Antibody specifically binding to GRP78 and use thereof

Abstract

The invention belongs to the field of antibody preparation, and particularly relates to an antibody specifically bound with GRP78 and application thereof. The present invention relates to an anti-GRP78 antibody comprising a heavy chain CDR and a light chain CDR of a specific sequence, or an antigen-binding fragment thereof. The anti-GRP78 antibody has very high specificity and affinity for GRP78, has a very significant growth-inhibiting effect, infiltration-inhibiting effect, and angiogenesis-inhibiting effect on colorectal cancer cell lines, and is expected to be effectively used as a composition for treating colorectal cancer, inhibiting metastasis, and inhibiting angiogenesis.

Description

Technical Field

[0001] This invention belongs to the field of antibody preparation, specifically relating to antibodies that specifically bind to GRP78 and their uses. Background Technology

[0002] 78-kDa glucose-regulated protein (GRP78) is an HSP70 family chaperone protein located in the endoplasmic reticulum (ER). It is a crucial factor in protein transport / transport into the ER and a key functional protein for maintaining intracellular calcium homeostasis. This protein consists of two domains: a nucleotide domain within the ER, more specifically, a domain that binds to ATP for degradation; and a substrate-binding domain (SBD) that binds to metabolic enzymes such as ATPase, metabolic-related proteins, and metabolic substrates (such as ADP). These domains regulate energy and calcium metabolism both within and outside the ER, based on intracellular calcium and ATP concentrations, by breaking down ATP during metabolism.

[0003] Pathologically, most colorectal cancers are adenocarcinomas, broadly classified by location as colon cancer and rectal cancer. The lower colon has the highest incidence, accounting for approximately 50% of cases in the workplace. Recent research indicates a significant increase in the incidence and mortality rates of colorectal cancer in South Korea due to changes in dietary habits. The causes of colon cancer are not fully understood, but considered factors include genetics, a diet high in fat and low in fiber, and inflammatory bowel disease. Colorectal cancer can occur at any age, but its frequency increases with age, frequently occurring in people in their 50s and 60s. The incidence is slightly higher in women with colon cancer and men with rectal cancer. Treatment for colorectal cancer is based on surgical resection, combined with chemotherapy and radiation therapy. Despite advancements in surgical, chemotherapy, and radiation therapies, the mortality rate is high because colorectal cancer is often asymptomatic, leading to metastasis to other organs and missed opportunities for surgery.

[0004] Recently, Imclone Systems Inc. developed Cetuximab, a recombinant mouse / human chimeric monoclonal antibody targeting EGFR (epidermal growth factor receptor). Although Cetuximab is widely used clinically to treat colorectal cancer, it is not effective as a monotherapy and is recommended for combination therapy with FOLFIR or FOLFOX. Another unmet medical need in the use of cetuximab for cancer treatment is its effectiveness only in a subset of colorectal cancer patients. Cetuximab is effective in approximately 10%–20% of colorectal cancer patients; the remaining patients exhibit cetuximab activity due to EGFR downstream gene mutations, including KRAS, PI3KCA (phosphatidyl-3-kinase alpha), PTEN (phosphatase and homolog), and BRAF. Therefore, identifying new therapeutic targets and developing new treatment regimens is crucial for improving clinical outcomes in colorectal cancer. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides an antibody that specifically binds to GRP78, comprising a light chain variable region and a heavy chain variable region; the light chain variable region includes light chain CDR1, light chain CDR2, and light chain CDR3; the heavy chain variable region includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; the amino acid sequence of light chain CDR1 is shown in SEQ ID NO:1; the amino acid sequence of light chain CDR2 is shown in SEQ ID NO:2; the amino acid sequence of light chain CDR3 is shown in SEQ ID NO:3; the amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO:4; the amino acid sequence of heavy chain CDR2 is shown in SEQ ID NO:5; and the amino acid sequence of heavy chain CDR3 is shown in SEQ ID NO:6.

[0006] The present invention also provides a nucleic acid molecule that encodes the aforementioned antibody.

[0007] The present invention also provides a recombinant expression vector comprising the above-mentioned nucleic acid molecules.

[0008] Furthermore, the recombinant expression vector also includes a promoter.

[0009] The present invention also provides a host cell, wherein the host cell comprises the above-mentioned nucleic acid molecule or the above-mentioned recombinant expression vector.

[0010] The present invention also provides a pharmaceutical composition comprising the above-described antibody, the above-described nucleic acid molecule, the above-described recombinant expression vector, or the above-described host cell.

[0011] The present invention also provides the use of the above-mentioned antibody, nucleic acid molecule, recombinant expression vector or host cell in the preparation of a composition for colorectal cancer diagnosis.

[0012] The present invention also provides a kit for detecting colorectal cancer, the kit comprising the aforementioned antibody.

