Peptides targeting STAT3 protein Tyr705 phosphorylation and their applications

Abstract

The present application discloses an antigenic peptide targeting the phosphorylation of Tyr705 of the STAT3 protein and its application. The antigenic peptide has an amino acid sequence as shown in SEQ ID NO. 6 to 9, or an amino acid homologous sequence with the same function as the sequence. The antibody prepared using the antigenic peptide can recognize the Tyr705 phosphorylation level of ΔS701 and FL subtypes, which will be of great significance for exploring the mechanism of the STAT3 protein ΔS701 subtype in cells, as well as the biological function research in animals and humans and drug development. In addition, the present application discloses a method for preparing an antibody against the phosphorylation of Tyr705 of the STAT3 protein. The antibody prepared by the method has strong specificity, high purity and good stability.

Description

Technical Field

[0001] The present application belongs to the technical field of STAT3 protein phosphorylation detection. The present application discloses an antigenic peptide targeting STAT3 protein Tyr705 phosphorylation and its application. Specifically, it relates to the ability to use the peptide as an antigen to prepare an antibody that targets and recognizes STAT3 protein Tyr705 phosphorylation and its application in detecting the phosphorylation level of STAT3 protein Tyr705. Background Art

[0002] Signal transducer and activator of transcription (STAT) proteins are a family of proteins with DNA-binding activity, including STAT1 to STAT6. STAT3 is the only embryonic-lethal nuclear transcription factor in this family, affecting the expression of more than 1,000 gene products.

[0003] The STAT3 protein structure consists of an N-terminal domain, a DNA-binding domain, a coiled-coil domain, an SH2 domain, and a C-terminal transcriptional activation domain, of which the SH2 domain is the most conserved. When cells are stimulated by nutrients, inflammation, or growth signals, the STAT3 protein becomes phosphorylated at Tyr705 by JAK kinases, leading to the dimerization of two STAT3 monomers and their translocation from the cytoplasm to the nucleus, where they participate in the transcriptional regulation of target genes. Therefore, by measuring the phosphorylation level of the STAT3 protein at Tyr705 in cells, biological processes such as cell growth, proliferation, metabolism, and inflammation can be indirectly explored.

[0004] Currently, existing technologies primarily use STAT3 protein Tyr705 phosphorylation antibodies (such as the Cat.9145 antibody disclosed by Cell Signaling Technology / CST) and their associated antibody kits to detect STAT3 protein Tyr705 phosphorylation levels. However, the NCBI database shows that STAT3 produces four distinct protein isoforms based on differential splicing of its gene introns: α, β, αΔS701, and βΔS701. Existing antibodies cannot effectively detect Tyr705 phosphorylation levels in the αΔS701 and βΔS701 isoforms of STAT3 protein. Summary of the Invention

[0005] On this basis, the inventors of this application conducted in-depth research on different subtypes of STAT3 protein (e.g. Figure 1As shown in Figure 2, the STAT3 gene has a hidden splice site in its 21st intron. The two protein isoforms produced by the alternative splicing of this site differ only in whether they contain serine 701 (Ser701), thereby further dividing the STAT3 protein into two isoforms: a subtype without Ser701 (hereinafter referred to as the ΔS701 subtype) and a subtype containing Ser701 (hereinafter referred to as the FL subtype). The STAT3ΔS701 subtype in human cells accounts for approximately 17% to 22% of the total STAT3. However, existing commercial antibodies (such as Cat.9145, Cell Signaling Technology, CST) cannot recognize the Tyr705 phosphorylation signal of the STAT3 protein ΔS701 subtype. To this end, the embodiments of the present application disclose at least the following technical solutions:

[0006] In a first aspect, the present invention discloses an antigenic peptide having immunogenicity targeting the Tyr705 phosphorylation site of the FL and ΔS701 isoforms of the STAT3 protein, wherein the antigenic peptide has an amino acid sequence as shown in at least one of (I) to (III):

[0007] (I) Amino acid sequences shown in SEQ ID NOs. 6 to 9;

[0008] (II) an amino acid sequence having at least 75% similarity to the sequence described in (I);

[0009] (III) An amino acid sequence obtained by substituting, deleting or adding one or more amino acid sequences to the amino acid sequence shown in (I) or (II), and having the same or similar functions as the amino acid sequence shown in (I) or (II);

[0010] (IV) The antigenic peptide described in (I), (II) or (III) is modified at some or all of the amino acid residues in its amino acid sequence.

[0011] In the second aspect, an antigen construct comprises the antigen peptide of the first aspect and a carrier for loading or coupling the antigen peptide of the first aspect.

[0012] In a third aspect, the present application also discloses the use of the antigenic peptide described in the first aspect and the antigenic construct described in the second aspect, wherein the use includes at least one of the following:

[0013] (1) Preparation of a STAT3 protein Tyr705 phosphorylation-specific antibody;

[0014] (2) Preparation of vaccines for diseases related to Tyr705 phosphorylation of STAT3 protein;

[0015] (3) Used as a drug for the treatment of inflammatory diseases;

[0016] (4) co-stimulatory molecule antagonists;

[0017] (5) Immunosuppressants.

[0018] In a fourth aspect, the embodiments of the present application disclose an antibody that specifically binds to the antigenic peptide described in the first aspect, or to the construct described in the second aspect.

[0019] In a fifth aspect, the embodiments of the present application disclose a method for preparing the antibody described in the fourth aspect, comprising the steps of immunizing an animal with the antigenic peptide described in the first aspect, or with the construct described in the second aspect, collecting antiserum and purifying the antiserum.

