Polypeptide and application thereof in resisting glomerular basement membrane disease
阅读说明：本技术 多肽及其在抗肾小球基底膜病中的用途 (Polypeptide and application thereof in resisting glomerular basement membrane disease ) 是由 赵明辉 崔昭 贾晓玉 史悦 于 2019-04-18 设计创作，主要内容包括：本公开提供了经修饰的多肽,其能够用于有效预防和治疗抗肾小球基底膜(抗GBM)病。本公开还提供了编码上述多肽的核苷酸序列和载体,包含多肽或载体的组合物,以及其在预防和治疗抗GBM病中的用途。(The present disclosure provides modified polypeptides that can be used for effective prevention and treatment of anti-glomerular basement membrane (anti-GBM) diseases. The disclosure also provides nucleotide sequences and vectors encoding the polypeptides, compositions comprising the polypeptides or vectors, and uses thereof in the prevention and treatment of anti-GBM disease.)
1. A polypeptide having the amino acid sequence:
a.TDIPPCPHGWXSLWKGFSFIMF (SEQ ID NO: 3); or
b.TDIPPCPHGWXSLWKGFSFIMF (SEQ ID NO:3), the fragment comprising CPHGWXSLWKGF(SEQID NO:4)，
Wherein X is any amino acid other than a hydrophobic amino acid.
2. The polypeptide of claim 1, wherein X is a hydrophilic amino acid.
3. The polypeptide of claim 2, wherein X is selected from G, N, Q, S, T, C and Y.
4. The polypeptide of claim 3, wherein X is S.
5. The polypeptide of any one of claims 1-4, wherein the fragment comprises at least 13 amino acids, such as at least 15 amino acids or at least 17 amino acids.
6. A nucleotide sequence encoding the polypeptide of any one of claims 1-5.
7. A vector comprising the nucleotide sequence of claim 6.
8. The vector of claim 7, wherein the vector is an expression vector.
9. A composition comprising the polypeptide of any one of claims 1-5 or the vector of claim 7 or 8.
10. Use of the polypeptide of any one of claims 1-5, the vector of claim 7 or 8, or the composition of claim 9 in the manufacture of a medicament for preventing or treating an anti-GBM disease in a subject.
Technical Field
The present disclosure relates to the field of immunotherapy. In particular, the disclosure relates to a modified polypeptide and its use in the prevention and treatment of anti-glomerular basement membrane (anti-GBM) diseases.
Technical Field
Anti-glomerular basement membrane (anti-GBM) disease is a group of autoimmune diseases characterized by the presence of anti-GBM antibodies in the circulation and/or the deposition of antibodies in the viscera. The kidney is usually affected by crescentic nephritis, which is characterized by acute onset and rapid progression, and can progress to end-stage nephropathy within a short period, and is the worst-case glomerulonephritis. The lung is usually suffered from lung bleeding, even fatal hemoptysis, which is a critical condition in internal medicine. Currently, there is no specific treatment for this disease. In addition, there are problems with plasmapheresis and immunosuppressants as existing therapeutic approaches: the former can only eliminate circulating autoantibodies and is expensive, while the latter can cause serious adverse reactions. Therefore, there is an urgent need in the art for new and effective therapeutic regimens against GBM disease.
Peptide Immunotherapy has been investigated as a potential intervention for some autoimmune diseases due to its ability to modulate and restore immune homeostasis (Larche M and Wraith DC., Peptide-based therapeutic regimens for alloergic and autoimmune diseases. Nat. Med. 2005; 11(4S): S69; mith EL and Peakman M., Peptide Immunotherapy for Type 1Diabetes-Clinical Advances. FrontImmunol.2018; 9: 392). For example, several peptides derived from autoantigens have been used in clinical trials for autoimmune diseases such as multiple sclerosis, type 1diabetes and celiac disease (Smith EL and Peakman m., as above), revealing the potential utility of peptide immunotherapy in the treatment of autoimmune diseases.
Early studies showed that the peptide fragment α 3-P14(α 3) located at amino acids 127-148 of the
Disclosure of Invention
The inventors surprisingly found that by reacting the alpha 3-P14 peptide (alpha 3)127-148:TDIPPCPHGWISLWKGFSFIMF, respectively; 1) or it comprises the core epitope CPHGW recognized by T cellsIReplacement of isoleucine (I) at position 137 of the
Accordingly, in one aspect, the present disclosure relates to a polypeptide having the amino acid sequence:
a.TDIPPCPHGWXSLWKGFSFIMF (SEQ ID NO: 3); or
b.TDIPPCPHGWXSLWKGFSFIMF (SEQ ID NO:3), the fragment comprising CPHGWXSLWKGF(SEQ ID NO:4)，
Wherein X is any amino acid other than a hydrophobic amino acid.