[0013] The present invention has the following beneficial effects: This invention relates to antibodies that specifically bind to GRP78 and their uses. More specifically, this invention relates to anti-GRP78 antibodies or antigen-binding fragments thereof comprising heavy chain CDRs and light chain CDRs of specific sequences. The aforementioned anti-GRP78 antibodies exhibit very high specificity and affinity for GRP78, and demonstrate significant inhibitory effects on the growth, invasion, and angiogenesis of colorectal cancer cell lines. They are expected to be effectively utilized as compositions for the treatment of colorectal cancer and for inhibiting metastasis and angiogenesis. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 ELISA analysis results used to screen for anti-GRP78scFv antibodies.

[0016] Figure 2 Sensitivity analysis results of anti-GRP78scFv antibody.

[0017] Figure 3 The result of inhibiting in vitro tube formation using HUVEC cells with anti-GRP78scFv antibody.

[0018] Figure 4 Results of the inhibition of cell extramigration by anti-GRP78scFv antibody.

[0019] Figure 5 Results of inhibition of cell wound healing activity by anti-GRP78scFv antibody.

[0020] Figure 6 The result of anti-GRP78scFv antibody increasing apoptosis in colon cancer cells. Detailed Implementation

[0021] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Example 1: Screening for anti-GRP78scFv antibodies.

[0027] 1. Run bio-panning Bioanalysis was performed using an OPAL library with a diversity of 7.6 × 10⁹. 10 μg of GRP78 antigen was immobilized on an epoxymagnetic bead and reacted with in vitro phage. The antigen-reacted phage was eluted and the output titer was measured. Input and output titers were measured each time, and biofeedback information was obtained to confirm successful execution. Each batch of endophage used phage at a concentration of ≥1 × 10¹² CFU / mL. The output was -4.14 × 10⁹ per batch. 7 Secondary -5.32×10 7 The cfu / mL reading indicates amplification as the bioassay proceeds. Therefore, it can be confirmed that the bioassay is proceeding normally.

[0028] 2. ELISA Analysis ELISA analysis was used to screen for highly sensitive and specific antibodies. The obtained bacteriophages were used to infect *E. coli* and plated onto LB plates supplemented with antibiotics. After culturing at 30°C for 16 h, colonies were randomly collected. Each colony was inoculated into LB medium, treated with IPTG to express scFv, and *E. coli* was lysed. The water-soluble fraction was then used for ELISA. First, 25 ng of GRP78 antigen was immobilized in a 96-well ELISA plate and blocked with PBS containing 3% BSA. After 1 hour, the obtained cell lysate was treated and reacted at 37°C for 2 hours. The plate was washed three times with PBS containing 0.1% Tween 20, and then HRP-conjugated anti-HA antibody was diluted 1:1000 in the blocking solution and reacted at 37°C for 1 hour. The plate was washed five times with PBS containing 0.1% Tween 20, then developed with TMB substrate, and the scFv antibody bound to the antigen was measured using an ELISA reader. Ninety-six antibodies were analyzed, and an OD value of 0.2 was randomly determined as the positive derivative and 0.1 as the negative derivative. Nine antibodies were then selected. Figure 1 ).

[0029] Example 2: Sequence Analysis of Anti-GRP78scFv Antibody Nine GRP78 scFv positive clones were screened using ELISA analysis, and individual clones were further sequenced through sequence analysis. Sequence analysis confirmed that eight of the nine positive clones were solitary clones.

[0030] Example 3: Purification and Sensitivity Analysis of Screened Anti-GRP78scFv Antibodies 1. Purification of anti-GRP78scFv antibody Anti-GRP78scFv antibodies with the highest antigen-binding activity were purified from eight previously screened species (Table 1). ER2738 *E. coli* cells transformed with the anti-GRP78scFv clone were cultured in 500 mL of SB medium, and then lysed with 1×TES buffer. Cell lysates were reacted with 0.5 mL of Niebead and eluted with imidazole.

[0031] Table 1 Sequence sequencing results

[0032] 2. Sensitivity analysis of purified anti-GRP78scFv antibody Sensitivity analysis of the purified anti-GRP78scFv antibody was performed. Sensitivity analysis was conducted using ELISA. First, different concentrations of GRP78 antigen were immobilized on ELISA plates, then treated with 10 μg / mL anti-GRP78scFv antibody. Analysis was performed using HRP-bound anti-HA antibody diluted 1:1000. The results showed that the EC50 of the anti-GRP78scFv antibody was 459 ng / mL. Figure 2 ).

[0033] Example 4: In vitro tube formation analysis of screened anti-GRP78scFv antibodies To investigate the effect of anti-GRP78scFv antibody on tube formation, 1×10⁶ EGM-2 cultured cells were subjected to... 4 HUVEC cells were dispensed onto Martigel-coated plates and cultured with anti-GRP78scFv antibody and 1 μg cefoperazone as positive controls. Images were acquired using a microscope, and tube formation was quantified by counting the branches in the tubes. The results showed that the anti-GRP78scFv antibody had a good inhibitory effect on tube formation. Figure 3 ).