[0020] In a sixth aspect, an embodiment of the present application discloses an ELISA kit, comprising the antibody described in the fourth aspect, and used to detect the phosphorylation level of the Tyr705 site of the ΔS701 and FL subtypes of the STAT3 protein.

[0021] In a seventh aspect, an embodiment of the present application discloses a pharmaceutical composition comprising the antibody described in the fourth aspect, and a pharmaceutically acceptable excipient or carrier.

[0022] Compared with the prior art, this application has at least one of the following beneficial effects:

[0023] The antigenic peptide disclosed in the examples of the present application has the immunogenicity of STAT3 protein Tyr705 phosphorylation, which can produce an immune response in vivo, and then produce a phosphorylated antibody that can simultaneously and specifically recognize various STAT3 protein isoforms pTyr705, thereby greatly improving the detection coverage of STAT3 protein pTyr705. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The presence of STAT3 protein αFL, αΔS701, βFL and βΔS701 isoforms in various species provided in this application.

[0025] Figure 2 This is a diagram showing the presence of STAT3 protein ΔS701 isoform and content analysis results in human cells provided in the examples of this application.

[0026] Figure 3 This is the HPLC purity determination result of the phosphorylated antigen peptide provided in the examples of this application.

[0027] Figure 4 This is the HPLC purity determination result of the non-phosphorylated antigen peptide provided in the examples of this application.

[0028] Figure 5 Electrophoresis diagram (a) of the expression plasmids of different STAT3 protein mutants provided in the examples of this application, sequencing comparison diagram (b), and Western Blot detection diagram (c) of different STAT3 protein isoforms detected by homemade antibodies and commercial antibodies;

[0029] Figure 5 In a, the lanes from left to right are Marker, pCDNA3.1-GFP, pCDNA3.1-GFP-STAT3-FL, pCDNA3.1-GFP-STAT3-ΔS701, and pCDNA3.1-GFP-STAT3-Y705F; Figure 5 b shows the sequencing alignment results of pCDNA3.1-GFP-STAT3-FL, pCDNA3.1-GFP-STAT3-ΔS701, and pCDNA3.1-GFP-STAT3-Y705F; Figure 5 In c, lanes 1, 2, and 3 show the WB detection results of the cell lysate obtained from Hela cells transfected with pCDNA3.1-GFP-STAT3-FL as the sample, lane 4 shows the WB detection results of the cell lysate obtained from Hela cells transfected with pCDNA3.1-GFP-STAT3-ΔS701 as the sample, and lane 5 shows the WB detection results of the cell lysate obtained from Hela cells transfected with pCDNA3.1-GFP-STAT3-Y705F as the sample. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is further described in detail below with reference to the following examples. It should be understood that the specific examples described herein are merely for the purpose of explaining this application and are not intended to limit this application. Reagents not described in detail in this application are all conventional reagents and can be obtained from commercial channels; methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0031] Based on the different splicing patterns of introns 21 and 23 of the STAT3 gene, the STAT3 protein can be divided into an α isoform containing serine 701 (referred to as STAT3αFL), an α isoform without serine 701 (referred to as STAT3αΔS701), a β isoform containing serine 701 (referred to as STAT3βFL), and a β isoform without serine 701 (referred to as STAT3βΔS701). To study the ratio of different STAT3 protein isoforms in human cells, the inventors of this application conducted the following experiments:

[0032] The experimental steps mainly include detecting the difference in the 701st serine (Ser701) site of the mRNA sequence of STAT3αFL (NCBI sequence number: NM_139276.3) and STAT3αΔS701 (NCBI sequence number: NM_003150.4), STAT3βFL (NCBI sequence number: NM_001369517.1) and STAT3βΔS701 (NCBI sequence number: NM_001369519.1) to determine the proportion of STAT3ΔS701 isoforms present in human cells. Taking advantage of the difference in serine 701 (Ser701) and the presence of a single recognition site (-AGCGCT-) for the restriction endonuclease (AfeⅠ) at Ser701, PCR amplification is performed to obtain a cDNA fragment containing the Ser701 region. The remaining DNA band after AfeI digestion is then distinguished from the digestion product by size. The schematic diagram of the digestion principle is shown in the figure. Figure 2 a.

[0033] 1. Plasmid AfeⅠ digestion

[0034] Based on the PCDNA3.1 plasmid, recombinant plasmids expressing STAT3αFL, STAT3αΔS701, STAT3βFL and STAT3βΔS701 were constructed respectively. Then, the recombinant plasmids were digested with AfeⅠ (New England Biolabs, NEB, Cat. R0652S). After adding reagents according to the enzyme digestion reaction system described in Table 1, the reaction was incubated at 37°C for 90 minutes and then treated at 65°C for 20 minutes to inactivate the enzyme. 0.2 μg of each sample before and after digestion was taken and electrophoresed on a 1.5% agarose gel at 135V for 45 minutes. The experiment was repeated three times. The results are shown in the figure below. Figure 2 As shown in b, AfeⅠ endonuclease can specifically recognize and fully cleave the FL isoform of STAT3, but cannot cleave the ΔS701 isoform of STAT3.

[0035] Table 1

[0036] Reagents Usage 10×Cutsmart 5μl DNA 1 μg AfeⅠ 1 μl <![CDATA[ddH2O]]> to 50μl

[0037] 2. AfeⅠ digestion of cell samples

[0038] L02, HepG2, A549, HEK-293T, and HeLa cells were passaged into six-well plates and cultured in DMEM supplemented with 10% fetal bovine serum in a 5% CO2 incubator. When the cells reached a density of approximately 90%, the medium was discarded and the cells were lysed with 1 mL of Trizol lysis buffer. RNA was extracted, and 1 μg of RNA was reverse-transcribed into cDNA. Cell cDNA was used as a DNA template for amplification using the following primer sequences: Afe I F: 5'-tccgtggaaccatacacaaa-3', as shown in SEQ ID NO. 1; Afe I cell R: 5'-ttatttccaaactgcatcaatgaa-3', as shown in SEQ ID NO. 2. The PCR reaction system is shown in Table 2, and the reaction procedure is shown in Table 3.

