serum for 1 hour;

c) sequentially diluting the nano antibody into different concentration gradients (0-125 nM) by using confining liquid, and incubating the nano antibody with cells for 1 hour;

d) pre-chilled PBS was washed 3 times, with blocking solution at 1: 100 dilution of anti-HA primary antibody, and incubating with cells for 1 hour;

e) precooled PBS was washed 3 times with blocking solution at 1: the Alexa 488-conjugated secondary antibody was diluted 1000 and incubated with the cells for 1 hour;

f) and washing with precooled PBS for 3 times, and detecting the binding condition of the cell surface nano antibody by using a full-automatic electrophoresis fluorescence immunoassay analyzer.

The binding force of the nanobodies with different concentrations to MKN45, TMK1, IM95 and CDH17 on the cell surface of AGS is detected as shown in FIG. 9, the nanobodies are observed to be positioned on MKN45 and the cell membrane surface of AGS under a laser confocal microscope as shown in FIG. 10, and it can be seen that the nanobodies have specific recognition capability to CDH17 protein on the cell membrane surface.

7) Nano antibody surface modification fluorescent molecule

Reacting the nano antibody with IR800-Mal for 2-3 h at room temperature according to a feeding ratio of 1: 0.5-2, and centrifuging by using a 15kD ultrafiltration tube to remove unreacted IR800-Mal to obtain the nano antibody-IR 800 conjugate. Then, SDS-PAGE gel analysis of purity is carried out, and the labeling effect of the protein is detected in a fluorescence mode.

8) And (3) constructing a mouse tumor model.

Recovering MKN45 cells, and after 3 passages, inoculating the cells into a xenograft subcutaneous tumor model, wherein the inoculation cell amount is 3x10⁶Each (100. mu.L), the tumor size reached 100mm after two weeks³And on the left and right, subsequent experiments can be carried out.

9) In vivo targeting verification

Tumor-bearing mice with similar tumor sizes are selected, 75 mu g of IR-800 labeled control nano antibody and E8 nano antibody are injected into tail veins, and the distribution condition of fluorescence signals in the mice is detected by a small animal living body imager at 24 h.

In this example, the fluorescent molecule IR800 is coupled to the nanobody by chemical reaction and injected into tumor-bearing mice, and after 24 hours, the results of fluorescence detection are shown in fig. 11 and 12. As can be seen in fig. 11, the fluorescence signal of the E8 nanobody gradually gathered to the tumor site, whereas the control nanobody C9 did not significantly enrich at the tumor site, and it can be seen that the E8 nanobody can image the tumor by recognizing CDH 17. In addition, the results of the fluorescence signal analysis of the control nanobodies C9 and E8 nanobodies at the tumor site are shown in FIG. 12, and it can be seen that the E8 nanobody can generate a significantly stronger fluorescence signal at the tumor site than the control C9.

The embodiment of the invention also discloses a nucleic acid, which is: nucleic acid encoding the above a1 nanobody or E8 nanobody, or a complementary sequence thereof.

The embodiment of the invention also discloses an expression vector which comprises the nucleic acid.

The embodiment of the invention also discloses a host cell which comprises the expression vector.

The embodiment of the invention also discloses application of the nano antibody targeting cadherin 17, and the A1 nano antibody and the E8 nano antibody are used as a detection reagent targeting cadherin 17, a living body imaging probe, a chimeric immune cell or a therapeutic antibody.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the present invention pertains, several simple deductions or substitutions can be made without departing from the concept of the present invention, and modified derivative antibodies such as addition, reduction or substitution on the original sequence of the a1 nm antibody or the E8 nm antibody should be considered as falling within the protection scope of the present invention.

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Claims

1. A nano-antibody targeting cadherin 17, characterized in that: the complementary determining region of the amino acid sequence of the nano antibody targeting cadherin 17 is CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 4 and CDR3 shown in SEQ ID NO. 6;

or CDR1 shown in SEQ ID NO. 9, CDR2 shown in SEQ ID NO. 11, and CDR3 shown in SEQ ID NO. 13.

2. The nanobody targeting cadherin 17 according to claim 1, characterized in that: the framework region of the amino acid sequence of the nano antibody targeting cadherin 17 comprises FR1 shown in SEQ ID NO. 1, FR2 shown in SEQ ID NO. 3, FR3 shown in SEQ ID NO. 5 and FR4 shown in SEQ ID NO. 7.

3. The cadherin 17-targeting nanobody of claim 2, characterized in that: the amino acid sequence of the nano antibody targeting cadherin 17 is shown in SEQ ID NO. 15.

4. The cadherin 17-targeting nanobody according to claim 1, characterized in that: the framework region of the amino acid sequence of the nano antibody targeting cadherin 17 comprises FR1 shown in SEQ ID NO. 8, FR2 shown in SEQ ID NO. 10, FR3 shown in SEQ ID NO. 12 and FR4 shown in SEQ ID NO. 14.

5. The Nanobody targeting cadherin 17 according to claim 4, characterized in that: the amino acid sequence of the nano antibody targeting cadherin 17 is shown in SEQ ID NO 16.

6. A nucleic acid, wherein said nucleic acid is: nucleic acid encoding the cadherin 17-targeting nanobody of any one of claims 1 to 5 or a complementary sequence thereof.

7. An expression vector comprising the nucleic acid of claim 6.

8. A host cell comprising the expression vector of claim 7.

9. Use of the cadherin 17-targeting nanobody of any one of claims 1 to 5 in the preparation of a cadherin 17-targeting detection reagent, a biopsy probe, a chimeric immune cell or a therapeutic antibody.