The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues and do not limit the minimum length of the product. Thus, the above terms encompass peptides, oligopeptides, polypeptides, dimers (heterologous and homologous), multimers (heterologous and homologous), and the like. The term also includes post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like.
As used herein, "amino acid" refers to the 20 naturally occurring amino acids and non-natural amino acid analogs. Amino acids may be referred to herein by their commonly known three letter symbols or by the one letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission. Amino acids can be divided into different groups, depending on their side chain properties, including:
basic amino acids: arginine (R), lysine (K), and histidine (H);
acidic amino acids: glutamic acid (E) and aspartic acid (D);
hydrophilic amino acids: glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), cysteine (C), and tyrosine (Y);
hydrophobic amino acids: alanine (a), valine (V), leucine (L), isoleucine (I), phenylalanine (F), tryptophan (W), methionine (M) and proline (P).
In the sequences of the polypeptides of the present disclosure, X may be any amino acid other than the hydrophobic amino acids (i.e., A, V, L, I, F, W, M and P). In some embodiments, X is a basic amino acid, e.g., selected from R, K and H; in other embodiments, X is an acidic amino acid, e.g., selected from E and D;in some embodiments, X is a hydrophilic amino acid, for example selected from G, N, Q, S, T, C and Y. In a preferred embodiment, X is S, i.e. the polypeptide has the amino acid sequence TDIPPCPHGWSSLWKGFSFIMF (SEQ ID NO:5), or TDIPPCPHGWSSLWKGFSFIMF (SEQ ID NO:5), the fragment comprising CPHGWSSLWKGF(SEQ ID NO:6)。
The polypeptides of the disclosure may have a structure as in TDIPPCPHGWXSLWKGFSFIMF (SEQ ID NO:3), as long as the fragment contains the core epitope CPHGWXSLWKGF (SEQ ID NO: 4). The fragments are, for example, at TDIPPCPHGWXSLWKGFSFIMF (SEQ ID NO:3), in the sequence of CPHGWXSLWKGF (SEQ ID NO:4) is a sequence in which one or more amino acids are extended at one or both ends.
The fragments may be of different lengths. In general, T cell epitopes can be divided into two groups, with CD8+ T cells typically recognizing epitopes containing 8-10 amino acids, and CD4+ T cells typically recognizing epitopes containing 13-17 amino acids. Accordingly, in some embodiments, the polypeptides of the disclosure have TDIPPCPHGWXSLWKGFSFIMF (SEQ ID NO:3), the fragment comprising CPHGWXSLWKGF (SEQ ID NO:4), wherein the fragment comprises at least 13 amino acids, such as at least 15 amino acids or at least 17 amino acids. For example, the fragment may be 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids in length.
In one aspect, the disclosure relates to a nucleotide sequence encoding any of the polypeptides described above. As used herein, the terms "nucleotide sequence," "polynucleotide," and "nucleic acid" are used interchangeably and refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single-or double-stranded form, or a combination of DNA or RNA, as well as polymers thereof. The term "nucleic acid" includes, but is not limited to, a gene, cDNA, or mRNA. In one embodiment, the nucleic acid molecule is synthetic (e.g., chemically synthesized or artificial) or recombinant.
The term "coding sequence" means a polynucleotide that encodes an amino acid sequence of a protein or polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame beginning with an initiation codon (e.g., ATG, GTG, or TTG) and ending with a stop codon (e.g., TAA, TAG, or TGA). The coding sequence may be derived from genomic DNA, or synthetic DNA, or a combination thereof.
Due to the degeneracy of the genetic code, several nucleic acids can encode polypeptides having the same amino acid sequence. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where a codon is identified as alanine, the codon can be replaced with any other codon encoding alanine without altering the encoded polypeptide. One of ordinary skill in the art will recognize that codons in a nucleic acid can be modified without changing the amino acid sequence of the protein or polypeptide it encodes.
In another aspect, the disclosure relates to a vector comprising a nucleotide sequence of the disclosure. In some embodiments, the vector is an expression vector. The term "vector" refers to a vector that can autonomously replicate in a host cell, and is preferably a multicopy vector. The vector may have a promoter and/or a terminator for expressing the introduced gene. The vector may be, for example, a vector derived from a bacterial plasmid, a viral vector, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, a phagemid or the like. The term "expression vector" refers to a vector that enables expression of a protein or polypeptide in a cell, and is typically a linear or circular DNA molecule comprising a polynucleotide encoding the protein or polypeptide and operably linked to an expression control sequence.
