WO2022007885A1 - 融合多肽和多肽二聚体及其用途 - Google Patents

融合多肽和多肽二聚体及其用途 Download PDF

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WO2022007885A1
WO2022007885A1 PCT/CN2021/105187 CN2021105187W WO2022007885A1 WO 2022007885 A1 WO2022007885 A1 WO 2022007885A1 CN 2021105187 W CN2021105187 W CN 2021105187W WO 2022007885 A1 WO2022007885 A1 WO 2022007885A1
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polypeptide
rearranged
fusion polypeptide
seq
interest
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PCT/CN2021/105187
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English (en)
French (fr)
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沈健
周家宏
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南京师范大学
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Priority claimed from CN202010651373.0A external-priority patent/CN113912735A/zh
Priority claimed from CN202010650886.XA external-priority patent/CN113912734A/zh
Application filed by 南京师范大学 filed Critical 南京师范大学
Priority to EP21838465.9A priority Critical patent/EP4186928A1/en
Priority to US18/004,634 priority patent/US20230322882A1/en
Publication of WO2022007885A1 publication Critical patent/WO2022007885A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention relates to fusion polypeptides and polypeptide dimers.
  • the present invention relates to modified immunomodulatory molecules and their use in the treatment of cancer.
  • cytokines such as activation of immune cells
  • modulation of the immune system by cytokines is a strategy in cancer immunotherapy.
  • certain sites on cytokines have an impact on their function (eg, immunomodulatory effects).
  • interleukin-2 can regulate the function of immune cells.
  • IL-2 binds to the receptors IL2R ⁇ (CD25), IL2R ⁇ (CD122) and IL2R ⁇ (CD132), and can activate downstream signaling pathways, including JAK1 and JAK3 kinases and the transcription factor STAT5, to stimulate immune cells (such as T cells and natural killers). (NK) cells) activation and proliferation.
  • IL-2 can not only conduct signal transduction through CD25/CD122/CD132 trimer, but also activate signaling pathway through CD122/CD132 dimer.
  • CD122/CD132 dimer is mainly expressed on the surface of CD8+ memory T cells and NK cells, while CD25/CD122/CD132 trimer is abundantly expressed on the surface of regulatory T cells (Treg) that play an immunosuppressive role. Therefore, the role of IL-2 is to stimulate or inhibit the activation of the immune system, depending on the activation of different immune cell types.
  • IL-2 is also a promising drug for cancer immunotherapy.
  • Recombinant IL-2 (aldesleukin) was approved by the U.S. Food and Drug Administration (FDA) in 1998 for the treatment of metastatic melanoma and kidney cancer, and is the only IL-2-based drug approved to date,
  • FDA U.S. Food and Drug Administration
  • Interleukin-15 can also activate immune responses and play an important role in the differentiation and proliferation of T cells and NK cells, as well as the development of dendritic cells.
  • IL-15 and IL-2 have similar working mechanism, activate downstream signaling pathways (JAK1/JAK3 and STAT3/STAT5) by binding CD122/CD132 receptor dimer, but its alpha receptor is different from IL-2 , is its unique IL-15R ⁇ (CD215) receptor.
  • IL-15 is presented to CD122 with high affinity in “trans” (trans, cell-cell contact) or "cis” (cis, on the same cell) after binding to the IL-15R ⁇ receptor on the cell membrane On the /CD132 receptor, it has also been found that IL-15 can also bind CD122/CD132 with moderate affinity and function without binding IL-15R ⁇ .
  • cytokines such as IL-2 and IL-15, have short in vivo half-lives and require continuous injection.
  • the present invention provides a fusion polypeptide comprising a carrier protein and a polypeptide of interest, wherein the carrier protein has a plurality of helical domains connected by loops, and the polypeptide of interest is inserted into the carrier protein In the loop, the carrier protein masks the site of interest on the polypeptide of interest, thereby blocking the accessibility of the site.
  • the polypeptide of interest is derived from a cytokine of the four alpha-helix bundle cytokine family, the cytokines comprising, from N-terminal to C-terminal, helical bundle 1 (H1 ), helical bundle 2 ( Four ⁇ -helix bundles of H2), helical bundle 3 (H3) and helical bundle 4 (H4).
  • the polypeptide of interest is a cytokine of the cyclically rearranged four alpha-helical bundle cytokine family comprising, from N-terminus to C-terminus, H2, H3, H4, and H1; H3, H4 , H1 and H2; or the four alpha-helical bundles of H4, H1, H2 and H3.
  • the amino acid corresponding to the N-terminus of the non-cyclically rearranged cytokine in the circulating-rearranged cytokine is linked by a linker to the amino acid corresponding to the C-terminus of the non-circulatingly rearranged cytokine.
  • the linker is a GS linker or a polyglycine linker of 1-10 amino acids in length.
  • the polypeptide of interest is selected from circulating rearranged IL-2 and circulating rearranged IL-15.
  • the cyclically rearranged IL-2 comprises four alpha-helical bundles, sequentially N-terminal to C-terminal, H3, H4, H1 and H2, or H4, H1, H2 and H3.
  • the cyclically rearranged IL-2 comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, or 5.
  • the site of interest is a CD25 binding site.
  • the cyclically rearranged IL-15 comprises four alpha-helical bundles from N-terminus to C-terminus, H3, H4, H1, and H2 in order.
  • the circulating rearranged IL-15 comprises the amino acid sequence of SEQ ID NO:7.
  • the site of interest is a CD215 binding site.
  • the carrier protein is albumin, preferably human serum albumin (HSA).
  • HSA human serum albumin
  • the ring is selected from the group consisting of: Loops for E400, K439-R445, E465-D471, P537-E542 and A561-T566, the positions are numbered with reference to SEQ ID NO:16.
  • the polypeptide of interest is cyclically rearranged IL-2, and the loop is selected from loops located at D56-L66, V293-L305, and A362-D365 of HSA.
  • the insertion site of the polypeptide of interest is selected from D56, A300, C361 and A362 of HSA.
  • the present invention provides a fusion polypeptide comprising a carrier protein HSA and circulating rearranged IL-2, wherein the circulating rearranged IL-2 comprises H3, H4 from N-terminus to C-terminus in order , H1 and H2, or four ⁇ -helical bundles of H4, H1, H2 and H3, and wherein the cyclically rearranged IL-2 is inserted into a loop of HSA selected from the group consisting of D56-L66, A92- P96, D129-E131, Q170-A172, K281-L283, V293-L305, E311-S312, E321-A322, A362-D365, L398-E400, K439-R445, E465-D471, P537-E542, A561-T566 Loop, the positions are numbered with reference to SEQ ID NO: 16, and the HSA masks the CD25 binding site of the circulating rearranged IL-2, thereby blocking the accessibility of
  • the cyclically rearranged IL-2 comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, or 5.
  • the loop is selected from the loops located at D56-L66, V293-L305, and A362-D365 of HSA.
  • the insertion site of the cyclically rearranged IL-2 is selected from the group consisting of D56, A300, C361 and A362 of HSA.
  • the present invention provides a fusion polypeptide comprising a carrier protein HSA and circulating rearranged IL-15, wherein the circulating rearranged IL-15 comprises H3, H4 from N-terminus to C-terminus in order four ⁇ -helix bundles of , H1 and H2, and wherein the cyclically rearranged IL-15 is inserted into a loop of HSA selected from the group consisting of D56-L66, A92-P96, D129-E131, Q170-A172 , K281-L283, V293-L305, E311-SS312, E321-A322, A362-D365, L398-E400, K439-R445, E465-D471, P537-E542 and A561-T566 loops, the positions being referenced to SEQ ID NO : 16, the HSA masks the CD215 binding site of the circulating rearranged IL-15, thereby blocking the accessibility of the site.
  • the HSA masks the CD
  • the present invention provides a fusion polypeptide comprising the amino acid sequence of one of SEQ ID NOs: 8-11.
  • the present invention also provides pharmaceutical compositions comprising the fusion polypeptides of the present invention.
  • the present invention provides a polypeptide dimer comprising a first polypeptide and a second polypeptide,
  • the first polypeptide comprises a first dimerization domain and a polypeptide of interest, wherein the polypeptide of interest is located at the first end of the first dimerization domain,
  • the second polypeptide comprises a second dimerization domain and a binding domain, wherein the binding domain is located at the first end of the second dimerization domain
  • first polypeptide and the second polypeptide form a dimer through the first dimerization domain and the second dimerization domain, and the first dimerization domain of the first dimerization domain The end is adjacent to the first end of the second dimerization domain, and the binding domain is capable of binding to the site of interest on the polypeptide of interest.
  • the polypeptide of interest is derived from a cytokine of the four alpha-helix bundle cytokine family, the cytokines comprising, from N-terminal to C-terminal, helical bundle 1 (H1 ), helical bundle 2 ( Four ⁇ -helix bundles of H2), helical bundle 3 (H3) and helical bundle 4 (H4).
  • the polypeptide of interest is a cytokine of the cyclically rearranged four alpha-helical bundle cytokine family comprising, from N-terminus to C-terminus, H2, H3, H4, and H1; H3, H4 , H1 and H2; or the four alpha-helical bundles of H4, H1, H2 and H3.
  • the amino acid corresponding to the N-terminus of the native cytokine in the circulating rearranged cytokine is linked by a linker to the amino acid corresponding to the C-terminus of the native cytokine.
