CN114163526B

CN114163526B - Nano antibody targeting glucose regulatory protein 78 and application thereof

Info

Publication number: CN114163526B
Application number: CN202111338662.6A
Authority: CN
Inventors: 徐承超; 李志杰; 王惠芳; 王继刚; 卓晓凤; 付春进; 袁吉民
Original assignee: Shenzhen Peoples Hospital
Current assignee: Shenzhen Peoples Hospital
Priority date: 2021-11-12
Filing date: 2021-11-12
Publication date: 2022-06-21
Anticipated expiration: 2041-11-12
Also published as: CN114163526A

Abstract

The invention provides a nanometer antibody targeting glucose regulatory protein 78 and application thereof, wherein the complementarity determining region of the amino acid sequence of the nanometer antibody targeting glucose regulatory protein 78 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 affinity of the nano antibody targeting the glucose regulatory protein 78 is in nanomolar level with the affinity of the glucose regulatory protein 78, the nano antibody has high affinity, and the nano antibody has specific recognition capability on GRP78 protein on the surface of a cell membrane.

Description

Nano antibody targeting glucose regulatory protein 78 and application thereof

Technical Field

The invention belongs to the technical field of immunology or molecular biology, and particularly relates to a nano antibody targeting glucose regulatory protein 78 and application thereof.

Background

Glucose regulatory protein 78 (glucose regulated protein 78 kDa, GRP 78), a protein with a molecular weight of 78 kDa found in glucose-free cultured chicken embryo fibroblasts by Ira Pastan et al in 1977, is completely identical to immunoglobulin heavy chain binding protein (BIP), so that it is also called immunoglobulin heavy chain binding protein (BiP) as an important endoplasmic reticulum chaperone, participates in endoplasmic reticulum stress and protein folding and transport, and is important for tumor proliferation, invasion, metastasis and angiogenesis. The GRP78 protein is composed of two functional domains in its full length, including a nucleotide-binding domain (NBD) capable of binding ATP and a substrate-binding domain (SBD) capable of binding polypeptide/protein. GRP78 is widely present in cytoplasm, but under stress conditions such as hypoxia, glucose deprivation, and pH drop in tumor microenvironment, it is transported from cytoplasm to membrane surface, i.e., surface GRP78 (cell surface GRP78, csGRP 78). The research finds that csGRP78 is highly expressed in various highly malignant tumors (such as TNBC, glioblastoma and gastric cancer); csGRP78 is mainly present on the plasma membrane in the form of a peripheral protein and interacts with other cell surface proteins (such as glycosylphosphatidylinositol anchor proteins) to mediate signal transduction by tumor cells.

In addition, the expression profile of GPRP78 gene showed that the expression level of csGRP78 was highest in highly malignant and invasive glioblastoma, and that high expression of lung cancer, breast cancer, stomach cancer, colon cancer, liver cancer and angiogenic endothelial cells was also reported among different cancer types. These findings demonstrate that csGRP78 can be present on both malignant and endothelial cells, but is rarely expressed on normal cells, suggesting that csGRP78 as a product of dysregulation of endoplasmic reticulum protein homeostasis can serve as a cancer cell-specific biomarker, a target for cancer imaging and therapy, and also a potential tool for selective drug delivery.

Monoclonal antibodies are large in molecular weight and poor in tissue permeability, and the effectiveness of the monoclonal antibodies in solid tumors is greatly limited. The variable domain (VHH) of a single-chain antibody (HCAb) found in camelidae is a nanobody (Nb). The relative molecular mass of the nano antibody is about 15 kDa, which is about 1/10 of the conventional antibody. The nano antibody has strong stability and good solubility, can be expressed by pronucleus, and in addition, the gene sequence of the nano antibody has 80 percent of homology with the sequence of the human VH gene family III, so the immunogenicity is lower. Finally, the nanobody still retains better affinity and specificity compared to the single-loving antibody scFv. Therefore, the nano antibody has unique potential in the fields of new drug development and disease diagnosis. However, reports on nanobodies with good binding property and high affinity with GRP78 protein are not found so far.

