Antibodies against phactr1 proteins and uses thereof

Abstract

The application provides an antibody against a PHACTR1 protein and an application thereof, and belongs to the technical field of antibody engineering and pharmacy. The antibody against the PHACTR1 protein provided by the application has high specificity, provides effective materials for immunodetection of the PHACTR1 protein, can play a therapeutic effect in related diseases, and has wide practical application value.

Description

Technical Field

[0001] This invention relates to the field of antibody engineering pharmaceutical technology, and in particular to antibodies against PHACTR1 protein and their applications. Background Technology

[0002] PHACTR1, short for phosphatase and actin regulator 1, belongs to the PHACTR family. It was first discovered by Patrick B. Allen's research group while screening for protein phosphatase 1 (PP1) interacting proteins in brain tissue. Its mRNA is highly expressed in rat brain, heart, and lung tissues. This gene is highly conserved evolutionarily, with over 95% homology between humans and mice, and over 90% homology between humans and chickens. The main functional regions of PHACTR1 include four RPEL motifs (one at the N-terminus and three at the C-terminus) and one nuclear localization sequence (NLS). The RPEL motif primarily mediates the binding of the protein to G-actin (globulin), while the NLS sequence suggests its potential for nucleus entry.

[0003] In recent years, numerous large-scale genome-wide association studies (GWAS) both domestically and internationally have indicated that multiple SNP sites on PHACTR1 are closely related to cardiovascular diseases such as atherosclerosis, hypertension, stroke, and diabetes. Existing techniques have clearly established the promoting effect of PHACTR1 in endothelial cells on atherosclerosis. Animal experiments have shown that PHACTR1 gene knockout significantly inhibits atherosclerotic plaque formation in Apoe knockout mice. Notably, when the adventitia, media, and intima of the aorta were isolated and analyzed, PHACTR1 was expressed only in the intima composed of vascular endothelial cells; and it was not expressed in mouse bone marrow or macrophages. Knockdown of PHACTR1 in human umbilical vein endothelial cells (HUVECs) using siRNA significantly inhibited endothelial cell inflammation under resting conditions and TNF-α stimulation, and reduced the expression of adhesion molecules such as ICAM-1 and VCAM-1, as well as the adhesion of THP-1 cells to HUVECs. Turbulent flow treatment of HUVEC cells (simulating hemodynamics that promote inflammation and atherosclerosis) revealed that overexpressed PHACTR1 was located in the HUVEC cell nucleus, suggesting its potential involvement in transcriptional regulation. Regarding the molecular mechanism, RNA-seq experiments using mRNA extracted from wild-type and PHACTR1 knockout mouse aortic endothelial cells showed that PHACTR1 knockout primarily affects genes related to inflammatory response and vascular development. Transcription factor analysis indicated that genes regulated by the transcription factor PPARγ were most significantly upregulated in ECs after PHACTR1 knockout. PPARγ is known to play a crucial protective role in endothelial function, and analysis of the PHACTR1 protein sequence revealed the presence of the LXXXIXXX(I / L) motif—a PPARγ transcriptional repressor motif—on both human and mouse PHACTR1. Further in vivo and in vitro experiments demonstrated that PHACTR1 inhibits the activity of the PPARγ transcription factor, and the PPARγ inhibitor GW9662 eliminated the protective effect of PHACTR1 knockout on endothelial cell inflammation and atherosclerosis. In summary, PHACTR1, located in the endothelial cell nucleus, acts as a transcriptional co-repressor molecule, regulating the transcription of PPARγ target genes, causing endothelial cell inflammation, and promoting the development of cardiovascular diseases such as atherosclerosis. However, there are currently no specific drugs targeting PHACTR1. Summary of the Invention

[0004] The purpose of this invention is to provide antibodies against PHACTR1 protein and their applications, providing effective materials for further research on related diseases.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides an antibody comprising VHCDR1, VHCDR2 and VHCDR3;

[0007] And includes VLCDR1, VLCDR2 and VLCDR3;

[0008] The amino acid sequence of VHCDR1 is shown in SEQ ID NO.3;

[0009] The amino acid sequence of VHCDR2 is shown in SEQ ID NO.4;

[0010] The amino acid sequence of VHCDR3 is shown in SEQ ID NO.5;

[0011] The amino acid sequence of VLCDR1 is shown in SEQ ID NO.7;

[0012] The amino acid sequence of VLCDR2 is GTN;

[0013] The amino acid sequence of VLCDR3 is shown in SEQ ID NO.9.