[0039] Table 2

[0040] Reagents Usage TaKaRa Taq Version 2.0 25 μl Template cDNA 4 μl AfeⅠF (10μM) 1 μl AfeⅠcellR (10μM) 1 μl <![CDATA[ddH2O]]> to 50μl

[0041] Table 3

[0042] step temperature time 1 98℃ 5min 2 98℃ 10sec 3 58℃ 30sec 4 72℃ 45sec Repeat steps 2-4 34 cycles 5 72℃ 5min 6 4℃ ∞

[0043] The PCR product was purified (TaKaRa, Cat. 9761) to test the purification effect. The purity was tested by agarose gel electrophoresis and the concentration was tested by ultra-micro nuclear protein analyzer. The purified product was digested with AfeⅠ (New England Biolabs, NEB, Cat. R0652S). The digestion reaction system and digestion result detection experimental method were the same as those for plasmid digestion. The experiment was repeated three times. Figure 2 Figure c shows the experimental results of AfeⅠ enzyme cleavage of STAT3 fragments in human cells. The experimental results show that STAT3αΔS701 / βΔS701 subtypes exist in human cells, and the proportion is not low.

[0044] 3. Real-time quantitative PCR (QPCR)

[0045] Based on the differences in the mRNA and cDNA sequences of STAT3αFL / βFL and STAT3αΔS701 / βΔS701 at Ser701, specific primers were designed. STAT3αFL / αΔS701 plasmid was used as a DNA template for QPCR experiments to test primer specificity. Each sample was repeated three times. The QPCR experiment was performed using the cDNA of the above cell samples as a DNA template. Each sample was repeated three times. The results are shown in Figure 2. Figure 2 As shown in d, the STAT3ΔS701 isoform exists in human cells and accounts for approximately 17% to 22% of the total.

[0046] The primer sequences used for QPCR were: AfeⅠF: 5'-tccgtggaaccatacacaaa-3', as shown in SEQ ID NO.1; FL QP R: 5'-ttcaggtatggggcagcgctac-3', as shown in SEQ ID NO.3; ΔS QP R: 5'-ttcaggtatggggcagcgcctg-3', as shown in SEQ ID NO.4; Total QP R: 5'-gctctctggccgacaatact-3', as shown in SEQ ID NO.5.

[0047] Table 4 QPCR reaction system (MonaBio, REF: MQ00401)

[0048] Reagents Usage MonAmpChemoHS qPCR Mix 10 μl Template cDNA 1 μl Forward primer (10 μM) 0.4 μl Reverse primer (10 μM) 0.4 μl High ROX Dye(100×) 0.2 μl Nuclease-Free water to 20μl

[0049] Table 5 QPCR amplification procedure

[0050] step temperature time 1 95℃ 30sec 2 95℃ 10sec 3 60℃ 30sec Repeat steps 2-3 39 cycles 4 95℃ 15sec 5 60℃ 15sec 6 95℃ 15sec

[0051] Furthermore, analysis of publicly available RNA-Seq data (GEO accession number GSE30611) revealed that the ΔS701 isoform accounts for 10–26% of total STAT3 in 16 human tissues. Although the STAT3ΔS701 isoform is less abundant than the FL isoform, the ratio of ΔS701 to total STAT3 remains relatively constant across human tissues. The constant presence of the STAT3ΔS701 isoform suggests that ΔS701 is essential for maintaining physiological functions. STAT3 transcriptional activity is achieved through phosphorylation at Tyr705, so studying changes in Tyr705 phosphorylation of the ΔS701 isoform after cytokine stimulation is crucial for uncovering its function. However, the most widely used STAT3Tyr705 phosphorylation antibody on the market (Cell Signaling Technology / CST, Cat.9145, cited in 1,669 articles) cannot recognize Tyr705 phosphorylation of the STAT3ΔS701 isoform in human cells, which seriously hinders the functional research of the STAT3ΔS701 isoform.

[0052] Antigenic peptide

[0053] To this end, the present invention discloses an antigenic peptide that mimics the Tyr705 phosphorylation epitope of the FL and ΔS701 isoforms of the STAT3 protein. This antigenic peptide is capable of generating an immune response in vivo, thereby producing an antibody that can simultaneously and specifically bind to various STAT3 protein isoforms bearing pTyr705, thereby significantly improving the detection coverage of STAT3 protein pTyr705.

[0054] This antigenic peptide has immunogenicity targeting the Tyr705 phosphorylation site of the STAT3 protein. Through in vivo immunization, a pTyr705-specific antibody that recognizes both the ΔS701 and FL isoforms of the STAT3 protein can be produced. Therefore, this antigenic peptide can be used to prepare vaccines targeting the Tyr705 phosphorylation site of the STAT3 protein and drugs for related inflammatory diseases. This antibody can be used to detect the phosphorylation level of the Tyr705 phosphorylation site of the STAT3 protein in cells, providing a detection method for exploring the molecular mechanisms of biological processes such as cell growth, proliferation, metabolism, and inflammation.