Disclosure of Invention

Aiming at the technical problems, the invention discloses a nano antibody targeting glucose regulatory protein 78 and application thereof, wherein the nano antibody has good binding property with the glucose regulatory protein 78 and high affinity, and can be used for specifically identifying the glucose regulatory protein 78.

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

a nanobody targeting glucose regulatory protein 78 has the amino acid sequence complementarity determining regions 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 INTGNPALV, the sequence of CDR2 shown in SEQ ID NO. 4 is SISGNTN, and the sequence of CDR3 shown in SEQ ID NO. 4 is KKLPFGS;

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 GFTLDYYAI, the sequence of CDR2 shown in SEQ ID NO. 11 is SSAGVLTN, and the sequence of CDR3 shown in SEQ ID NO. 13 is AAADARQLKVRQCLSSNAYTY.

As a further improvement of the invention, the framework region of the amino acid sequence of the nanobody targeting glucose regulatory protein 78 includes 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 the glucose regulatory protein 78 is shown in SEQ ID NO. 15.

As a further improvement of the invention, the framework region of the amino acid sequence of the nanobody targeting glucose regulatory protein 78 includes 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 nanobody targeting the glucose regulatory protein 78 is shown in SEQ ID NO. 16.

The nano antibody of the technical scheme of the invention is an antibody of a specific target glucose regulatory protein 78, can be combined with the target glucose regulatory protein 78, and has high antigen binding property, high affinity, low immunogenicity and stronger tissue penetration.

The invention also discloses a nucleic acid, which is: nucleic acid encoding a nanobody targeting glucose regulatory protein 78 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 nano antibody targeting the glucose regulatory protein 78, which is used as a detection reagent targeting the glucose regulatory protein 78, 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 nano antibody targeting the glucose regulatory protein 78 has good binding activity with the glucose regulatory protein 78, has nanomolar affinity with the glucose regulatory protein 78, has high affinity, and has specific recognition capability on the glucose regulatory protein 78. 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 a graph showing the results of expressing GRP78 protein on cancer cell membranes and cancer cell membranes of tumor tissues in examples of the present invention, wherein (a) shows the results of expressing GRP78 protein on cell membranes of breast cancer tumor cells 4T1, (b) shows the results of staining GRP78 in tumor tissues of 4T1 mice, and (c) shows the results of staining tumor tissues of stomach cancer cells MKN45 mice.

FIG. 2 is an analytical diagram of the purification of GRP78 protein in an example of the present invention; wherein (a) is an electrophoretogram of a full-length protein GRP78, and (b) is an immunoblot of a full-length protein GRP 78; (c) the electrophoretogram for purification of GRP78 NBD structural domain protein, and the electrophoretogram for purification of GRP78 SBD structural domain protein.

FIG. 3 is a preliminary result of ELISA verification in the present example; wherein (a) is the primary result of full-length protein phage ELISA verification, (b) the primary result of NBD protein phage ELISA verification, and (c) the primary result of SBD protein phage ELISA verification.

FIG. 4 is a graph showing the results of deep sequencing of a library after screening three proteins of GRP78 according to an embodiment of the present invention, wherein (a) is a frequency distribution diagram of deep sequencing sequences of a library obtained from a full-length GRP78 protein, (b) is a frequency distribution diagram of deep sequencing sequences of a library after screening a GRP78 NBD domain, and (c) is a frequency distribution diagram of deep sequencing sequences of a library after screening a GRP78 SBD domain.

FIG. 5 shows the results of ELISA-verified comparison of NBD1 nanobody and SBD C5 nanobody of the present invention with a comparative nanobody, wherein (a) is NBD1 nanobody, (b) is SBD C5 nanobody, (C) is NBD C1 nanobody, (d) is SBD2 nanobody, and (e) is NGS3 nanobody.