[0014] The present invention also provides an antibody, wherein the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.2;

[0015] The amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO.6.

[0016] The present invention also provides an antigen-binding fragment that binds to the PHACTR1 antigen protein, comprising VHCDR1, VHCDR2 and VHCDR3;

[0017] And includes VLCDR1, VLCDR2 and VLCDR3;

[0018] The amino acid sequence of VHCDR1 is shown in SEQ ID NO.3;

[0019] The amino acid sequence of VHCDR2 is shown in SEQ ID NO.4;

[0020] The amino acid sequence of VHCDR3 is shown in SEQ ID NO.5;

[0021] The amino acid sequence of VLCDR1 is shown in SEQ ID NO.7;

[0022] The amino acid sequence of VLCDR2 is GTN;

[0023] The amino acid sequence of VLCDR3 is shown in SEQ ID NO.9.

[0024] The present invention also provides the coding gene of the antibody, comprising the heavy chain variable region coding gene as shown in SEQ ID NO.10 and the light chain variable region coding gene as shown in SEQ ID NO.14.

[0025] The present invention also provides the application of the antibody, antigen-binding fragment or encoding gene in the preparation of reagents or kits for detecting PHACTR1 protein.

[0026] The present invention also provides a reagent or kit for detecting PHACTR1 protein, containing the antibody or the antigen-binding fragment.

[0027] The present invention also provides the use of the antibody, antigen-binding fragment, or encoding gene in the preparation of products that specifically bind to the PHACTR1 protein in a test sample.

[0028] The present invention also provides the use of the antibody, antigen-binding fragment or encoding gene in the preparation of products that specifically bind to PHACTR1 protein subtype A and / or protein subtype D in the test sample.

[0029] The present invention also provides the use of the antibody, antigen-binding fragment, or encoding gene in the preparation of medicaments for the treatment or adjuvant treatment of diseases for which PHACTR1 protein is a molecular target.

[0030] The present invention also provides the use of the antibody, antigen-binding fragment or encoding gene in the preparation of a medicament for the treatment or adjuvant treatment of atherosclerosis.

[0031] The beneficial effects of this invention are:

[0032] The antibody against PHACTR1 protein provided by this invention has high specificity, providing an effective material for the immunoassay of PHACTR1 protein, and can exert therapeutic effects in related diseases, thus having broad practical application value. Attached Figure Description

[0033] Figure 1 The Western blot results for antibody recognition of recombinant antigen protein, where 1: protein molecular weight standard, 2: recombinant antigen protein;

[0034] Figure 2 The image shows the ELISA results for antibody recognition of recombinant proteins of antigens.

[0035] Figure 3The image shows the Western blot results for antibody recognition of human and mouse PHACTR1 protein. In the image, the blue arrows indicate human PHACTR1 protein isoform A / mouse PHACTR1 protein isoform 4, and the red arrows indicate human PHACTR1 protein isoform D / mouse PHACTR1 protein isoform 5. 1: Lysate of 293FT cells overexpressing human PHACTR1 protein isoform A, 2: Lysate of 293FT cells overexpressing human PHACTR1 protein isoform D, 3: Wild-type mouse brain tissue, 4: PHACTR1 knockout mouse brain tissue, 5: Wild-type mouse lung tissue, and 6: PHACTR1 knockout mouse lung tissue. Detailed Implementation

[0036] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0037] SEQ ID NO.1:

[0038] VAQHHHTVLPSQIQHQLQYGSHGQHLPSTTGSLPMHPSGCRMIDELNKTLAMTMQRLESSEQRVPCSTSYHSSGLHSGDGVTKAGPMGLPEIRQVPTVVIECDDNKENVPHESDYEDSSCLYTREEEEEEEDEDDDSSLYTSSLAMKVCRKDSLA IKLSNRPSKRELEEKNILPRQTDEERLELRQQIGTKLTRRLSQRPTAEELEQRNILKPRNEQEEQEEKREIKRRLTRKLSQRPTVEELRERKILIRFSDYVEVADAQDYDRRADKPWTRLTAADKAAIRKELNEFKSTEMEVHELSRHLTRFHRP

[0039] SEQ ID NO.2:

[0040] EVKLLESGGGLVQPGGSLKLSCAASGFDFSGYWMSWVRQAPGKGLEWIGEIIPDSSTINYSPSLKDKFIISRDNAKKTLYLQMSNVRSEDTALYYCARPSYYSSTFAYWGQGTLVTVSA

[0041] SEQ ID NO.3:

[0042] GFDFSGYW

[0043] SEQ ID NO.4:

[0044] IIPDSSTI

[0045] SEQ ID NO.5:

[0046] ARPSYYSSTFAY

[0047] SEQ ID NO.6:

[0048] QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCVLWYSNLWVFGGGTKLTVL

[0049] SEQ ID NO.7:

[0050] TGAVTTSNY

[0051] SEQ ID NO.8:

[0052] GTN

[0053] SEQ ID NO.9:

[0054] VLWYSNLWV

[0055] SEQ ID NO.10:

[0056] GAGGTGAAGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTGGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGAAAAGGGCTAGAATGGATTGGAGAGATTATTCCAGATAGCAGTACGATAAACTATTCGCCATCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAAGACGCTGTACCTGCAAATGAGCAATGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGACCTAGTTACTACAGTTCTACCTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0057] SEQ ID NO.11:

[0058] GGATTCGATTTTAGTGGATACTGG

[0059] SEQ ID NO.12:

[0060] ATTATTCCAGATAGCAGTACGATA

[0061] SEQ ID NO.13:

[0062] GCAAGACCTAGTTACTACAGTTCTACCTTTGCTTAC

[0063] SEQ ID NO.14:

[0064] CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGTTCTATGGTACAGCAACCTTTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA

[0065] SEQ ID NO.15:

[0066] ACTGGGGCTGTTACAACTAGTAACTAT

[0067] SEQ ID NO.16:

[0068] GGTACCAAC

[0069] SEQ ID NO.17:

[0070] GTTCTATGGTACAGCAACCTTTGGGTG

[0071] Example

[0072] I. Antigen Design

[0073] Due to the presence of different splicing variants, the PHACTR1 protein comprises multiple isoforms; human PHACTR1 has five isoforms: A, B, C, D, and E. This invention selects a polypeptide segment from a common region of the five isoforms, specifically positions 271-580 of PHACTR1 isoform A, as an antigen to prepare anti-PHACTR1 monoclonal antibodies. The amino acid sequence is shown in SEQ ID NO.1.

[0074] II. Antigen Preparation

[0075] First, RNA was extracted from human umbilical vein endothelial cells and reverse transcribed into a cDNA library. Specific primers were designed and synthesized to amplify the PHACTR1 gene, obtaining the full-length cDNA sequence of the human PHACTR1 gene. Using this cDNA as a template, primers were designed to amplify the gene fragment encoding the polypeptide (antigen) at positions 271-580 of human PHACTR1. This gene fragment was cloned into a Q vector (from Abmart), and the cloned plasmid was amplified in E. coli TOP10, followed by expression in E. coli Rosetta, yielding the prokaryotic recombinant protein of the human PHACTR1 polypeptide at positions 271-580. The antigen was then purified by Ni column affinity chromatography.