[0055] To this end, the antigenic peptide disclosed in the examples of the present application has immunogenicity targeting the Tyr705 phosphorylation site of the STAT3 protein, wherein the antigenic peptide is at least one of (I) to (IV):

[0056] (I) Amino acid sequences shown in SEQ ID NOs. 6 to 9;

[0057] (II) an amino acid sequence having at least 75% similarity to the sequence described in (I);

[0058] (III) An amino acid sequence obtained by substituting, deleting or adding one or more amino acid sequences to the amino acid sequence shown in (I) or (II), and having the same or similar functions as the amino acid sequence shown in (I) or (II);

[0059] (IV) The antigenic peptide described in (I), (II) or (III) is modified at some or all of the amino acid residues in its amino acid sequence.

[0060]

[0046] The terms "polypeptide," "peptide," and "protein" are used herein interchangeably and are defined to mean a biomolecule composed of amino acids linked by peptide bonds.

[0061] The term "peptide" is a chain of amino acids (usually L-amino acids) connected at their alpha carbons by a peptide bond formed by a condensation reaction between the carboxyl group of the alpha carbon of one amino acid and the amino group of the alpha carbon of another amino acid. The terminal amino acid at one end of the chain (i.e., the amino terminus) has a free amino group, while the terminal amino acid at the other end of the chain (i.e., the carboxyl terminus) has a free carboxyl group. Thus, the term "amino terminus" (abbreviated as N-terminus) refers to the free alpha amino group on the amino terminal amino acid of a peptide, or to the alpha amino group (imino group when involved in a peptide bond) of an amino acid at any other position within the peptide. Similarly, the term "carboxyl terminus" (abbreviated as C-terminus) refers to the free carboxyl group on the carboxyl terminal amino acid of a peptide, or to the carboxyl group of an amino acid at any other position within the peptide.

[0062] As used herein, "some or all of the amino acid residues in its amino acid sequence are modified." Such modified peptides can be prepared by any method known in the art. For example, modified peptides can be prepared by modifying the functional groups of the side chains of the amino acid residues constituting the peptide, such modifications as esterification, alkylation, halogenation, phosphorylation, sulfonation, or amidation. Furthermore, a variety of substances can be bound to the peptide at the N- and / or C-termini. For example, amino acids, peptides, or their analogs can be bound to the peptide. When such substances are bound to the "antigenic peptide described in (I), (II), or (III)" above, they can be removed by any method, such as by in vivo enzymatic reactions or intracellular processing, ultimately producing the antigenic peptide. Such "modifications" can be intended to regulate peptide solubility; improve peptide stability, such as protease resistance; deliver the peptide to specific tissues or organs; or increase peptide uptake by antigen-presenting cells. For example, such "modifications" can also be intended to enhance the immunogenicity of the antigenic peptide or promote its specific binding to the phosphorylated epitope at Tyr705 of the STAT3 protein.

[0063] The term "functionally identical or similar amino acid sequence" or "functional fragment" as used herein refers to a functional peptide fragment having substantially the same (biological) activity as the peptide defined herein (e.g., as shown in SEQ ID NOs. 6 to 9, respectively), i.e., the fragment is still capable of eliciting a highly specific immune response (i.e., having immunogenic activity) in an organism, but particularly in an animal, especially a mammal or a human, to produce an antibody that can specifically recognize and bind to the Tyr705 phosphorylation epitope of the FL isoform and the ΔS701 isoform of the STAT3 protein.

[0064] The term "residue" is used herein to refer to an amino acid that is incorporated into a peptide via an amide bond. Thus, the amino acid can be a naturally occurring amino acid or, unless otherwise limited, can include known analogs of natural amino acids that function in a manner similar to naturally occurring amino acids (i.e., amino acid analogs).

[0065] In order to maintain the immunogenicity of the STAT3 protein Tyr705 phosphorylation epitope, a small number (e.g., 1, 2, or several) or a small percentage of amino acids can be modified (added, deleted, and / or substituted). Here, the term "several" means 7 or fewer amino acids, such as 6 or 5 or fewer. The percentage of amino acids to be modified is preferably 20% or less, more preferably 15% or less, more preferably 10% or less, and even more preferably 1-5%.

[0066] It will be understood by those skilled in the art that altering a single amino acid or individual additions, deletions, or substitutions of a small percentage of amino acids in an amino acid sequence results in the retention of the properties of the original amino acid side chains. This is therefore referred to as a "conservative substitution" or "conservative modification," meaning that the alteration replaces an amino acid with a chemically similar amino acid, where the alteration to the protein results in a protein with similar function. Conservative substitution tables providing functionally similar amino acids are well known in the art. Items 1) to 8) below each contain amino acids that are conservative substitutions for each other:

[0067] 1) Alanine (A), Glycine (G);

[0068] 2) Serine (S), threonine (T);

[0069] 3) Aspartic acid (D), glutamic acid (E);

[0070] 4) Asparagine (N), glutamine (Q);

[0071] 5) Arginine (R), Lysine (K);

[0072] 6) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0073] 7) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); and

[0074] 8) Cysteine ​​(C), methionine (M).

[0075] Such conservatively modified peptides are also considered peptides of the present invention. However, the peptides of the present invention are not limited thereto and may include non-conservative modifications as long as the peptide retains the immunogenicity of the original peptide at the phosphorylation site Tyr705 of the STAT3 protein.

[0076] In certain embodiments of the present application, the amino acid sequences of the antigenic peptides are shown in SEQ ID NOs. 6 to 9, as shown in Table 6. Also shown in Table 6 are the amino acid sequences of control peptides used for comparative experiments, as shown in SEQ ID NOs. 10 to 13. In Table 6, "p-" indicates that the amino acid residue at that site is phosphorylated, and "-NH2" indicates that the amino acid residue at that site is amidated.