  • the linker is a GS linker or a polyglycine linker of 1-10 amino acids in length.
  • the polypeptide of interest is circulating rearranged IL-2 or IL-15.
  • the cyclically rearranged IL-2 comprises four alpha-helical bundles, sequentially N-terminal to C-terminal, H3, H4, H1 and H2, or H4, H1, H2 and H3.
  • the circulating rearranged IL-2 comprises the amino acid sequence of SEQ ID NO:21.
  • the site of interest is a CD25 binding site and the binding domain is the extracellular domain of CD25.
  • the cyclically rearranged IL-15 comprises four alpha-helical bundles from N-terminus to C-terminus, H3, H4, H1, and H2 in order.
  • the cyclically rearranged IL-15 comprises the amino acid sequence of SEQ ID NO:22.
  • the site of interest is a CD215 binding site and the binding domain is the extracellular domain of CD215.
  • the first and second dimerization domains comprise the heavy chain constant regions CH2 and CH3 of an immunoglobulin (Ig).
  • Ig is a human Ig, eg, human IgGl.
  • the first dimerization domain and the second dimerization domain form the Fc region of human IgGl.
  • the Fc region of the human IgGl is modified.
  • the first dimerization domain comprises the amino acid sequence of SEQ ID NO:23
  • the second dimerization domain comprises the amino acid sequence of SEQ ID NO:24
  • the first The dimerization domain comprises the amino acid sequence of SEQ ID NO:24
  • the second dimerization domain comprises the amino acid sequence of SEQ ID NO:23.
  • the first end of the first dimerization domain is C-terminal, and the first end of the second dimerization domain is C-terminal.
  • polypeptide dimers of the invention comprise a first polypeptide and a second polypeptide
  • the first polypeptide comprises the first chain of the Fc region of human IgG1 and circulating rearranged IL-2, the circulating rearranged IL-2 is C-terminal to the first chain of the Fc region of the human IgG1 connect,
  • the second polypeptide comprises the second chain of the Fc region of human IgG1 and the CD25 extracellular domain, the CD25 extracellular domain is linked to the C-terminus of the second chain of the Fc region of human IgG1, and
  • the cyclically rearranged IL-2 comprises four ⁇ -helix bundles which are H3, H4, H1 and H2; or H4, H1, H2 and H3 in order from the N-terminus to the C-terminus.
  • the circulating rearranged IL-2 comprises the amino acid sequence of SEQ ID NO:21.
  • polypeptide dimers of the invention comprise a first polypeptide and a second polypeptide
  • the first polypeptide comprises the first chain of the Fc region of human IgG1 and circulating rearranged IL-15, the circulating rearranged IL-15 and the C-terminus of the first chain of the Fc region of the human IgG1 connect,
  • the second polypeptide comprises the second chain of the Fc region of human IgG1 and the CD215 extracellular domain, the CD215 extracellular domain is linked to the C-terminus of the second chain of the Fc region of human IgG1, and
  • the cyclically rearranged IL-15 comprises four ⁇ -helix bundles from N-terminus to C-terminus, H3, H4, H1 and H2 in sequence.
  • the cyclically rearranged IL-15 comprises the amino acid sequence of SEQ ID NO:22.
  • the first strand comprises the amino acid sequence of SEQ ID NO:23 and the second strand comprises the amino acid sequence of SEQ ID NO:24; or the first strand comprises the amino acid sequence of SEQ ID NO:24 amino acid sequence, and the second strand comprises the amino acid sequence of SEQ ID NO:23.
  • the present invention also provides pharmaceutical compositions comprising the polypeptide dimers of the present invention.
  • the present invention also provides a method of treating cancer or activating immune cells or increasing the proliferation of immune cells (eg T cells or NK cells), comprising administering to a subject in need thereof an effective amount of a fusion polypeptide of the present invention, A polypeptide dimer or a pharmaceutical composition of the present invention.
  • immune cells eg T cells or NK cells
  • the present invention also provides the use of the fusion polypeptide, polypeptide dimer or pharmaceutical composition of the present invention in the preparation of a medicament for the treatment of cancer and in the preparation of a method for activating immune cells or increasing immune cells (eg T cells or NK cells). Use in proliferative drugs.
  • fusion polypeptides, polypeptide dimers, or pharmaceutical compositions of the invention are also provided for use in treating cancer or activating immune cells or increasing the proliferation of immune cells (eg, T cells or NK cells).
  • immune cells eg, T cells or NK cells.
  • the present invention also provides polynucleotides and vectors encoding the fusion polypeptides or polypeptide dimers of the present invention, and host cells comprising the polynucleotides and vectors.
  • Figure 1 shows a schematic diagram of the three-dimensional structure of wild-type IL-2 binding to its receptor (based on PDB number 2ERJ), wherein “loop 1" corresponds to S95-L100 of native IL-2, and “loop 2" corresponds to native IL-2 N50-P54.
  • positions referring to IL-2 herein are numbered with reference to the IL-2 precursor sequence of SEQ ID NO: 1 (UniProt P60568), wherein amino acid residues 1-21 are the signal peptide sequence, and the The sequence is amino acid residues 22-153 of SEQ ID NO:1.
  • Figure 2 shows a schematic diagram of the three-dimensional structure of wild-type IL-15 binding to its receptor (based on PDB number 4GS7), where the "opened loop" corresponds to S102-A105 of native IL-15.
  • positions referenced herein to IL-15 are numbered with reference to the IL-15 precursor sequence of SEQ ID NO: 6 (UniProt P40933), where residues 1-48 are the signal peptide, and the sequence of native IL-15 is Amino acid residues 49-162 of SEQ ID NO:6.
  • Figure 3 shows a schematic diagram of the three-dimensional structure of HSA.
  • Figures 4-7 show the expression of fusion polypeptides of the invention and the structural model of HSA masking the CD25 binding site on IL-2.
  • Figures 8 and 9 show the structural model of the polypeptide dimer of the present invention and its expression, and the amino acid sequence of cyclically rearranged IL-2 is shown in SEQ ID NO: 21.
  • Figures 10-13 show in vitro activity assays of fusion polypeptides of the present invention.
  • Figures 14 and 15 show the results of in vitro activity assays of the polypeptide dimers of the present invention, and the amino acid sequence of circulating rearranged IL-2 is shown in SEQ ID NO: 21.
  • Figure 16 shows the experimental results of injecting the fusion polypeptide or polypeptide dimer of the present invention into tumor-bearing mice, A: tumor 27 days after the first injection; B: curve of tumor volume over time; and C: mouse body weight curve over time.
  • Figure 17 shows the results of immunohistochemical staining of tumors from different groups of mice, A: mean optical density; and B: representative imaging results.
  • peptide refers to a chain of at least two amino acids linked by peptide bonds.
  • polypeptide is used interchangeably herein with the term “protein” and refers to a chain containing ten or more amino acid residues. All peptide and polypeptide formulas or sequences herein are written left to right, indicating the orientation from the amino terminus to the carboxy terminus.
  • dihedral angle in a peptide chain refers to the angle by which two adjacent peptide bond planes can rotate about the alpha carbon atom between the two peptide bond planes.
  • amino acid In the context of peptides, the terms "amino acid”, “residue” and “amino acid residue” are used interchangeably and include both naturally occurring amino acids and unnatural amino acids in proteins.
  • amino acid residue The one-letter and three-letter names of amino acids naturally occurring in proteins use names commonly used in the art, as found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • nucleic acid molecule includes DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA) and analogs of DNA or RNA produced using nucleotide analogs.
  • the nucleic acid molecule may be single-stranded or double-stranded, preferably double-stranded DNA.
  • the nucleic acid can be synthesized using nucleotide analogs or derivatives (eg, inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acids with altered base-pairing ability or increased nuclease resistance.
  • the term "encode” refers to a polynucleotide that directly specifies the amino acid sequence of its protein product.
  • the boundaries of the coding sequences are generally defined by open reading frames, which typically begin with the ATG start codon or additional start codons such as GTG and TTG and end with stop codons such as TAA, TAG and TGA.
  • the coding sequence may be a DNA, cDNA or recombinant nucleotide sequence.
  • hybridize is a nucleotide that is at least about 90%, preferably at least about 95%, more preferably at least about 96%, more preferably at least 98% homologous to each other under given stringent hybridization and washing conditions Sequences generally remain hybridized to each other.
  • percent amino acid identity or “percent amino acid sequence identity” refers to comparing the amino acids of two polypeptides that, when optimally aligned, have approximately the specified percentage of amino acids identical.
  • 95% amino acid identity refers to comparing the amino acids of two polypeptides which, when optimally aligned, are 95% identical.
  • the sequences are aligned for optimal comparison (eg gaps can be introduced in the first amino acid or nucleic acid sequence to match the second amino acid sequence) or nucleic acid sequences for optimal alignment).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.
  • percent identity number of identical positions/total number of positions (ie, overlapping positions) x 100).
  • the two sequences are the same length.
  • Conservative amino acid substitutions generally have minimal effect on the activity of the resulting protein. This substitution is described below.
  • a conservative substitution is the replacement of an amino acid with an amino acid that is similar in size, hydrophobicity, charge, polarity, steric characteristics, aromaticity, etc. Such substitutions are usually conservative when it is desired to fine-tune the properties of the protein.
  • homologous amino acid residues refer to amino acid residues that have similar chemical properties related to hydrophobicity, charge, polarity, steric characteristics, aromatic characteristics, and the like.