[0076] III. Animal Immunization

[0077] Two healthy female BALB / c mice (8-12 weeks old) were immunized with purified antigen in a routine four-times, two-week interval. For the initial immunization, 30 μg of antigen with complete Freund's adjuvant was injected subcutaneously at multiple sites. For the second to fourth immunizations, the antigen dose remained the same, but incomplete Freund's adjuvant was added and injected subcutaneously at multiple sites. Seven days after the third immunization, blood was collected from the mice to determine the ELISA titer of the antiserum (recombinant protein antigen coated plate, 25 ng / well). Mice with an antiserum titer greater than 8K were then immunized a fourth time, entering the fusion phase three days post-immunization.

[0078] IV. Fusion and Subcloning

[0079] SP2 / 0 myeloma cells were resuscitated and passaged ahead of schedule. On the day of fusion, spleen cells from eligible immunized mice were ground through a cell sieve and resuspended in 1640 medium to obtain a single-cell suspension. BALB / c mice aged 6-10 weeks were euthanized and disinfected, and frozen fructose solution was injected into the peritoneal cavity. After gently massaging the abdomen several times, the cells were aspirated to obtain peritoneal cells, which were then used as feeder cells. Spleen cells from immunized mice were mixed with SP2 / 0 myeloma cells at a ratio of 10:1 in 1640 medium, and 50% polyethylene glycol was slowly added dropwise for fusion. After fusion, the cells were diluted and seeded in 384-well plates, and cultured for 10-14 days in HAT selective medium. When colonies were larger than 3 mm or covered half the bottom of the well, the supernatant from each well was collected for ELISA to detect the antigen response and to rank cells by affinity. Subclones with high affinity were selected. Selected subclonal cells were seeded into 96-well plates and cloned using the limiting dilution method. After 8-9 days of culture, when clones were visible to the naked eye, the supernatant in each well was tested again by ELISA for the antigen. Cells with high affinity were selected for the next round of subcloning. The above steps were repeated until the positive rate of cell lines in the wells reached 100% (recognizing the corresponding antigen). At this point, monoclonal antibodies were considered to have been obtained, and the cell line was successfully established.

[0080] V. Preparation of Monoclonal Antibodies

[0081] The successfully established hybridoma cell lines were passaged in 1640 medium for large-scale expansion. Monoclonal antibodies were mass-produced using an in vivo ascites induction method. Balb / c mice were intraperitoneally injected with Freund's incomplete adjuvant one week prior to inoculation, and the hybridoma cell suspension was injected into the mice's peritoneum one week later. Ascites fluid was collected 10-14 days after inoculation and centrifuged to obtain a supernatant rich in anti-PHACTR1 monoclonal antibodies.

[0082] VI. Antibody Identification

[0083] 1. Western blot detection of the antibody's recognition of the PHACTR1 recombinant antigen: Recognition of 2 ng of recombinant protein is one of the important parameters for determining whether an antibody is a good antibody. Western blot detection was performed against the corresponding antigen: 2 ng of recombinant protein was loaded, transferred to a membrane, stained with Ponceau S, and the transfer results were observed. If successful, proceed to the next step. The NC membrane was placed in 5% skim milk powder, blocked at room temperature for 1 hour, and then incubated overnight at 4°C on a shaker. The milk powder needed to be fresh, pre-dissolved, and centrifuged at 3500 rpm for 5 min to remove insoluble particles. The membrane was incubated with diluted primary antibody at room temperature for 2 hours, washed 4 times with TBST, and then incubated with secondary antibody at room temperature for 45 min, washed 3 times with TBST. The membrane was washed again. ECL reagent was added, and the signal was detected after 1 minute of reaction. The results showed that the antibody could successfully recognize the antigen, such as... Figure 1 As shown.

[0084] 2. ELISA detection sensitivity of the antibody against the antigen: The protein antigen coating concentration was 1 μg / ml, blocked with 5% milk PBS-T. The antibody was diluted as follows: 1: 3.125K / 6.25K / 12.5K / 25K / 50K / 100K. Primary antibody was incubated at 37°C for 1 hour; secondary antibody was incubated at 37°C for 45 minutes; chromogenic solution was added, and the reaction was stopped at 37°C for 15 minutes. The absorbance at 450 nm was measured using an ELISA reader. The results showed that the antibody bound well to the antigen, such as... Figure 2 As shown.