[0077] Table 6

[0078] Antigenic peptide Sequence information Phospho-peptide 1 <![CDATA[CDPGAAP(p-Y)LKTKF-NH2, as shown in SEQ ID NO.6]]> Phospho-peptide 2 ADPGAAP(pY)LKTKFIC, as shown in SEQ ID NO.7 Phospho-peptide 3 <![CDATA[CEADPGAAP(p-Y)LKTKF-NH2, as shown in SEQ ID NO.8]]> Phospho-peptide 4 <![CDATA[CDPGAAP(p-Y)LKTKFIC-NH2, as shown in SEQ ID NO.9]]> Control peptide 1 <![CDATA[CDPGAAPYLKTKF-NH2, as shown in SEQ ID NO.10]]> Control peptide 2 ADPGAAPYLKTKFIC, as shown in SEQ ID NO. 11 Control peptide 3 <![CDATA[CEADPGAAPYLKTKF-NH2, as shown in SEQ ID NO. 12]]> Control peptide 4 <![CDATA[CDPGAAPYLKTKFIC-NH2, as shown in SEQ ID NO.13]]>

[0079] On the other hand, the present invention discloses an antigenic construct comprising the antigenic peptide of the first aspect and a carrier for loading or coupling the antigenic peptide of the first aspect. The carrier particularly also has the functionality of an adjuvant for producing a supramolecular antigenic construct. In certain embodiments, the antigenic peptide according to the first aspect is modified by attachment to, for example, liposomes or reconstitution in, for example, liposomes to produce the "supramolecular antigenic construct" described in WO Publication WO2005 / 081872, the description of which is incorporated herein by reference in its entirety. The "supramolecular antigenic construct" thus obtained exhibits unique antigenic peptide presentation on its surface, which results in enhanced antigen exposure and ultimately leads to the production of antibodies exhibiting high conformational sensitivity. Specifically, the antigenic peptide according to the present invention is modified by binding to a lipophilic or hydrophobic moiety that facilitates insertion into the lipid bilayer of the liposome carrier / immune adjuvant, particularly by acting as an anchor for the peptide in the liposome bilayer and having a lipophilic or hydrophobic moiety of a size that causes the peptide to be positioned and stabilized close to the liposome surface.

[0080] In some embodiments, the lipophilic or hydrophobic moiety is a fatty acid, triglyceride, or phospholipid, particularly one containing a carbon chain between C12 and C24, but particularly palmitic acid.

[0081] In some embodiments, the antigenic peptide of the present application is modified by covalently binding at least two molecules of palmitic acid to the N-terminus and C-terminus of the antigenic peptide and reconstituted in a liposome carrier.

[0082] In some embodiments, the peptides in the conjugate are each coupled to four molecules of palmitic acid; thus, they are tetrapalmitoylated.

[0083] In some embodiments, two molecules of palmitic acid are coupled to the N-terminal end of the peptide and two molecules of palmitic acid are coupled to the C-terminal end of the peptide or fragment.

[0084] In some embodiments, the present application provides an antigenic peptide according to the present application, which is modified by conjugation with a lipophilic or hydrophobic moiety, such as palmitic acid, and reconstituted in liposomes, wherein the liposome preparation may further comprise an adjuvant for generating a supramolecular antigen construct, such as lipid A, alum, calcium phosphate, interleukin 1 and / or microcapsules of polysaccharides and proteins, in particular detoxified lipid A, such as monophosphoryl or diphosphoryl lipid A, or alum.

[0085] In one embodiment of the present application, the present application relates to a supramolecular construct, wherein each carrier molecule comprises one or more antigenic peptides described in the present application, especially two or more antigenic peptides.

[0086] In one embodiment of the present application, the carrier molecule is a liposome.

[0087] In some embodiments, each carrier molecule of the supramolecular constructs involved in the present application and the supramolecular constructs described herein comprises a combination of two or more antigenic peptides of any one of SEQ ID NOs. 6 to 9.

[0088] In the "supramolecular antigen construct" of the present application, the liposome can have a dual function, which can be used as a carrier containing the supramolecular construct described herein, and at the same time act as an adjuvant to increase or stimulate the immune response in the target animal or human to be treated with the therapeutic vaccine of the present application. It should also be understood that the supramolecular antigen construct composition of the present application may further comprise other adjuvants, including but not limited to keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), chicken ovalbumin (OVA), bovine thyroglobulin (THY) and other adjuvants, such as lipid A, alum, calcium phosphate, interleukin 1, and / or microcapsules of polysaccharides and proteins, but especially detoxified lipid A, such as monophosphoryl or diphosphoryl lipid A, or alum; other preservatives; diluents; emulsifiers; stabilizers; and other ingredients known and used in vaccines in the prior art. In addition, any adjuvant system known in the art can be used in the composition of the present application. Such adjuvants include, but are not limited to, Freund's incomplete adjuvant; Freund's complete adjuvant; polydisperse β-(1,4) linked acetylated mannan; polyoxyethylene-polyoxypropylene copolymer adjuvants; modified lipid adjuvants; saponin derivative adjuvants; inactivated pertussis toxin; hemocyanin; group B meningococcal outer membrane protein; Pseudomonas aeruginosa exotoxin A; cholera toxin B subunit; bacterial outer membrane protein; Escherichia coli heat-labile enterotoxin; pneumolysin; gonococcal pilus protein; lipopolysaccharide (LPS) of Gram-negative bacteria; large polymeric anions, such as dextran sulfate; and inorganic gels, such as alum, aluminum hydroxide, or aluminum phosphate.

[0089] In certain embodiments of the present application, the peptide described in the first aspect can be synthesized according to synthetic methods and / or biosynthesized according to commonly used methods in the field of chemistry.