  • amino acids that are homologous to each other include positively charged lysine, arginine, histidine, negatively charged glutamic acid, aspartic acid, hydrophobic glycine, alanine, valine, leucine acid, isoleucine, proline, phenylalanine, polar serine, threonine, cysteine, methionine, tryptophan, tyrosine, asparagine, glutamine , aromatic phenylalanine, tyrosine, tryptophan, serine and threonine with chemically similar side chain groups, or glutamine and asparagine, or leucine and isoleucine.
  • Examples of conservative amino acid substitutions in proteins include: Ser for Ala, Lys for Arg, Gln or His for Asn, Glu for Asp, Ser for Cys, Asn for Gln, Asp for Glu, Pro for Gly, Asn or Gln for His, Leu or Val replaces Ile, Ile or Val replaces Leu, Arg or Gln replaces Lys, Leu or Ile replaces Met, Met, Leu or Tyr replaces Phe, Thr replaces Ser, Ser replaces Thr, Tyr replaces Trp, Trp or Phe replaces Tyr, and Ile or Leu replaces Val.
  • expression includes any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • cytokine refers to the production of immune cells (eg, monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) etc.) are stimulated to synthesize and secrete a class of small molecular proteins with a wide range of biological activities. Cytokines generally regulate cell growth and differentiation, as well as immune responses, by binding to the corresponding receptors.
  • immune cells eg, monocytes, macrophages, T cells, B cells, NK cells, etc.
  • non-immune cells endothelial cells, epidermal cells, fibroblasts, etc.
  • Cytokines generally regulate cell growth and differentiation, as well as immune responses, by binding to the corresponding receptors.
  • four alpha-helix bundle cytokine family refers to cytokines comprising four alpha-helix bundles in their tertiary structure.
  • the natural (wild-type) four ⁇ -helix bundle cytokine family consists of helical bundle 1 (H1), helical bundle 2 (H2), helical bundle 3 (H3) and helical bundle 4 from N-terminal to C-terminal The four ⁇ -helical bundles of (H4).
  • Cytokines of the four-alpha-helix bundle cytokine family include, but are not limited to, IL-2, IL-4, IL-6, IL-7, IL-9, IL-15, IL-21, G-CSF, and GM-CSF .
  • cyclic rearrangement refers to changing the order in which the four alpha-helix bundles are arranged in the primary structure of cytokines of the four-alpha-helix bundle cytokine family.
  • circulating rearranged cytokines comprise four alpha-helical bundles of H2, H3, H4, and H1; H3, H4, H1, and H2; or H4, H1, H2, and H3, in order from N-terminus to C-terminus.
  • Cyclic rearrangement involves the design of a polypeptide by fusing (either directly or via a linker) the N-terminus to the C-terminus of the polypeptide (the original polypeptide, such as a wild-type four-alpha-helix bundle cytokine) to form a The circular molecule is opened (cut or broken) between H2, H2 and H3, or H3 and H4, forming a new linear polypeptide with different N- and C-termini than the original polypeptide. Cyclic rearrangement preserves the sequence, structure and function of the polypeptide (except for the optional presence of linkers) while creating new C- and N-termini at different positions. Cyclic rearrangement also includes any process that produces the cyclically rearranged linear molecules described herein. Often, cyclically rearranged polypeptides are expressed directly as linear molecules without actually going through the steps of cyclization and opening.
  • linker refers to an amino acid sequence covalently linked to the N and/or C terminus of a polypeptide.
  • Linkers can be used to link the N- and C-termini of the same polypeptide (as in cyclic rearrangements), or it can be agreed to link the N- and C-termini of different polypeptides to form fusion polypeptides.
  • a linker also refers to a polynucleotide encoding the amino acid sequence of the linker.
  • linkers have no specific biological activity. However, the amino acids that make up the linker can be selected based on certain properties of the linker or the resulting molecule, such as flexibility, hydrophilicity, net charge or whether it is proteolytically sensitive, and lacks immunogenicity.
  • wild-type polypeptide refers to a naturally occurring polypeptide.
  • modification refers to a modification of a polynucleotide or polypeptide sequence, including, but not limited to, substitution, deletion, insertion, and/or addition of one or more nucleotides or amino acids. Modifications also include chemical modifications that do not alter the sequence of the polynucleotide or polypeptide, such as methylation of polynucleotides, glycosylation of polypeptides, and the like. In this context, modifications also include cyclic rearrangements as described above.
  • opening site refers to the position in a cyclic molecule at which peptide bonds are eliminated to form new amino and carboxyl termini during cyclic rearrangement, or the corresponding position in a polynucleotide encoding the polypeptide .
  • the opening site is specified by the position of a pair of amino acids between the amino and carboxy termini of the wild-type polypeptide, which become the new amino and carboxy termini of the cyclically rearranged polypeptide.
  • the new N-terminus corresponds to the residue at position 97 of native IL-2, and the new C-terminus corresponds to the residue at position 96 of native IL-2; in IL-2 -15 (105/102), the new N-terminus corresponds to the residue at position 105 of native IL-15, and the new C-terminus corresponds to the residue at position 102, positions 103 and 104 of native IL-15 base is removed.
  • the term "receptor” is to be understood as a protein present on the cell surface that binds a ligand, and also covers soluble receptors that are not present on the cell surface, which have a corresponding cell surface receptor or are associated with a corresponding cell surface receptor receptor related.
  • Cell surface receptors typically consist of distinct domains or subunits with distinct functions, such as an extracellular domain, a transmembrane domain, and an intracellular effector domain, which contain regions that interact with ligands, The transmembrane domain anchors the receptor in the cell membrane, while the intracellular effector domain generates cellular signals (signal transduction) in response to ligand binding.
  • Soluble receptors typically consist of one or more extracellular domains that are proteolytically cleaved from the membrane anchor region.
  • variant refers to a polypeptide that differs from a reference polypeptide but retains essential properties.
  • a typical variant of a polypeptide differs in its primary amino acid sequence from the reference polypeptide. Often, the differences are limited such that the sequences of the reference polypeptide and the variant are generally very similar and identical in many regions.
  • the amino acid sequence of a variant and a reference polypeptide may differ by one or more modifications (eg, substitutions, additions and/or deletions).
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • Variants of polypeptides can be naturally occurring, such as allelic variants; they can also be artificially produced variants. Additionally, the term "variant” as used herein includes cyclically rearranged variants of the polypeptide.
  • carrier protein refers to a protein that is fused to a molecule of interest (eg, a polypeptide or hapten) and has functions such as aiding in the delivery of the protein of interest, prolonging its half-life, making the hapten immunogenic, etc. .
  • the carrier protein itself does not possess the biological activity of the molecule of interest.
  • the carrier protein used in the present invention has a plurality of helical structures connected by loops, that is, a "helix-loop-helix” structure, for example, albumin such as human serum albumin (HSA), albumin-binding protein, and the like.
  • HSA human serum albumin
  • the "insertion site" of a polypeptide of interest in a carrier protein refers to the position of the carrier protein closest to the N-terminus of the polypeptide of interest in the primary structure after the polypeptide of interest is inserted into the carrier protein. position of residues.
  • a carrier protein "shadows" a site of interest, meaning that the carrier protein sterically hinders the binding of the site of interest to its receptor, ie, the carrier protein sterically clashes with the receptor.
  • accessibility of a site refers to the ability of a site to contact and bind with its binding partner. When the accessibility of the site is blocked, it cannot contact and bind with its binding partner.
  • immunoglobulin refers to globulins that have antibody activity or chemical structure and are similar to antibody molecules.
  • Natural immunoglobulin is a tetrapeptide chain structure composed of two identical light chains and two identical heavy chains connected by interchain disulfide bonds.
  • Fc fragment or "Fc region” is the portion of an immunoglobulin that papain hydrolyzes Ig, which cleaves the Ig into two identical Fab fragments and an Fc fragment.
  • the Fc fragment of a native antibody comprises two identical polypeptides linked by disulfide bonds. Each polypeptide contains two heavy chain constant regions (CH2 and CH3, eg, the Fc region of IgG), or three heavy chain constant regions (CH2, CH3, and CH4, eg, the Fc region of IgM and IgE).
  • signal peptide refers to a short peptide that directs the transfer of newly synthesized proteins to the secretory pathway.
  • the signal peptide is located at the N-terminus of the newly synthesized protein and is, for example, 5-30 amino acid residues in length. Signal peptides can be removed during protein processing so that mature proteins do not contain signal peptides.
  • treatment refers to a method of obtaining beneficial or desired results, including but not limited to eradication or amelioration of the underlying disease being treated.
  • therapeutic benefit is obtained by eliminating or ameliorating one or more physiological symptoms associated with the underlying disease; thus, improvement is observed in the subject despite the fact that the subject may still have the underlying disease.
  • terapéuticaally effective amount and “therapeutically effective dose” refer to the amount of active ingredient that, when administered in a single dose or in repeated doses, achieves a measurable beneficial effect, including but not limited to , the effect on any symptom, aspect, measured parameter or characteristic of a disease or disorder.
  • a dose refers to an amount administered to a subject once (unit dose) or two or more times within a defined time interval.
  • a dose can refer to an amount administered (eg, by one administration, or two or more administrations) over one day, two days, one week, two weeks, three weeks, or one or more months.