FIG. 6 is a graph of surface plasmon resonance analysis of the affinity of NBD1 nanobody, SBD C5 nanobody and full-length GRP78, wherein (a) is NBD1 nanobody, and (b) is SBD C5 nanobody.

Detailed Description

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

1. In vitro detection of GRP78 membrane expression

Inoculating the breast cancer cells 4T-1 on a high content 96 micro-porous plate according to the number of 10,000-20,000/hole; after the cells are attached to the wall, washing the cells for 3 times by PBS and fixing the cells for 10 min by 0.25 percent paraformaldehyde; blocking with 5% BSA for 1 h; the anti-GRP78 antibody (1: 200) is diluted by PBST and incubated for 1 h at room temperature; PBST was washed 3 times, diluted with PBST and incubated for 1 h at room temperature with a fluorescent secondary antibody (1: 1000); PBST was washed 3 times, mounted with DAPI-containing mounting medium, and examined for membrane expression of GRP78 using a high content cellular imager.

2. In vivo detection of GRP78 membrane expression

Constructing breast cancer 4T1 and gastric cancer MKN45 tumor models, and determining whether the tumor volume reaches 300 mm³The tumor tissue is collected, a 10 mu m frozen section is prepared, and the membrane expression condition of the GRP78 protein in the tumor tissue is detected by immunofluorescence.

In this example, membrane expression of GRP78 on cancer cells and frozen cancer tissue was examined, and GRP78 protein staining was performed after fixing breast cancer cells 4T1 and 4T1 and MKN45 mouse tumor tissue with low-concentration paraformaldehyde, as shown in FIG. 1. As can be seen in FIG. 1 (a), the cell membrane of breast cancer tumor cell 4T1 is highly expressing GRP 78; as can be seen in fig. 1 (b), staining of 4T1 mouse tumor tissue GRP78 showed high expression of GRP78 in the tumor cell membrane; as can be seen in FIG. 1 (c), the tumor tissue staining of mouse with gastric cancer cells MKN45 GRP78 also suggests that the cancer cell membrane highly expresses GRP 78. Overall, GRP78 was highly expressed in cancer cell membranes, suggesting that GRP78 may serve as a potential therapeutic target for tumors.

3. Expression purification of full-Length GRP78 and NBD, SBD Domain

The full-length GRP78, NBD and SBD gene sequences were cloned into the prokaryotic expression vector PET-14 b. The expression purification steps are as follows:

a) and (3) transformation: adding the plasmid into BL21 (DE3) competent cells, standing on ice for 20 min, thermally shocking for 90 s, adding LB culture medium, shaking for 1 h, coating the bacterial liquid on an ampicillin plate, and performing inverted culture;

b) large-scale induction: selecting a monoclonal for amplification, adding isopropyl-beta-D-thiogalactoside (IPTG) with a certain concentration, and inducing overnight at 16 ℃;

c) centrifugally collecting the bacteria, and breaking the bacteria by a high-pressure bacteria breaker;

d) 12000 g, centrifuging for 1 h at 4 ℃, taking the supernatant and incubating with Ni-NTA resin for 1 h at 4 ℃;

e) purifying by using a Ni column; f) further separating and purifying by using a molecular sieve (AKTA purifier);

g) protein purity was identified by SDS-PAGE electrophoresis.

GRP78 contains two domains, NBD and SBD, as shown in FIG. 2. this example expresses and purifies three proteins, full-length (FIG. 2 (a), FIG. 2 (b)), NBD domain (FIG. 2 (c)), and SBD domain (FIG. 2 (d)), with molecular weights of about 80KD, 45KD and 35KD, respectively.

4. Nanobody library screening

The natural phage nanobody library constructed by 103 alpaca peripheral blood is used, and the capacity of the phage display library is 2 multiplied by 10⁹. Screening was performed using the immunotube method. The screening steps are as follows:

a) coating the target protein on an immune tube according to the concentration of 50 mu g/mL, and carrying out 3 rounds of elutriation and enrichment;

b) using a third round of phage eluate plating, randomly picking 96 single clones for ELISA verification, and taking the positive standard that the ELISA reading is 3 times larger than the BSA reading and the reading is more than 0.5;

c) positive single clones identified by 2 Phase-ELISA were sent to the company for sequencing.