[0090] As used herein, the term "isolated" refers to being substantially or essentially free from components that normally accompany it as found in its native state. Thus, the peptides described herein do not contain materials normally associated with their native environment. For example, the isolated immunogenic peptides described herein are at least about HPLC 90% (pure) as measured by band intensity on a silver-stained gel.

[0091] Protein purity or homogeneity can be demonstrated by many methods known in the art, such as staining followed by polyacrylamide gel electrophoresis of a protein sample. For some purposes, high resolution will be required for purification using HPLC or similar means.

[0092] When the immunogenic peptides are relatively short in length (i.e., less than about 50 amino acids), they are typically synthesized using standard chemical peptide synthesis techniques. For example, solid phase synthesis methods well known to those skilled in the art are employed. Solid phase synthesis is a preferred method for chemically synthesizing the immunogenic peptides described herein, wherein the C-terminal amino acid of the sequence is attached to an insoluble support, and the remaining amino acids in the sequence are then added sequentially.

[0093] For example, in one embodiment, referring to the classic Fmoc solid phase synthesis method provided in "Study on Chemical Synthesis of O-phosphorylated Peptides Based on FmOc Strategy [J] Journal of Chemistry of Universities, No. s1, 2001", phosphoramidite was used as a phosphorylation reagent, and the phosphorylated amino acids having the amino acid sequences shown in SEQ ID NOs. 6 to 13 were synthesized by a monomer phosphorylation method, and the amidated amino acids at the carbon end were synthesized with reference to "New Synthesis Process of L-Prolinamide [J] Shandong Industrial Technology, No. 9, 2016", and then the polypeptides having the amino acid sequences shown in SEQ ID NOs. 6 to 13 were synthesized in sequence by the Fmoc solid phase synthesis method, and the lyophilized products with HPLC purity greater than 90% were purified (the results are shown in FIG. Figure 3 and Figure 4 shown).

[0094] Alternatively, the antigenic peptides described herein can be obtained by combining biosynthetic methods of recombinant expression with chemical modification, which are well known to those skilled in the art. Generally speaking, this involves generating a nucleic acid sequence encoding the peptide, placing the nucleic acid in an expression cassette under the control of a specific promoter, expressing the peptide in a host, isolating the expressed peptide, and renaturing the peptide as needed. Techniques sufficient to guide technicians in completing such methods are found in the literature. It is well known in the art that immunogenic peptides expressed by a host can be purified by standard methods including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like. For use as therapeutic agents, substantially pure compositions of about 50% to 95% homogeneity are preferred, and most preferably 80% to 95% or greater homogeneity.

[0095] Antibody

[0096] On the other hand, the present application also discloses an antibody that can react specifically with the antigenic peptide disclosed in the above embodiments. Specifically, the antibody can be produced by injecting the antigenic peptide or antigenic construct as an antigen into the body to induce an immune response.

[0097] In some embodiments, the preparation of the antibody comprises the following steps:

[0098] 1. Preparation of Antigen

[0099] In this example, phosphorylated peptides 1-4 as shown in SEQ ID NOs. 6-9 in Table 6 were conjugated to keyhole limpet hemocyanin (KLH) (using SμLfo-SMCC as the coupling agent) as immunizing antigens. Phosphorylated peptides 1-4 and control peptides 1-4 in Table 6 were conjugated to bovine serum albumin (BSA) (using glutaraldehyde as the coupling agent) as detection antigens. The conjugation method was described in "Study on the immunogenicity and arthrogenicity of synthetic cyclic citrullinated protein short peptides [J] Chinese Journal of Immunology, 2017, Vol. 1". The prepared antigens were diluted to 1 mg / mL in phosphate-buffered saline (PBS) and stored in aliquots at -20°C.

[0100] 2. Animal immunization

[0101] On days 1, 15, 29, and 43, 1 mL of each antigen was added to 1 mL of Freund's complete adjuvant and emulsified (to test the emulsification level: drop a drop of emulsified antigen into normal saline; if it does not disperse, the desired level has been achieved). Immunization was performed subcutaneously at multiple sites (at least 8) on the nape of the neck. Two New Zealand white rabbits (Jiangsu Ai Ling Fei) were immunized with each antigen. On day 53, blood was drawn from the carotid artery to collect large quantities of antiserum. The rabbit blood was refrigerated at 4°C overnight. The next day, aseptically, the blood was aliquoted into 50 mL centrifuge tubes and centrifuged at 10,000 rpm / min at 4°C for 30 minutes. The supernatant, the post-immunization antiserum, was collected and stored at -20°C.

[0102] 3. Antibody Purification

[0103] Affinity purification columns were prepared using phosphorylated and non-phosphorylated peptides, respectively, to affinity purify phosphorylation-specific antibodies.

[0104] (1) Phosphorylated peptides 1 to 4 shown in SEQ ID NOs. 6 to 9 were respectively connected to activated Sulfolink Resin (Cat. No. 20401, Thermo Fisher Scientific) to prepare antigen affinity columns, with 1 mL of Sulfolink Resin coupled to 1 mg of peptide.

[0105] (2) Equilibrate the affinity column with 10 column volumes of PBS and drain the solution; filter the rabbit serum through a 0.45 μm filter membrane.

[0106] (3) The serum is passed through the antigen affinity column, and the solution is drained away and the flow-through is collected.

[0107] (4) Equilibrate with 10 column volumes of PBS and drain out the solution.

[0108] (5) Add 5 mL of antibody eluate and collect the eluate in separate tubes, 1 mL per tube.

[0109] (6) The absorbance of the collected eluate at 280 nm was measured, and the fractions with absorbance greater than 1.0 were combined and dialyzed against PBS.