  • half-life refers to the time it takes for the in vivo concentration of a target molecule to decrease by 50%.
  • the half-life of the target molecule will increase if it remains in the biological matrix (blood, serum, plasma, tissue) for a longer period of time compared to an appropriate control.
  • the half-life can be increased by 10%, 20%, 30%, 40%, 50% or more compared to an appropriate control.
  • Cyclic rearrangement refers to changing the order in which the four ⁇ -helix bundles are arranged in the primary structure of cytokines of the four ⁇ -helix bundle cytokine family.
  • a cyclically rearranged four alpha-helix bundle cytokine contains four alphas from N-terminal to C-terminal, H2, H3, H4, and H1; H3, H4, H1, and H2; or H4, H1, H2, and H3 - Helical bundles.
  • Cyclic rearrangement involves the design of a polypeptide by fusing (either directly or via a linker) the N-terminus to the C-terminus of the polypeptide (the original polypeptide, such as a wild-type four-alpha-helix bundle cytokine) to form a The circular molecule is opened (cut or broken) between H2, H2 and H3, or H3 and H4, forming a new linear polypeptide with different N- and C-termini than the original polypeptide.
  • the cyclically rearranged polypeptides provide optimized ends for fusion with other polypeptides while retaining the biological activity of the original polypeptide. If the new terminus interrupts critical regions of the original polypeptide, it may lose activity. Likewise, cyclically rearranged polypeptides cannot retain biological activity if ligation of the original ends would destroy activity.
  • a protein to produce active cyclic rearrangements: 1) ligation of the ends of the original polypeptide does not destroy its biological activity; 2) there must be at least one "open site" in the original polypeptide at which Dots can form new ends without disrupting regions critical for their folding and biological activity.
  • candidate polypeptides undergoing cyclic rearrangement are in their native folded state (original protein) with the original N and C termini in close proximity, e.g., the N and C termini of the original protein are less than or equal to
  • the location of the new end is geometrically, structurally and functionally advantageous (relative to the native end) for fusion to the desired polypeptide fusion partner and to reduce the length of the desired linker.
  • FIG. 1 The structure of IL-2 is shown in Figure 1, where loops 1 and 2 are examples of where new ends are formed.
  • Figure 2 shows the structure of IL-15, where the "opened loop" is an example of where new ends are formed.
  • recombinant constructs are engineered by linking the native N-terminus and C-terminus of IL-2 by a linker and at amino acid residues A93-R103 or N50 -L56 opens the cyclic molecule to form a linear molecule with new N and C termini.
  • a new N-terminus is formed at amino acid residue R103 and a new C-terminus is formed at A93, and amino acid residues Q94-P102 are deleted, ie IL-2 (R103/A93).
  • the circulating rearranged IL-2 is IL-2(L56/N50), IL-2(L56/K55), or IL-2(N53/K52).
  • a new N-terminus is formed at N97 and a new C-terminus is formed at K96, ie IL-2 (N97/K96).
  • the cyclically rearranged IL-2 comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, or 5.
  • recombinant constructs are engineered by linking the native N- and C-termini of IL-15 through a linker to form a circular molecule, and at amino acid residue S102-
  • the cyclic molecule is opened between A105 to form a linear molecule with new N and C termini.
  • a new C-terminus is formed at amino acid residues S102, G103 or D104
  • a new N-terminus is formed at G103, D104, A105 or S106.
  • the circulating rearranged IL-15 is IL-15(G103/S102), IL-15(D104/S102), IL-15(A105/S102), IL-15(S106/S102) ), IL-15(S106/G103), IL-15(A105/G103), IL-15(D104/G103), IL-15(A105/D104), IL-15(S106/D104), IL-15 (S106/A105).
  • the length of the linker used to join the N and C termini of the original polypeptide is related to the distance of the N and C termini in the original protein.
  • the linker used to join the N and C termini of the original polypeptide is 1-10 amino acids in length, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In some embodiments, the linker is greater than 10 amino acids in length.
  • a 3 amino acid linker such as GSG is used to link the N and C termini of native IL-2; a four amino acid linker such as GGGG (SEQ ID NO: 17) is used to link the N and C termini of native IL-15.
  • residue T23 of native IL-2 ie, T2 of SEQ ID NO: 1
  • residue C145 of native IL-2 C124 of SEQ ID NO: 1
  • the cyclically rearranged IL-2 comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, or 5.
  • the circulating rearranged IL-15 comprises the amino acid sequence of SEQ ID NO:7.
  • the circulating rearranged IL-2 comprises the amino acid sequence set forth in SEQ ID NO:23.
  • the circulating rearranged IL-15 comprises the amino acid sequence set forth in SEQ ID NO:24.
  • the fusion polypeptide of the present invention comprises a carrier protein and a polypeptide of interest, wherein the carrier protein has multiple helical domains connected by loops, the polypeptide of interest is inserted into the loop of the carrier protein, and the carrier protein shields the site of interest on the polypeptide of interest, thereby blocking the accessibility of the site.
  • the linker such as its sequence and/or length
  • the polypeptide of interest is derived from a cytokine of the four alpha-helical bundle cytokine family, including but not limited to IL-2, IL-4, IL6, IL-7, IL-9, IL15 and IL21.
  • the polypeptide of interest is a cytokine of the circulating rearranged four alpha-helix bundle cytokine family.
  • the polypeptide of interest is circulating rearranged IL-2 or IL-15.
  • Native IL-2 has binding sites for CD25, CD122 and CD132 (referred to as IL-2 receptor alpha, beta and gamma, respectively).
  • IL-2 can either bind to the CD25/CD122/CD132 trimer, thereby activating T regulatory cells (Treg) expressing the trimer to suppress the immune response, or it can bind to the CD122/CD132 dimer, thereby activating the expression of The dimerized immune cells (such as CD8+ memory T cells and NK cells) and stimulated their proliferation. Binding of CD25 to IL-2 changes the conformation of IL-2, increasing its affinity for the CD122/CD132 dimer.
  • the polypeptide of interest is circulating rearranged IL-2
  • the site of interest is the CD25 binding site
  • the fusion polypeptide has equivalent or improved native IL-2 Activity, such as activation of JAK1/JAK3 and STAT3/STAT5 signaling pathways.
  • IL-15 can also activate immune responses and play an important role in the differentiation and proliferation of T cells and NK cells, as well as the development of dendritic cells.
  • IL-15 and IL-2 have similar working mechanism, activate downstream signaling pathways (JAK1/JAK3 and STAT3/STAT5) by binding CD122/CD132 receptor dimer, but its alpha receptor is different from IL-2 , is its unique IL-15R ⁇ (CD215).
  • IL-15 binds to CD122/CD132 with high affinity after binding to IL-15R ⁇ , while IL-15 binds to CD122/CD132 with moderate affinity without binding to IL-15R ⁇ .
  • the polypeptide of interest is circulating rearranged IL-15
  • the site of interest is the CD215 binding site
  • the fusion polypeptide has equivalent or improved native IL-15 Activity, such as activation of JAK1/JAK3 and STAT3/STAT5 signaling pathways.
  • a carrier protein suitable for use in the present invention is capable of binding to the neonatal Fc receptor (FcRn) (eg albumin), or the carrier protein binds to a protein capable of binding FcRn (eg albumin binding protein).
  • FcRn neonatal Fc receptor
  • albumin a protein capable of binding FcRn
  • Human serum albumin is one of the most stable and most abundant proteins in human serum (35-50g/L), accounting for half of the protein in serum. Its main function is to transport substances (such as hormones, fatty acids, etc.), maintain pH and osmotic pressure, etc. HSA herein includes wild-type HSA and modified HSA.
  • HSA is a holo- ⁇ -helical protein with a molecular weight of 66.5 kDa and consists of three similar domains (DI, DII, DIII) forming a "heart-shaped" structure ( Figure 3).
  • HSA can bind to human FcRn under acidic conditions (pH ⁇ 6.5), and is subsequently recovered to the cell surface and released back into the blood, thereby preventing HSA from entering lysosomes for degradation.
  • FcRn binds mainly to DIII and part of DI.
  • HSA The precursor sequence of HSA is shown in SEQ ID NO: 16 (Uniprot P02768), and all positions involving HSA herein are numbered with reference to SEQ ID NO: 16. Residues 1-24 in the precursor sequence of HSA are the signal peptide, and native HSA comprises residues 25-609 of SEQ ID NO: 16.
  • the carrier protein is HSA.
  • the insertion of the polypeptide of interest does not affect the binding of HSA to FcRn.
  • the polypeptide of interest is inserted into a loop of HSA selected from the group consisting of: The rings of S312, E321-A322, A362-D365, L398-E400, K439-R445, E465-D471, P537-E542, A561-T566, preferably the rings at D56-L66, V293-L305 and A362-D365.
  • the insertion site for the polypeptide of interest is amino acid residue D56, A300, C361 or A362 of HSA.
  • the relative position between the carrier protein and the polypeptide of interest can be controlled by deleting one or more amino acids in the loop of the carrier protein, such as HSA, or not deleting amino acids in the loop of the carrier protein.
  • HSA can also be modified to improve its properties.
  • free cysteine in wild-type HSA can be substituted with other amino acids, such as serine.
  • the HSA comprises the amino acid substitution C58S.
  • the carrier protein has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16 and has a The same or similar structure as type HSA.
  • the N- and C-termini of the polypeptide of interest are linked to the carrier protein by linkers.