The three proteins are used, a nano antibody phage library constructed by 103 alpaca peripheral blood is screened by an immune tube method, after three rounds of screening, 96 clones are respectively picked from a full-length protein library, an NBD protein library and an SBD protein library, and phage ELISA preliminary verification is carried out, wherein the result is shown in figure 3. As can be seen in FIG. 3 (a), the full-length protein phage ELISA showed 7 potential positive clones but with lower affinity, and as can be seen in FIGS. 3 (b) and 3 (c), the phage ELISA for NBD and SBD yielded one clone with better potential affinity, respectively.

The phage ELISA-based verification experiment can only detect a limited number of clones, and cannot reflect the frequency distribution of clone sequences in the library after the whole screening, so that in this embodiment, deep sequencing is performed on the nano library obtained by screening three proteins of GRP78, the sequencing result is analyzed, more than two million independent nano antibody sequences are obtained in total, and the obtained nano antibody sequences are sequenced according to the occurrence frequency, as shown in fig. 4. The more frequent sequences were selected for the next protein expression and purification comparison experiments, where five sequences with frequency over 1% of the full length of GRP78, 1 sequence with NBD domain close to 1% of frequency, and four sequences with SBD over 1% of frequency were used.

Through a large number of experiments, the NBD1 nano antibody and the SBD C5 nano antibody are finally selected.

Wherein the framework region of the amino acid sequence of the NBD1 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 FR4 shown in SEQ ID NO. 7; the complementarity determining region is CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 4, and CDR3 shown in SEQ ID NO. 6; the amino acid sequence of the antibody is shown in SEQ ID NO. 15. Namely the sequence of the NBD1 nanobody is: MAVQLVESGGGSVQAGGSLTLSCAASINTGNPALVGWYRQAPGKQREMVAMISISGNTNYAPSVKGRFTISRDNANKTIFLQMNSLTPEDTAVYYCKKLPFGSWGQGTQVTVSS are provided.

The framework region of the amino acid sequence of the SBD C5 nanobody is FR1 shown as SEQ ID NO. 8, FR2 shown as SEQ ID NO. 10, FR3 shown as SEQ ID NO. 12, 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 SBD C5 nanobody is: MAVQLVESGGGLVQAGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSAGVLTNYVDSVKGRFTISRDNAKGAVYLQMNDLKPEDAALYFCAAADARQLKVRQCLSSNAYTYWGQGTQVTVSS are provided.

5. Expression and purification of the nanobody:

and (3) constructing a coding region sequence of the nano antibody obtained by sequencing on a plasmid (added with an HA tag and a His tag), and expressing and purifying on escherichia coli. The expression purification step is similar to the antigen protein purification step. And HA tags are added in the sequence of the nano antibody for the detection of subsequent experiments, the size of the obtained nano antibody is about 15KD to 18KD, and the purity is over 90 percent.

6. ELISA affinity detection

Using full-length GRP78 to coat an ELISA plate, while using BSA protein as a control antigen, different concentrations of nanobodies were incubated with the antigen, and then using HRP-labeled HA secondary antibody for signal amplification, and observing color development, the nanobodies included NBD1 nanobody and SBD C5 nanobody, and NBD C1 nanobody, SBD2 nanobody, NGS3 nanobody as a comparison. The method comprises the following specific steps:

a) GRP78 protein was coated on Elisa plates at a concentration of 10. mu.g/mL overnight at 4 ℃;

b) blocking with 5% BSA at room temperature for 2 h;

c) PBST is washed twice, and a nanometer antibody solution diluted by times is added to be incubated for 1 h at room temperature;

d) PBST is washed for three times, anti-HA HRP (1: 3000) is added into each hole, and the mixture is incubated for 1 h at room temperature;

e) PBST is washed for three times, TMB reading matter is added, and color change is observed;

f) 1M HCl is added in time to stop the reaction, and the light absorption value is read at 450 nm by using an enzyme-linked immunosorbent assay.