[0110] 4. Antibody titer detection

[0111] Protein concentration was determined by ultraviolet absorption, and antibody titer was determined by enzyme-linked immunosorbent assay. New Zealand white rabbits were immunized with antigens prepared from the phosphorylated peptides 1 to 4 shown in SEQ ID NOs. 6 to 9, and the antibodies were purified as described above. The antibodies were diluted with coating diluent to ratios of 1:1250, 1:2500, 1:5000, 1:10000, 1:20000, 1:40000, and 1:80000, and the following steps (1) to (6) were followed.

[0112] (1) Coating: Dilute the coating antigen to 1 μg / ml with coating buffer, add 100 μl to each well of the ELISA plate, and incubate at 4°C overnight.

[0113] (2) Blocking: Discard the coating solution, add 200 μl of blocking solution (5% skim milk powder) to each well, and incubate at 37°C for 1.5 hours.

[0114] (3) Add the sample to be tested: discard the blocking solution, add the sample (serum or antibody), add 100ul to each well of the ELISA plate, and incubate at 37℃ for 1 hour; rinse 10 times with washing buffer and pat dry the liquid in the well.

[0115] (4) Secondary antibody incubation: Dilute the enzyme-labeled goat anti-rabbit secondary antibody to the working concentration with blocking buffer, add 100 μl to each well of the enzyme-labeled plate, and incubate at 37°C for 30 minutes; rinse 10 times with washing buffer and pat dry the liquid in the well.

[0116] (5) Color development: Add TMB color development substrate: add 100 μl to each well of the ELISA plate and incubate at 37°C for 15 minutes.

[0117] (6) Termination and reading: Add 50 μl of 2M H2SO4 to each well to terminate the reaction, and read the value at OD450 nm using a microplate reader.

[0118] 5. Results

[0119] The titers of the generated antibodies were tested using detection antigens prepared using the peptides shown in SEQ ID NOs. 6 to 13 coupled to bovine serum albumin. The results are shown in Table 7. Ag-1 to 4 are detection antigens prepared using the phosphorylated peptides shown in SEQ ID NOs. 6 to 9 coupled to BSA, and Ag-control-1 to 4 are detection antigens prepared using the control peptides shown in SEQ ID NOs. 10 to 13 coupled to BSA. Ab-1 to 4 are phosphorylated antibodies obtained by animal immunization and antibody purification using phosphorylated peptides 1 to 4 shown in SEQ ID NOs. 6 to 9 coupled to hemocyanin. The results showed that the ELISA titer of the STAT3 protein Tyr705 phosphorylation-specific antibody against the phosphorylated peptide was greater than 1:80,000.

[0120] Table 7 OD450nm values

[0121]

[0122]

[0123] application

[0124] To this end, the embodiments of the present application also substantially disclose applications of the antigenic peptide described in the first aspect and the antigenic construct described in the second aspect. The applications include at least one of the following:

[0125] (1) Preparation of a STAT3 protein Tyr705 phosphorylation-specific antibody;

[0126] (2) preparing drugs for diseases related to Tyr705 phosphorylation of STAT3 protein;

[0127] (3) Used as a drug for the treatment of inflammatory diseases;

[0128] (4) co-stimulatory molecule antagonists;

[0129] (5) Immunosuppressants.

[0130] In addition, the antibodies prepared by the methods provided in the above examples specifically bind to peptides 6 to 9 that mimic phosphorylation at Tyr705 of the STAT3 protein. The present examples also provide a kit for detecting phosphorylation of the STAT3 protein at Tyr705 using such antibodies. Furthermore, the present examples disclose a kit comprising the antibodies of the present application, which can specifically bind to the Tyr705 phosphorylation epitope of the ΔS701 and FL isoforms of the STAT3 protein to detect the Tyr705 phosphorylation level of the STAT3 protein in a sample. By targeting the Tyr705 site of the STAT3 protein in cells, tissues, or organisms, the antibody can be used to prepare an immunoblotting kit or an ELISA kit for detection.

[0131] In some embodiments of the present application, the detection kit comprises an ELISA plate coated with an antibody against Tyr705 phosphorylation of the STAT3 protein ΔS701 and FL isoforms, the enzyme-labeled antibody, a wash buffer, a blocking solution, a coating buffer, a TMB color development solution, and a stop solution. In one embodiment, the ELISA detection kit for detecting Tyr705 phosphorylation of the STAT3 protein ΔS701 and FL isoforms of the present application specifically comprises the following components:

[0132] STAT3 protein FL isoform and ΔS701 Tyr705 phosphorylation antibodies (provided in the above examples);

[0133] enzyme-labeled antibodies;

[0134] Washing buffer: PBST buffer: 1000 mL 0.01 mol / L PBS + 0.5 mL Tween-20;

[0135] Blocking solution: PBS buffer containing 5% skim milk powder;

[0136] Coating buffer: Contains 0.015 M Na2CO3 and 0.035 M NaHCO3, pH 9.6;

[0137] TMB stock solution: 10 mg TMB is fully dissolved in 5 mL of anhydrous ethanol;

[0138] TMB color development solution: contains 0.5 mL TMB stock solution, 10 mL substrate buffer and 2.1 μL 30% H2O2 aqueous solution, and is freshly prepared; and

[0139] Stop solution: 2M H2SO4 solution.

[0140] The above-mentioned kit can be used to identify Tyr705 phosphorylation of various STAT3 protein isoforms, for example, using an ELISA method to detect STAT3 FL isoform and ΔS701 isoform proteins or peptides thereof phosphorylated at Tyr 705. In one embodiment, the preparation method of the detection kit further includes the step of coating an ELISA plate with antibodies against STAT3 protein FL isoform and ΔS701 Tyr705 phosphorylation.