  • the N-terminus of the polypeptide of interest is linked to the carrier protein through a linker, and the C-terminus of the polypeptide of interest is directly linked to the carrier protein.
  • the N-terminus of the polypeptide of interest is directly linked to the carrier protein, and the C-terminus of the polypeptide of interest is linked to the carrier protein through a linker.
  • the N- and C-termini of the polypeptide of interest are directly linked to the carrier protein.
  • the linker has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids.
  • the linker is selected from helical linkers, or flexible linkers (eg, GS linkers and polyglycine linkers).
  • the fusion polypeptide of the present invention comprises the carrier protein HSA and cyclically rearranged IL-2, wherein the cyclically rearranged IL-2 comprises H3, H4, H1 and H2 in order from N-terminus to C-terminus , or the four ⁇ -helix bundles of H4, H1, H2, and H3, and wherein the cyclically rearranged IL-2 is inserted into a loop of HSA selected from the group consisting of D56-L66, A92-P96, D129- Rings for E131, Q170-A172, K281-L283, V293-L305, E311-S312, E321-A322, A362-D365, L398-E400, K439-R445, E465-D471, P537-E542 and A561-T566, described Positions are numbered with reference to SEQ ID NO: 16, and the HSA masks the CD25 binding site of the circulating rearranged IL-2, thereby blocking
  • the cyclically rearranged IL-2 comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, or 5.
  • the loop is selected from the loops located at D56-L66, V293-L305, and A362-D365 of HSA.
  • the insertion site of the cyclically rearranged IL-2 is selected from the group consisting of D56, A300, C361 and A362 of HSA.
  • the cyclically rearranged IL-2 comprises the amino acid sequence of SEQ ID NO:2.
  • the loop is a loop located at A362-D365 of HSA, preferably, the insertion site is C361 of HSA.
  • the N-terminus of the cyclically rearranged IL-2 is linked to C361 of HSA through linker EAAAKAEAAA (SEQ ID NO: 19), the C-terminus is linked to D365 of HSA through linker GS, residue 362 of HSA -364 is removed.
  • the circulating rearranged IL-2 comprises the amino acid sequence of SEQ ID NO:4.
  • the loop is a loop located at D56-L66 of HSA, preferably, the insertion site is D56 of HSA.
  • the N-terminus of the cyclically rearranged IL-2 is linked to D56 of HSA through a linker AAAAAK (SEQ ID NO:20), and the C-terminus is directly linked to E57 of HSA.
  • the circulating rearranged IL-2 comprises the amino acid sequence of SEQ ID NO:5.
  • the loop is a loop located at V293-L305 of HSA, preferably, the insertion site is A300 of HSA.
  • the N-terminus of the cyclically rearranged IL-2 is directly linked to A300 of HSA, and the C-terminus is linked to D301 of HSA through linker G.
  • the cyclically rearranged IL-2 consists of the amino acid sequence of SEQ ID NO:4.
  • the loop is a loop located at A362-D365 of HSA, preferably, the insertion site is A362 of HSA.
  • the N-terminus of the cyclically rearranged IL-2 is directly linked to A362 of HSA and the C-terminus is directly linked to A363 of HSA.
  • the present invention provides a fusion polypeptide comprising a carrier protein HSA and circulating rearranged IL-15, wherein the circulating rearranged IL-15 comprises H3, H4 from N-terminus to C-terminus in order four ⁇ -helix bundles of , H1 and H2, and wherein the cyclically rearranged IL-15 is inserted into a loop of HSA selected from the group consisting of D56-L66, A92-P96, D129-E131, Q170-A172 , K281-L283, V293-L305, E311-SS312, E321-A322, A362-D365, L398-E400, K439-R445, E465-D471, P537-E542 and A561-T566 loops, the positions are referred to SEQ ID NO : 16, the HSA masks the CD215 binding site of the circulating rearranged IL-15, thereby blocking the accessibility of the site.
  • the HSA masks the CD
  • fusion polypeptides of the invention comprise the amino acid sequence of one of SEQ ID NOs: 8-11. In some embodiments, fusion polypeptides of the invention comprise at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence of one of SEQ ID NOs: 8-11 The amino acid sequence of sequence identity, wherein the fusion polypeptide comprises a native CD25 binding site, which has comparable or improved activity to native IL-2, such as the activity of activating JAK1/JAK3 and STAT3/STAT5 signaling pathways.
  • the fusion polypeptides of the invention have a more durable immunostimulatory effect. In some embodiments, the fusion polypeptides of the present invention have a higher safety profile.
  • the half-life of the fusion polypeptides of the invention is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% compared to native IL-2 %, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more. In some embodiments, the half-life of the fusion polypeptides of the invention is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% compared to native IL-15 %, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more.
  • the polypeptide dimer of the present invention comprises a first polypeptide and a second polypeptide
  • the first polypeptide comprises a first dimerization domain and a polypeptide of interest, wherein the polypeptide of interest is located at the first end of the first dimerization domain,
  • the second polypeptide comprises a second dimerization domain and a binding domain, wherein the binding domain is located at the first end of the second dimerization domain
  • first polypeptide and the second polypeptide form a dimer through the first dimerization domain and the second dimerization domain, and the first dimerization domain of the first dimerization domain The end is adjacent to the first end of the second dimerization domain, and the binding domain is capable of binding to the site of interest on the polypeptide of interest.
  • the polypeptide of interest is derived from a cytokine of the four alpha-helical bundle cytokine family, including but not limited to IL-2, IL-4, IL6, IL-7, IL-9, IL15 and IL21.
  • the polypeptide of interest is a cytokine of the circulating rearranged four alpha-helix bundle cytokine family.
  • the polypeptide of interest is cyclically rearranged IL-2 or IL-15, as previously described.
  • Native IL-2 has binding sites for CD25, CD122 and CD132 (referred to as IL-2 receptor alpha, beta and gamma, respectively).
  • IL-2 can either bind to the CD25/CD122/CD132 trimer, thereby activating T regulatory cells (Treg) expressing the trimer to suppress the immune response, or it can bind to the CD122/CD132 dimer, thereby activating the expression of The dimerized immune cells (such as CD8+ memory T cells and NK cells) and stimulated their proliferation. Binding of CD25 to IL-2 changes the conformation of IL-2, increasing its affinity for the CD122/CD132 dimer.
  • the polypeptide of interest is circulating rearranged IL-2
  • the site of interest is a CD25 binding site
  • the binding domain is the extracellular domain of CD25
  • the polypeptide dimer has comparable or improved activity to native IL-2, such as the activity of activating JAK1/JAK3 and STAT3/STAT5 signaling pathways.
  • IL-15 can also activate immune responses and play an important role in the differentiation and proliferation of T cells and NK cells, as well as the development of dendritic cells.
  • IL-15 and IL-2 have similar working mechanism, activate downstream signaling pathways (JAK1/JAK3 and STAT3/STAT5) by binding CD122/CD132 receptor dimer, but its alpha receptor is different from IL-2 , is its unique IL-15R ⁇ (CD215).
  • IL-15 binds to CD122/CD132 with high affinity after binding to IL-15R ⁇ , while IL-15 binds to CD122/CD132 with moderate affinity without binding to IL-15R ⁇ .
  • the polypeptide of interest is circulating rearranged IL-15
  • the site of interest is a CD215 binding site
  • the binding domain is the extracellular domain of CD215
  • the polypeptide dimer has comparable or improved activity to native IL-15, such as the activity of activating JAK1/JAK3 and STAT3/STAT5 signaling pathways.
  • the site of interest and the binding domain are located in different (first and second) polypeptides.
  • the first and second dimerization domains form a dimer such that the site of interest and the binding domain are brought into proximity.
  • the first and second dimerization domains form dimers by, for example but not limited to, covalent linkage, hydrogen bonding, electrostatic interactions and/or van der Waals forces, preferably covalent linkage.
  • the first and second dimerization domains form dimers through disulfide bonds.
  • the first and second dimerization domains comprise heavy chain constant regions CH2 and CH3 of an immunoglobulin (Ig), eg, human Ig (eg, human IgGl).
  • Ig immunoglobulin
  • the first dimerization domain and the second dimerization domain form the Fc region of human IgGl.
  • Different mutations can be introduced in the Fc segment to achieve different functions, for example, increase the affinity of Fc to FcRn under acidic conditions; decrease or increase the affinity of Fc to different Fc ⁇ receptors and C1q to weaken or enhance antibody-dependent cells Toxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), etc.
  • ADCC antibody-dependent cells Toxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • the Fc region in the polypeptide dimers of the invention comprises Fc silent mutations, such as L234A+L235A+P329G, to reduce ADCC, ADCP and CDC, wherein the amino acid position numbering of the Fc region follows the IMGT EU numbering convention ( http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGH Gnber.html).
  • the Fc region is a homodimer, ie, the first dimerization domain is identical in sequence to the second dimerization domain. In some embodiments, the Fc region is heterodimeric, ie, the first dimerization domain is different in sequence from the second dimerization domain. In some embodiments, the Fc region is knob-hole modified.
  • a mutation T366Y (the first-generation knob-and-hole structure) or S354C+T366W (the second-generation knob-and-hole structure) is introduced into one polypeptide chain to form a knob chain; in the other polypeptide chain, Y407T (the first-generation knob-and-hole structure) or Y349C +T366S+L368A+Y407V (the second-generation pestle-hole structure), forming a hole chain, the numbering rule is the same as above.