The results of ELISA verification of the nanobodies using human GRP78 full-length protein and bovine serum albumin as controls are shown in FIG. 3, and it can be seen that SBD C5, NBD1, NBD C1, SBD2 and NGS3 nanobodies are all combined with GRP78 to different degrees, wherein the binding activity of SBD C5 and NBD1 antibodies is optimal.

The following experiments will further detect the protein affinity of NBD1 nanobody, SBD C5 nanobody with GRP78 using Surface Plasmon Resonance (SPR) technique.

7. Identification of affinity constants of Nanobodies with GRP78 by Surface Plasmon Resonance (SPR)

GRP78 protein was adjusted to 1 mg/mL using the Biacore T200 system, dissolved in sodium acetate buffer at pH 5.5, and the procedure was set to flow the protein solution through the chip, thereby immobilizing it on the negatively charged chip by amino carboxyl coupling. And then, flowing the nano antibody solution diluted by times through the chip, and calculating the mutual binding affinity according to the response value, wherein the affinity KD is obtained by dividing kinetic constants ka and KD.

By adopting the steps, the NBD1 nano antibody and SBD C5 nano antibody are subjected to surface plasmon resonance technology to determine the affinity with GRP78 full-length protein, and as shown in FIG. 4, the affinity of NBD1 nano antibody with GRP78 is 3.455 nM, and the affinity of SBD C5 nano antibody with GRP78 is 92.45 nM, which reach nanomolar level.

The embodiment of the invention also discloses a nucleic acid, which is: nucleic acid encoding NBD1 nanobody, SBD C5 nanobody or complementary sequences 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 the glucose regulatory protein 78, which is used as a detection reagent targeting the glucose regulatory protein 78, 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 sequences of NBD1 nanobody and SBD C5 nanobody should be considered as falling within the scope of the present invention.

Sequence listing

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Claims

1. The nanometer antibody of target glucose regulatory protein 78, characterized in that: the complementarity determining region of the amino acid sequence of the nanobody targeting glucose regulatory protein 78 is composed of CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 4 and CDR3 shown in SEQ ID NO. 6.

2. The nanobody targeting glucose regulatory protein 78 according to claim 1, characterized in that: the framework region of the amino acid sequence of the nanobody targeting glucose regulatory protein 78 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 nanobody targeting glucose regulatory protein 78 according to claim 2, characterized in that: the amino acid sequence of the nano antibody of the targeted glucose regulatory protein 78 is shown in SEQ ID NO. 15.

4. Nanobodies targeting glucose regulatory protein 78, characterized in that: the complementarity determining region of the amino acid sequence of the nanobody targeting glucose regulatory protein 78 is composed of CDR1 shown in SEQ ID NO. 9, CDR2 shown in SEQ ID NO. 11 and CDR3 shown in SEQ ID NO. 13.

5. The nanobody targeting glucose regulatory protein 78 according to claim 4, characterized in that: the framework region of the amino acid sequence of the nanobody targeting glucose regulatory protein 78 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.

6. The nanobody targeting glucose regulatory protein 78 according to claim 5, characterized in that: the amino acid sequence of the nano antibody of the targeted glucose regulatory protein 78 is shown in SEQ ID NO 16.

7. A nucleic acid, wherein said nucleic acid is: nucleic acid encoding the nanobody targeting glucose regulatory protein 78 of any of claims 1 to 6, or a complementary sequence thereof.

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

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

10. Use of the nanobody targeting glucose regulatory protein 78 of any one of claims 1 to 6 in the preparation of a detection reagent targeting glucose regulatory protein 78, a biopsy probe, a chimeric immune cell or a therapeutic antibody.