[0141] In one embodiment, the step of detecting phosphorylation of the STAT3 protein Tyr705 site using the antibody prepared above comprises:

[0142] (1) Construction of expression vector

[0143] Refer to the method shown in "Construction of PCDNA3.1-GFP-LC3B eukaryotic expression vector [J] Anhui Agricultural Sciences, 2015, No. 6." to construct the STAT3 protein expression vector pCDNA3.1-GFP-STAT3-FL containing Ser701, the STAT3 protein expression vector pCDNA3.1-GFP-STAT3-ΔS701 lacking Ser701, and the mutant pCDNA3.1-GFP-STAT3-Y705F that cannot undergo Tyr705 phosphorylation. The agarose gel electrophoresis and sequencing alignment results of these expression plasmids are shown in Figure 2. Figure 5 a and Figure 5 As shown in b.

[0144] (2) Construction of eukaryotic expression cells

[0145] Hela cells were seeded into six-well plates, cultured in DMEM medium containing 10% fetal bovine serum, and placed in a 5% CO2 incubator. When the cells grew to a density of approximately 60-70%, the above-mentioned expression vectors were taken and transfected into the Hela cells respectively by conventional liposome transfection. 24 hours after transfection, the experimental groups were stimulated with 10 ng / mL IL-6 for 30 minutes, and the cells were collected to obtain recombinant cells expressing the FL isoform, ΔS701 isoform, and Y705F mutant of the STAT3 protein, respectively.

[0146] (3) Immunoblotting

[0147] Each of the above cell cultures was collected and lysed using 500 μl of RIPA lysis buffer (Beyotime Biotech, Cat. P0013C) supplemented with protease inhibitors. Samples were then prepared for immunoblotting according to the instructions for PPase (New England Biolabs, NEB, Cat. P0753S). A control group consisted of cell samples digested with PPase, and the digested samples were then used for immunoblotting (WB).

[0148] The results are as follows Figure 5 As shown in Figure c, the commercial pTyr705 antibody, taking CST as an example, cannot recognize the pTyr705 signal of the STAT3 protein ΔS701 isoform, while the homemade antibody can recognize the tyrosine phosphorylation signal at position 705 of the FL isoform and ΔS701 isoform of the STAT3 protein.

[0149] Pharmaceutical composition

[0150] To this end, the present application also discloses a pharmaceutical composition comprising the antibody provided in the above embodiments and a pharmaceutically acceptable excipient or carrier.

[0151] The term "pharmaceutically acceptable" means approved by the relevant regulatory agencies or listed in the generally recognized pharmacopoeia for use in animals, more particularly for humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's complete and incomplete adjuvant), excipient, or vehicle used together with the reagent. Such a carrier can be a sterile liquid, such as water and oil, including those of petroleum, animal, plant, or synthetic origin, including, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. When intravenously administering drugs and / or diagnostic compositions, water is a common carrier. Saline solutions and aqueous solutions of glucose and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, anhydrous skim milk, glycerol, propylene, ethylene glycol, water, ethanol, etc. Other examples of pharmaceutically acceptable carriers, excipients, and stabilizers include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin and gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other sugars including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™, as known in the art. In addition to the above ingredients, the pharmaceutical and / or diagnostic compositions of the present invention may also include lubricants, wetting agents, sweeteners, flavorings, emulsifiers, suspending agents, and preservatives. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like.

[0152] In summary, the present application relates to a STAT3 protein Tyr705 phosphorylated antigen peptide and its application. The antibody prepared using the antigen peptide can recognize the phosphorylation level of the Tyr705 site of ΔS701 and FL subtypes. The preparation method of the antibody provided in the present application is simple to operate, and the antibody prepared by this method has strong specificity, high purity and good stability.

[0153] The above is only a preferred specific implementation method of the present application, but the scope of protection of the present application is not limited thereto. Any changes or replacements that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed in this application should be covered by the scope of protection of the present application.

Claims

1. An isolated antigenic peptide having immunogenicity targeting the Tyr705 phosphorylation site of STAT3 protein, wherein the antigenic peptide is selected from at least one of the following phosphorylated peptides 1 to 4: The amino acid sequence of phosphorylated peptide 1 is CDPGAAP(pY)LKTKF-NH2; The amino acid sequence of phosphorylated peptide 2 is ADPGAAP(pY)LKTKFIC; The amino acid sequence of phosphorylated peptide 3 is CEADPGAAP(pY)LKTKF-NH2; The amino acid sequence of phosphorylated peptide 4 is CDPGAAP(pY)LKTKFIC-NH2; in, "p-" indicates that the amino acid residue at that site is phosphorylated, and "-NH2" indicates that the amino acid residue at that site is amidated.

2. An antigen construct, comprising the antigen peptide according to claim 1 and a carrier for loading or coupling the antigen peptide according to claim 1.

3. The antigen construct according to claim 2, wherein The carrier is a liposome.

4. The antigen construct according to claim 2, wherein The carrier is selected from one of keyhole limpet hemocyanin, bovine serum albumin, chicken ovalbumin, bovine thyroglobulin, inactivated pertussis toxin, group B meningococcal outer membrane protein, Pseudomonas aeruginosa exotoxin A, cholera toxin B subunit, bacterial outer membrane protein, Escherichia coli heat-sensitive enterotoxin, pneumolysin, gonococcal pilin and lipopolysaccharide of Gram-negative bacteria.

5. Use of the antigenic peptide according to claim 1 or the antigenic construct according to any one of claims 2 to 4 for preparing a specific antibody targeting the Tyr705 phosphorylation site of the STAT3 protein.

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