  • the first polypeptide comprises a knob chain and the second polypeptide comprises a hole chain.
  • the first polypeptide comprises a hole chain and the second polypeptide comprises a knob chain.
  • the knob comprises or has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the sequence with SEQ ID NO:23 identical amino acid sequences.
  • the hole chain comprises or has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the sequence with SEQ ID NO:24 identical amino acid sequences.
  • the first dimerization domain comprises or is at least 70%, 80%, 90%, 95%, 96%, 97%, 98% identical to SEQ ID NO:23 or an amino acid sequence of 99% sequence identity
  • the second dimerization domain comprises or has at least 70%, 80%, 90%, 95%, 96% with SEQ ID NO:24 , 97%, 98%, or 99% sequence identity
  • the first dimerization domain comprises SEQ ID NO:24 or has at least 70%, 80%, 90% with SEQ ID NO:24 , 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences
  • said second dimerization domain comprises SEQ ID NO:23 or has at least 70% with SEQ ID NO:23 , 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences.
  • the polypeptide of interest is C-terminal to the first dimerization domain (ie, the first chain of the Fc region), and the binding domain is located in the first dimerization domain (ie, the first chain of the Fc region) C-terminus of the dimerization domain (ie the second chain of the Fc region).
  • the polypeptide of interest is N-terminal to the first dimerization domain (ie, the first chain of the Fc region), and the binding domain is located in the first dimerization domain (ie, the first chain of the Fc region) N-terminus of the dimerization domain (ie the second chain of the Fc region).
  • the polypeptide of interest is linked to the first dimerization domain by a linker, and the binding domain is linked to the second dimerization domain by a linker.
  • the linker is a flexible linker.
  • the linker has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids.
  • the linker has 1-20, 2-18, 3-16, 5-15, 6-12, or 8-10 amino acids.
  • the linker has the amino acid sequence GGGGSGGGGS (SEQ ID NO:30).
  • the polypeptide dimer of the invention comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the first chain of the Fc region of human IgG1 and circulating rearranged IL-2,
  • the circulating rearranged IL-2 is linked to the C-terminus of the first chain of the Fc region of human IgG1, wherein the second polypeptide comprises the second chain of the Fc region of human IgG1 and the CD25 extracellular domain,
  • the CD25 extracellular domain is linked to the C-terminus of the second chain of the Fc region of human IgG1, and wherein the circulating rearranged IL-2 is as previously described, eg, the circulating rearranged IL-2 consists of amino acids.
  • the sequence SEQ ID NO:21 consists of.
  • the first strand consists of the amino acid sequence of SEQ ID NO:23 and the second strand consists of the amino acid sequence of SEQ ID NO:24; or the first strand consists of the amino acid sequence of SEQ ID NO:24; 24, and the second strand consists of the amino acid sequence of SEQ ID NO: 23.
  • the polypeptide dimer of the invention comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the first chain of the Fc region of human IgG1 and circulating rearranged IL-15,
  • the circulating rearranged IL-15 is linked to the C-terminus of the first chain of the Fc region of human IgG1, wherein the second polypeptide comprises the second chain of the Fc region of human IgG1 and the CD215 extracellular domain,
  • the CD215 extracellular domain is linked to the C-terminus of the second chain of the Fc region of human IgG1, and wherein the circulating rearranged IL-15 is as previously described, eg, the circulating rearranged IL-15 comprises amino acids Sequence SEQ ID NO:22.
  • the first strand comprises the amino acid sequence of SEQ ID NO:23 and the second strand comprises the amino acid sequence of SEQ ID NO:24; or the first strand comprises the amino acid sequence of SEQ ID NO:24 amino acid sequence, and the second strand comprises the amino acid sequence of SEQ ID NO:23.
  • modifications can be made to the amino acid sequence of a cyclically rearranged polypeptide without reducing its biological activity. Such modifications are well known to those skilled in the art and include the addition of residues such as methionine at the amino terminus to provide an initiation site, or addition of additional amino acids at either terminus to protect the protein from exopeptidases of destruction.
  • amino acid substitutions such as conservative substitutions, can be made without affecting the activity of the protein.
  • non-essential regions of the molecule can be shortened or completely eliminated.
  • regions of the molecule that themselves are not involved in the activity of the molecule they can be eliminated or replaced by shorter fragments that serve only to maintain the correct spatial relationship between the active components of the molecule.
  • the circulating rearranged IL-2 comprises at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:21
  • the circulating rearranged IL-15 comprises at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:22 A specific amino acid sequence, wherein the first polypeptide comprises a native CD215 binding site, and the polypeptide dimer has comparable or improved activity to native IL-15.
  • the first polypeptide comprises the amino acid sequence of SEQ ID NO:25 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:26.
  • the first polypeptide comprises an amino acid having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:25 sequence
  • the second polypeptide comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 26, wherein the The first polypeptide comprises a native CD25 binding site, and the polypeptide dimer has comparable or improved activity to native IL-2.
  • the first polypeptide comprises the amino acid sequence of SEQ ID NO:27 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:28.
  • the first polypeptide comprises an amino acid having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:27 sequence
  • the second polypeptide comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 28, wherein the The first polypeptide comprises a native CD25 binding site, and the polypeptide dimer has comparable or improved activity to native IL-2.
  • the half-life of the polypeptide dimers of the invention is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% compared to native IL-2 , 100%, 150%, 200% or more.
  • the half-life of the polypeptide dimers of the invention is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% compared to native IL-15 , 100%, 150%, 200% or more.
  • the present invention also provides a polynucleotide encoding the fusion polypeptide of the present invention.
  • the present invention also provides polynucleotides encoding the polypeptide dimers of the present invention.
  • the first polypeptide and the second polypeptide are encoded by a single polynucleotide. In some embodiments, the first polypeptide and the second polypeptide are encoded by different polynucleotides.
  • Nucleic acid molecules of all or part of the nucleic acid sequences of the present invention can be isolated by polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based on the sequence information contained in the sequences.
  • PCR polymerase chain reaction
  • the polynucleotides of the present invention can be amplified according to standard PCR amplification techniques using cDNA, mRNA or genomic DNA as template and suitable oligonucleotide primers.
  • the nucleic acid thus amplified can be cloned into a suitable vector and characterized by DNA sequence analysis.
  • polynucleotides of the present invention can be prepared by standard synthetic techniques, eg, using an automated DNA synthesizer.
  • the present invention also relates to complementary strands of the nucleic acid molecules described herein.
  • a nucleic acid molecule that is complementary to another nucleotide sequence is one that is sufficiently complementary to that nucleotide sequence so that it can hybridize to the other nucleotide sequence to form a stable duplex.
  • polynucleotides of the present invention do not include polynucleotides that hybridize only to a poly A sequence (such as the 3' end poly(A) of mRNA) or to a complementary stretch of poly T (or U) residues.
  • the present invention also provides vectors, preferably expression vectors, comprising the polynucleotides of the present invention. Further, the present invention also provides host cells comprising the polynucleotides or vectors (preferably, expression vectors) of the present invention. In some embodiments, the polynucleotides of the invention integrate into the genome of the host cell. In some embodiments, the polynucleotides of the invention do not integrate into the genome of the host cell.
  • a single expression vector is used to express the first polypeptide and the second polypeptide of the fusion polypeptides of the invention.
  • different expression vectors are used to express the first polypeptide and the second polypeptide of the fusion polypeptides of the invention.
  • the choice of expression vector will depend on the host cell used to express the fusion polypeptide or polypeptide dimer.
  • Host cells that can be used to express the fusion polypeptides of the present invention include, but are not limited to, bacteria (including E. coli), yeast, insect cells, and mammalian cells, such as COS, CHO, HeLa, and 293-6E cells. Expression vectors suitable for use in various host cells are known in the art.
  • suitable expression vectors for bacteria include, but are not limited to, pET vectors (eg, pET-28a, pET-30a, pET-32a, and pET-40a, etc.), pEX vectors (eg, pEX-1), pGH112, pUC118, and pEZZ18;
  • Expression vectors for yeast include but are not limited to pESP vectors (such as pESP-1, pESP-2, pESP-3, etc.), pDR196, pHiSi, p53his, pSH47 and pYCP211; expression vectors suitable for insect cells include but are not limited to pCoBlast, pIEX/Bac-3, pIEXBac-c-EGFP-4, pFastBac1-His-C, pIEXBac-c-EGFP-3, pFastBac1-GST-N and pIEXBac-c-EGFP-2;
  • the host cell is a 293-6E cell.
  • the expression vector is pcDNA3.4.
  • the polynucleotides of the present invention further comprise a sequence encoding a signal peptide.
  • the signal peptide comprises the amino acid sequence of METDTLLLWVLLLWVPGSTG (SEQ ID NO: 18).
  • the polynucleotides or vectors of the invention can be transferred (transfected) into selected host cells by methods known in the art, such as calcium chloride transformation and calcium phosphate treatment for E. coli, electroporation, lipofection Dysamine treatment or PEI treatment for mammalian cells.
  • Cells transformed with the vector can be screened for antibiotic resistance genes such as amp, gpt, neo and hyg genes in the vector.
  • recombinant fusion proteins can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity chromatography, column chromatography using ionic or hydrophobic resins, gel electrophoresis, and the like. Substantially pure compositions with a purity of at least about 90 to 95% are preferred, and 98 to 99% or higher are most preferred for pharmaceutical use.