[0034] Example 5: In vitro cell migration analysis of screened anti-GRP78scFv antibodies 1. Migration assay To examine the effect of anti-GRP78scFv antibody on cell migration, colon cancer cells (HT29) were cultured at 1×10⁻⁶ cells per well. 5Cells were cultured in serum-free medium, and medium containing 10% FBS was added to the lower chamber. After 48 hours, to measure cell migration, the upper chamber was wiped with a PBS-soaked swab and fixed for 30 minutes at room temperature by adding 1 ml of 4% paraformaldehyde. Cells were stained with 500 μl of 1% crystal violet for 30 minutes, washed with water, and then the cell count was quantified under a microscope. The results showed that the anti-GRP78scFv antibody had a good inhibitory effect on cell infiltration. Figure 4 ).

[0035] 2. Wound-healing assay To investigate the effect of anti-GRP78scFv antibody on cell wound healing, colon cancer cells (HT29) were divided into 1×10⁻⁶ cells. 5 Cells were cultured in 37°C medium containing 5% carbon dioxide. After 24 hours, the cells were scraped and treated with different concentrations of anti-GRP78scFv, followed by incubation. After 48 hours, 1 ml of 4% paraformaldehyde was added for fixation at room temperature for 30 minutes to measure cell wound healing motility. Cells were then stained with 500 μl of 1% crystal violet for 30 minutes, washed with water, and wound healing motility was measured under a microscope. The results showed that the anti-GRP78scFv antibody effectively inhibited cell wound healing motility in a concentration-dependent manner. Figure 5 ).

[0036] Example 6 analyzes the effect of the screened anti-GRP78 scFv antibody on colon cancer cell death. 1. MTT assay To investigate the effect of anti-GRP78scFv antibody on the growth of colon cancer cells (HT29), colon cancer cells were aliquoted into 96-well plates, with 1 × 10⁶ cells per well. 4 The cells were then cultured at 37°C in a medium containing 5% carbon dioxide. After 24 hours, they were treated with different concentrations of anti-GRP78scFv and then further incubated under the same conditions. After the culture was completed, MTT{3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol ethidium bromide} solution was added, and the reaction was carried out for 3 hours. The culture medium was then removed, and 100 μL of dimethyl sulfide (DMSO) was added to each cell. The cells were shaken for 15 minutes, and then the absorbance was measured at 570 nm using an ELISA reader. The results showed that the concentration-dependent increase in anti-GRP78scFv antibody-induced apoptosis (…) Figure 6 ).

[0037] 2. LDH assay To investigate the effect of anti-GRP78scFv antibody on the growth of colon cancer cells, colon cancer cells were aliquoted into 96-well plates, with 1 × 10⁶ cells per well. 4Cells were cultured at 37°C in a medium containing 5% carbon dioxide for 24 hours. Afterward, they were treated with different concentrations of anti-GRP78scFv and then further incubated under the same conditions. After incubation, the cultures were transferred to 96-well plates, 50 μl of LDH reagent solution was added, and the plates were incubated at room temperature for 30 min. 50 μl of Stop solution was added, and absorbance was measured at 490 nm using an ELISA reader. As shown in Table 2, cell death increased in a concentration-dependent manner with anti-GRP78scFv.

[0038] Table 2 Absorbance Measurement Results

[0039] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An antibody that specifically binds to GRP78, characterized in that, It includes a light chain variable region and a heavy chain variable region; the light chain variable region includes light chain CDR1, light chain CDR2, and light chain CDR3; the heavy chain variable region includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; the amino acid sequence of light chain CDR1 is shown in SEQ ID NO:1; the amino acid sequence of light chain CDR2 is shown in SEQ ID NO:2; the amino acid sequence of light chain CDR3 is shown in SEQ ID NO:3; the amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO:4; the amino acid sequence of heavy chain CDR2 is shown in SEQ ID NO:5; and the amino acid sequence of heavy chain CDR3 is shown in SEQ ID NO:

6.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody as described in claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule as described in claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector also includes a promoter.

5. A host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 2 or the recombinant expression vector of claim 3 or 4.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody of claim 1, the nucleic acid molecule of claim 2, the recombinant expression vector of claim 3, or the host cell of claim 4.

7. The use of the antibody of claim 1, the nucleic acid molecule of claim 2, the recombinant expression vector of claim 3, or the host cell of claim 4 in the preparation of a composition for colorectal cancer diagnosis.

8. A reagent kit, characterized in that, The kit is for detecting colorectal cancer, and the kit includes the antibody as described in claim 1.

Images