The term "expression control sequence" means a nucleic acid sequence necessary for expression of a polynucleotide encoding a mature polypeptide. Each expression control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide, or native or foreign with respect to one another. Such expression control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. In general, expression control sequences include at least a promoter, transcriptional and translational stop signals.
In one aspect, the present disclosure relates to a composition comprising any of the polypeptides or vectors of the present disclosure, and optionally one or more pharmaceutically acceptable vehicles, excipients, and/or diluents. The phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. As used herein, the phrase "pharmaceutically acceptable vehicle, excipient, and/or diluent" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, medium, encapsulating material, manufacturing aid, or solvent encapsulating material, that maintains the stability, solubility, or activity of the polypeptide or carrier of the present disclosure.
In some embodiments, the composition is for prophylactic use, e.g., for administration to a patient at risk of developing anti-GBM disease to prevent the development of anti-GBM disease. In other embodiments, the composition is for therapeutic use, e.g., for administration to a patient having an anti-GBM disease to treat an already existing anti-GBM disease.
In some embodiments, the composition further comprises one or more additional anti-GBM disease therapeutic agents, such as an immunosuppressive agent.
In another aspect, the disclosure relates to a method of preventing or treating an anti-GBM disease in a subject, the method comprising the step of administering to the subject a polypeptide, vector or composition of the disclosure. In some embodiments, the method further comprises administering to the subject one or more additional anti-GBM disease treatments, such as plasmapheresis and immunosuppressive agents.
In one aspect, the disclosure relates to the use of a polypeptide, vector or composition of the disclosure for preventing or treating an anti-GBM disease in a subject. In some embodiments, the polypeptides, vectors, or compositions of the present disclosure are used in combination with one or more additional anti-GBM disease treatments, such as plasmapheresis and immunosuppressants.
In another aspect, the disclosure relates to a polypeptide, vector or composition of the disclosure for use in the manufacture of a medicament for preventing or treating an anti-GBM disease in a subject. In some embodiments, the medicament further comprises one or more additional anti-GBM disease therapeutic agents, such as an immunosuppressive agent.
Drawings
FIG. 1 shows the sequence and homology alignment of the α 1-P14 and α 3-P14 polypeptides, as well as the designed m-P14 polypeptide.
Figure 2 shows the results of modeling the binding of α 3-P14 polypeptide and m-P14 polypeptide to HLA-DRB1 x 1501 molecule using the protein docking software Rossetta.
FIG. 3 shows the effect of α 1-P14, α 3-P14 and m-P14 polypeptides in immunizing animals to induce anti-GBM disease. FIG. 3A shows the level of circulating antibodies against the corresponding antigens in each group of animals after immunization, which was measured by ELISA; FIGS. 3B-3D show 24 hour proteinuria levels, BUN levels, and blood creatinine levels, respectively, in each group of animals; FIGS. 3E-3H show the results of pathological IgG deposition and renal damage in renal tissue from various groups of animals; figure 3M shows the percentage of crescent formation in the tissues of each group of animals.
FIG. 4 shows a schematic of an experimental protocol for inducing anti-GMB disease in animals by using an α 3-P14 polypeptide and testing the prophylactic and therapeutic effects of m-P14 polypeptide on disease.
FIG. 5 shows the prophylactic effect of m-P14 polypeptide on α 3-P14 polypeptide-induced anti-GBM disease. FIG. 5A shows levels of circulating antibodies to the α 3-P14 polypeptide in groups of animals following immunization with the α 3-P14 polypeptide; FIG. 5B shows the percentage of crescent formation in the tissues of each group of animals; FIGS. 5C-5E show 24 hour proteinuria levels, BUN levels, and blood creatinine levels, respectively, in each group of animals; fig. 5F-5I show the results of pathological IgG deposition and kidney damage in kidney tissue of various groups of animals.
FIG. 6 shows the therapeutic effect of m-P14 polypeptide on α 3-P14 polypeptide-induced anti-GBM disease. FIG. 6A shows the percentage of crescent formation in the tissues of each group of animals; FIGS. 6B-6D show 24 hour proteinuria levels, BUN levels, and blood creatinine levels, respectively, in each group of animals; fig. 6E-6G show the results of pathological IgG deposition and kidney damage in kidney tissue of various groups of animals.
Figure 7 shows the levels of circulating and kidney-eluted anti- α 3-NC1 antibody in each group of animals. FIGS. 7A and 7B show the levels of circulating and kidney-eluted anti- α 3-NC1 antibody in animals of the disease group, the prevention group, and the negative control group, respectively; fig. 7C and 7D show the levels of circulating and kidney-eluted anti- α 3-NC1 antibody in the disease group, treatment group, and negative control group animals, respectively.
Detailed Description
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