  • the fusion polypeptide of the present invention may also contain a tag sequence, including but not limited to His6 tag, FLAG tag and the like.
  • fusion polypeptides of the invention comprise the amino acid sequence of one of SEQ ID NOs: 12-15. In some embodiments, fusion polypeptides of the invention comprise at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence of one of SEQ ID NOs: 12-15 The amino acid sequence of sequence identity, wherein the fusion polypeptide comprises a native CD25 binding site, which has comparable or improved activity to native IL-2, such as the activity of activating JAK1/JAK3 and STAT3/STAT5 signaling pathways.
  • the polynucleotides or vectors of the invention encode the amino acid sequence of SEQ ID NO:31, and the amino acid sequence of SEQ ID NO:32.
  • the polynucleotide or vector of the invention such as an expression vector, encodes at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of SEQ ID NO:31 Amino acid sequences of sequence identity and amino acid sequences having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 32, wherein the expressed
  • the first polypeptide comprises a native CD25 binding site, and the polypeptide dimer has comparable or improved activity to native IL-2, such as an activity to activate JAK1/JAK3 and STAT3/STAT5 signaling pathways.
  • the polynucleotides or vectors of the invention encode the amino acid sequence of SEQ ID NO:33 and the amino acid sequence of SEQ ID NO:34.
  • the polynucleotides or vectors of the invention encode at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:33 Amino acid sequences of sequence identity and amino acid sequences having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 34, wherein the expressed
  • the first polypeptide comprises a native CD25 binding site, and the polypeptide dimer has comparable or improved activity to native IL-2, such as an activity to activate JAK1/JAK3 and STAT3/STAT5 signaling pathways.
  • the present invention provides pharmaceutical compositions comprising the fusion polypeptides of the present invention.
  • the pharmaceutical composition comprises a fusion polypeptide of the present invention and at least one pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be prepared by combining the fusion polypeptide of the present invention with a pharmaceutically acceptable carrier according to known methods.
  • the present invention also provides pharmaceutical compositions comprising the polypeptide dimers of the present invention.
  • the pharmaceutical composition comprises the polypeptide dimer of the present invention and at least one pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be prepared by combining the polypeptide dimer of the present invention with a pharmaceutically acceptable carrier according to known methods.
  • Pharmaceutically acceptable carriers include, but are not limited to, solvents, emulsifiers, buffers, stabilizers, and the like.
  • the solvent includes water, aqueous solutions, non-aqueous solvents (eg vegetable oils).
  • compositions of the present invention may be administered by any suitable route, including subcutaneous, intramuscular, intraarticular, intravenous, intradermal, intraperitoneal, intranasal, intracranial, parenteral administration.
  • the pharmaceutical composition of the present invention is administered intravenously. It will be appreciated that the route of administration may vary with the therapeutic agent, the condition and age of the recipient, and the disease being treated.
  • compositions of the present invention may be in the form of solutions or lyophilized formulations.
  • the pharmaceutical compositions of the present invention are provided as lyophilized powders for reconstitution prior to administration.
  • the pharmaceutical compositions of the present invention can also be provided in liquid form, which can be administered directly to a patient.
  • the composition is provided in a prefilled syringe in liquid form.
  • compositions of the present invention are encapsulated in liposomes.
  • the liposomes can be coated with a flexible water-soluble polymer that avoids uptake by the organs of the mononuclear phagocyte system, primarily the liver and spleen.
  • Hydrophilic polymers suitable for coating liposomes include, but are not limited to, PEG, polyvinylpyrrolidone, polyvinylmethyl ether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl oxazoline oxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethyl cellulose hydroxyethyl amide, hydrophilic polyvinyl alcohol, etc.
  • compositions of the present invention may be administered one or more times, depending on the dosage and frequency required and tolerated by the patient.
  • the administered pharmaceutical composition should provide a sufficient amount of the protein of the invention to effectively treat the patient.
  • the present invention also provides methods of treating diseases, such as diseases involving immunosuppression, using the fusion polypeptides of the present invention.
  • a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a fusion polypeptide or pharmaceutical composition of the invention.
  • cancers include, but are not limited to, lung cancer, liver cancer, kidney cancer, head and neck cancer, colorectal cancer, gastric cancer, nasopharyngeal cancer, glioma, melanoma, and osteosarcoma.
  • a method of activating immune cells or increasing the proliferation of immune cells comprising administering to a subject in need thereof an effective amount of a fusion polypeptide of the present invention or a pharmaceutical composition of the present invention.
  • the immune cells are T cells or NK cells.
  • a fusion polypeptide or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of cancer.
  • cancers include, but are not limited to, lung cancer, liver cancer, kidney cancer, head and neck cancer, colorectal cancer, gastric cancer, nasopharyngeal cancer, glioma, melanoma, and osteosarcoma.
  • a fusion polypeptide or pharmaceutical composition of the invention in the manufacture of a medicament for activating immune cells or increasing the proliferation of immune cells.
  • the immune cells are T cells or NK cells.
  • the present invention also provides methods of treating diseases, such as diseases involving immunosuppression, using the polypeptide dimers of the invention.
  • a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide dimer or pharmaceutical composition of the invention.
  • cancers include, but are not limited to, lung cancer, liver cancer, kidney cancer, head and neck cancer, colorectal cancer, gastric cancer, nasopharyngeal cancer, glioma, melanoma, and osteosarcoma.
  • a method of activating immune cells or increasing the proliferation of immune cells comprising administering to a subject in need thereof an effective amount of a polypeptide dimer of the present invention or a pharmaceutical composition of the present invention.
  • the immune cells are T cells or NK cells.
  • a polypeptide dimer or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of cancer.
  • cancers include, but are not limited to, lung cancer, liver cancer, kidney cancer, head and neck cancer, colorectal cancer, gastric cancer, nasopharyngeal cancer, glioma, melanoma, and osteosarcoma.
  • the immune cells are T cells or NK cells.
  • a polypeptide dimer comprising a first polypeptide and a second polypeptide
  • the first polypeptide comprises a first dimerization domain and a polypeptide of interest, wherein the polypeptide of interest is located at the first end of the first dimerization domain,
  • the second polypeptide comprises a second dimerization domain and a binding domain, wherein the binding domain is located at the first end of the second dimerization domain
  • first polypeptide and the second polypeptide form a dimer through the first dimerization domain and the second dimerization domain, and the first dimerization domain of the first dimerization domain The end is adjacent to the first end of the second dimerization domain, and the binding domain is capable of binding to the site of interest on the polypeptide of interest.
  • polypeptide dimer of embodiment P1 wherein the polypeptide of interest is derived from a cytokine of the family of four alpha-helical bundles of cytokines comprising, in order from N-terminus to C-terminus, helical bundle 1 (H1 ), helical bundle 2 (H2), helical bundle 3 (H3) and four ⁇ -helix bundles of helical bundle 4 (H4).
  • polypeptide dimer of embodiment P2 wherein the polypeptide of interest is a cytokine of the cyclically rearranged four alpha-helix bundle cytokine family comprising H2, H3, H4 from N-terminus to C-terminus and H1; H3, H4, H1 and H2; or the four alpha-helical bundles of H4, H1, H2 and H3.
  • polypeptide dimer of embodiment P3 wherein the amino acid corresponding to the N-terminus of the native cytokine in the circulating rearranged cytokine is linked by a linker to the amino acid corresponding to the C-terminus of the native cytokine.
  • polypeptide dimer of embodiment P4 wherein the linker is a GS linker or a polyglycine linker of 1-10 amino acids in length.
  • polypeptide dimer of embodiment P6 wherein the cyclically rearranged IL-2 comprises four alphas in order from N-terminus to C-terminus H3, H4, H1 and H2, or H4, H1, H2 and H3 - Helical bundles.
  • polypeptide dimer of embodiment P7, wherein the circulating rearranged IL-2 comprises the amino acid sequence of SEQ ID NO:21.
  • polypeptide dimer of embodiment P6, wherein the cyclically rearranged IL-15 comprises four alpha-helical bundles in sequence from N-terminus to C-terminus H3, H4, H1 and H2.
  • polypeptide dimer of embodiment P10, wherein the circulating rearranged IL-15 comprises the amino acid sequence of SEQ ID NO:4.
  • polypeptide dimer of embodiment P14, wherein the Ig is human IgGl.
  • P16 The polypeptide dimer of any one of embodiments P1-P15, wherein the first dimerization domain and the second dimerization domain form the Fc region of human IgG1.
  • P17 The polypeptide dimer of any one of embodiments P1-P16, wherein the first dimerization domain comprises the amino acid sequence of SEQ ID NO: 21, and the second dimerization domain comprises SEQ ID NO: : the amino acid sequence of 22; or the first dimerization domain comprises the amino acid sequence of SEQ ID NO:22, and the second dimerization domain comprises the amino acid sequence of SEQ ID NO:21.
  • P18 The polypeptide dimer of any one of embodiments P13-P17, wherein the first end of the first dimerization domain is C-terminal and the first end of the second dimerization domain is C-terminal end.