[0085] 3. Western blot analysis of the antibody's activity against human and mouse PHACTR1 protein: The obtained monoclonal antibody was identified using full-length human PHACTR1 protein and brain and lung tissues from wild-type or PHACTR1 knockout mice. Western blot results showed that the antibody effectively recognized overexpressed human PHACTR1 protein as well as PHACTR1 protein in mouse brain and lung tissues, with significantly better recognition specificity than commercially available PHACTR1 antibody (Santa Cruz Biotech, catalog number SC-514800). Figure 3 As shown.

[0086] VII. Identification of the variable region sequence of the antibody

[0087] The variable region of this monoclonal hybridoma was sequenced using next-generation sequencing (NGS). Hybridoma cell lines were lysed using Trizol lysis buffer, and total RNA was extracted using HiPure RNA Mini Columns (Magen). mRNA was reverse transcribed into a cDNA library using SMART Scribe reverse transcriptase (Takara) with oligo-dT primers and template-changing oligonucleotide (TSO) primers. The first round of PCR amplification was then performed using upstream primers anchored to TSO and downstream primers targeting the heavy or light chain constant regions to obtain heavy or light chain gene fragments, respectively. After purification with magnetic beads, the PCR products were amplified a second time using the Illumina TruSeq DNA library preparation kit with tagged primers to form a TruSeq dual-tagged library. Next-generation sequencing was performed using an Illumina MiSeq PE300. The sequencing results were processed and analyzed using the cutadapt (v1.9.1) and Pandaseq (v2.10) software packages to obtain the gene sequences. The variable region sequences, particularly the CDR regions of the heavy and light chains, were analyzed using NCBI-IgBLAST (v1.17.0). The amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO.2; the amino acid sequences of the three complementarity-determining regions CDR1, CDR2, and CDR3 on the heavy chain are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively. The amino acid sequence of the light chain variable region is shown in SEQ ID NO.6; the amino acid sequences of the three complementarity-determining regions CDR1, CDR2, and CDR3 on the heavy chain are shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively. The nucleotide sequence of the variable region of the heavy chain is shown in SEQ ID NO. 10; the nucleotide sequences of the three complementarity-determining regions CDR1, CDR2, and CDR3 on the heavy chain are shown in SEQ ID NO. 11, SEQ ID NO. 12, and SEQ ID NO. 13, respectively. The nucleotide sequence of the variable region of the light chain is shown in SEQ ID NO. 14; the nucleotide sequences of the three complementarity-determining regions CDR1, CDR2, and CDR3 on the light chain are shown in SEQ ID NO. 15, SEQ ID NO. 16, and SEQ ID NO. 17, respectively.

[0088] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antibody against PHACTR1 protein, characterized in that, Includes VHCDR1, VHCDR2 and VHCDR3; And includes VLCDR1, VLCDR2 and VLCDR3; The amino acid sequence of VHCDR1 is shown in SEQ ID NO.3; The amino acid sequence of VHCDR2 is shown in SEQ ID NO.4; The amino acid sequence of VHCDR3 is shown in SEQ ID NO.5; The amino acid sequence of VLCDR1 is shown in SEQ ID NO.7; The amino acid sequence of VLCDR2 is GTN; The amino acid sequence of VLCDR3 is shown in SEQ ID NO.9; The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.2; The amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO.

6.

2. The encoding gene of the antibody according to claim 1, characterized in that, It includes the heavy chain variable region coding gene as shown in SEQ ID NO.10 and the light chain variable region coding gene as shown in SEQ ID NO.

14.

3. The use of the anti-PHACTR1 protein antibody of claim 1 or the encoding gene of claim 2 in the preparation of a medicament for the treatment or adjuvant treatment of atherosclerosis.

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