  • a polypeptide dimer comprising a first polypeptide and a second polypeptide
  • the first polypeptide comprises the first chain of the Fc region of human IgG1 and circulating rearranged IL-2, the circulating rearranged IL-2 is C-terminal to the first chain of the Fc region of the human IgG1 connect,
  • the second polypeptide comprises the second chain of the Fc region of human IgG1 and the CD25 extracellular domain, the CD25 extracellular domain is linked to the C-terminus of the second chain of the Fc region of human IgG1, and
  • the cyclically rearranged IL-2 comprises four ⁇ -helix bundles which are H3, H4, H1 and H2; or H4, H1, H2 and H3 in order from the N-terminus to the C-terminus.
  • polypeptide dimer of embodiment P19, wherein the circulating rearranged IL-2 comprises the amino acid sequence of SEQ ID NO:2.
  • a polypeptide dimer comprising a first polypeptide and a second polypeptide
  • the first polypeptide comprises the first chain of the Fc region of human IgG1 and circulating rearranged IL-15, the circulating rearranged IL-15 and the C-terminus of the first chain of the Fc region of the human IgG1 connect,
  • the second polypeptide comprises the second chain of the Fc region of human IgG1 and the CD215 extracellular domain, the CD215 extracellular domain is linked to the C-terminus of the second chain of the Fc region of human IgG1, and
  • the cyclically rearranged IL-15 comprises four ⁇ -helix bundles from N-terminus to C-terminus, H3, H4, H1 and H2 in sequence.
  • polypeptide dimer of embodiment P21, wherein the circulating rearranged IL-15 comprises the amino acid sequence of SEQ ID NO:22.
  • a pharmaceutical composition comprising the polypeptide dimer of any one of embodiments P1-P23.
  • P26 Use of the polypeptide dimer of any one of embodiments P1-P23 in the manufacture of a medicament for activating immune cells or increasing the proliferation of immune cells.
  • P28 One or more isolated polynucleotides encoding the polypeptide dimer of any one of embodiments P1-P23.
  • One or more expression vectors comprising the polynucleotide of embodiment P28.
  • a host cell comprising the polynucleotide of embodiment P28 or the vector of embodiment P29.
  • Embodiment 1 the construction of expression vector
  • the construction of the expression vector was completed by Nanjing GenScript Biotechnology Co., Ltd., including: synthesizing nucleic acids encoding the amino acid sequences of SEQ ID NOs: 12-15 and 31-34, the nucleic acids additionally comprising a signal peptide encoding SEQ ID NO: 18
  • the nucleic acid is constructed into mammalian cell expression vector pcDNA 3.4 by molecular cloning method, as well as plasmid amplification and purification, etc.
  • 293-6E cells were transfected with nucleic acids encoding fusion polypeptides for eukaryotic expression.
  • the expression and purification of fusion polypeptides were completed by Nanjing GenScript Biotechnology Co., Ltd. The process is briefly described as follows:
  • -293-6E cells were grown in serum-free FreeStyle TM 293 Expression medium (Thermo Fisher Scientific, Carlsbad, CA, USA) in Erlenmeyer Erlenmeyer flasks (Corning Inc., Acton, MA) in a shaking incubator ( Cultures were performed on VWR Scientific, Chester, PA) at 37°C and 5% CO 2 .
  • the plasmid mixture (mass ratio of plasmids encoding the first and second polypeptides is 1:1) and transfection reagents (such as polyetherimide, Polyetherimide, PEI) in a suitable ratio (such as 1 : 3 mass ratio) were mixed in the medium, and then added to the cell culture medium for transfection, and the target protein was secreted and expressed.
  • transfection reagents such as polyetherimide, Polyetherimide, PEI
  • the cell survival rate is about 50%-80%, and the supernatant is obtained by centrifugation, which contains the target protein (the expressed polypeptide does not contain the signal peptide).
  • the molecular weight, purity and sequence coverage of the purified proteins were analyzed by SDS-PAGE, Western blotting (results not shown), high performance liquid chromatography coupled with molecular sieves (SEC-HPLC) and liquid chromatography mass spectrometry (LC-MS) methods evaluated.
  • the primary antibody used in western blotting was mouse anti-His-tag antibody (GenScript, cat. No. A00186) or mouse anti-FLAG antibody (GenScript, cat. No. A00187), and the secondary antibody was horseradish peroxidase-modified goat anti-mouse IgG antibody ( GenScript, Cat. No. A00160).
  • Figure 4 Opening of IL-2 in loop 1 of IL-2 to form a cyclic rearrangement, the insertion site is at C361 of HSA (SEQ ID NO: 12);
  • Figure 5 In IL-2 Cyclic rearranged IL-2 is opened in loop 2 of -2, and the insertion site is at D56 of HSA (SEQ ID NO: 13);
  • Figure 6 Cyclic rearranged IL-2 is opened in loop 2 of IL-2 2, the insertion site is at A300 of HSA (SEQ ID NO: 14);
  • Figure 7 Opening of IL-2 in loop 1 of IL-2 to form a circular rearrangement, the insertion site is at A362 of HSA (SEQ ID NO: 14); :15).
  • Figure 8 Circulating rearranged IL-2 fused to the knob chain of the knob-in-hole modified human IgG1 Fc region (SEQ ID NO: 31), the extracellular domain of CD25 was Knob-hole modified human IgG1 Fc region hole chain fusion (SEQ ID NO: 32);
  • Figure 9 Circulating rearranged IL-2 fused to a knob-hole modified human IgG1 Fc region hole chain fusion (SEQ ID NO: 32) NO: 33), the extracellular domain of CD25 was fused to the knob chain of the knob-hole structure-modified human IgG1 Fc region (SEQ ID NO: 34).
  • Panel A shows a structural model of HSA shielding the CD25 binding site on IL-2
  • Panel B shows the SDS-PAGE results of reduced and non-reduced fusion polypeptides
  • Panel C shows the results of molecular sieve analysis.
  • Panel A shows the structural model of the polysaccharide dimer of the present invention
  • Panel B shows the SDS-PAGE results of reduced and non-reduced polypeptide dimers
  • Panel C shows the results of molecular sieve analysis.
  • HEK-Blue TM IL-2 cell line (InvivoGen) was used to detect the activity of these fusion proteins in activating downstream signaling pathways at the cellular level.
  • the HEK-Blue TM IL-2 cell line is constructed by stably transfecting human embryonic kidney 293 cells (HEK-293) with genes encoding molecules required for the IL-2 signaling pathway, including CD25, CD122, and CD132
  • the receptor molecule, the human JAK3 kinase, and the transcription factor STAT5 were additionally transferred into the STAT5-regulated secreted embryonic alkaline phosphatase (SEAP) reporter gene.
  • SEAP STAT5-regulated secreted embryonic alkaline phosphatase
  • Serum inactivation Take 45mL of serum (FBS) into a 50mL centrifuge tube, heat inactivate it in a 56°C water bath for 30min (mixing once every 10min), and temporarily store it at 4°C.
  • FBS serum
  • Growth medium DMEM+10%FBS+1%PS+100 ⁇ g/mL Normocin+1 ⁇ g/ml puromycin+1X HEK-Blue TM CLR Selection; test medium: DMEM+10%FBS (inactivated)+1%PS+ 100 ⁇ g/mL Normocin; freezing medium: DMEM+20%FBS+10%DMSO.
  • the cells were placed in growth medium, cultured and passaged at 37 °C, 5% CO 2 incubator;
  • HEK-Blue IL-2 cell suspension gently rinse the cells twice with pre-warmed DPBS, and resuspend in pre-warmed DPBS to form a single cell suspension. After counting the cells, centrifuge at 200 ⁇ g for 5 min; Resuspend in pre-warmed test medium at a cell density of 2.8 ⁇ 10 5 cells/mL;
  • Human IL-2 protein positive control, AcroBiosystems, Beijing Baipsis Biotechnology Co., Ltd., product number IL2-H4113
  • HSA negative control
  • human IgG1 Fc segment negative control, Beijing Baipsis Biosystems
  • step 3 was used to detect the activity of the fusion polypeptide and polypeptide dimer prepared in Example 2, wherein each fusion polypeptide was also prepared serially diluted samples according to the method in step 3.2.
  • Figure 10 Opening of IL-2 in loop 1 of IL-2 to form a cyclic rearrangement, the insertion site is at C361 of HSA (SEQ ID NO: 12);
  • Figure 11 In IL-2 Cyclic rearranged IL-2 is opened in loop 2 of -2, and the insertion site is at D56 of HSA (SEQ ID NO: 13);
  • Figure 12 Cyclic rearranged IL-2 is opened in loop 2 of IL-2 2, the insertion site is at A300 of HSA (SEQ ID NO: 14);
  • Figure 13 Opening of IL-2 forming a circular rearrangement in loop 1 of IL-2, the insertion site is at A362 of HSA (SEQ ID NO: 14); :15).
  • the first polypeptide comprises a knob chain fusion of circulating rearranged IL-2 to a knob-hole modified human IgG1 Fc region (SEQ ID NO: 31), the second polypeptide comprises the extracellular domain of CD25 and Hole chain fusion of a knob-and-hole modified human IgG1 Fc region (SEQ ID NO: 32);
  • Figure 15 The first polypeptide comprises a hole chain fusion of a circularly rearranged IL-2 to a knob-and-hole modified human IgG1 Fc region (SEQ ID NO: 32) SEQ ID NO: 33), the second polypeptide comprises the extracellular domain of CD25 fused to a knob-and-hole structure-modified human IgG1 Fc region knob chain (SEQ ID NO: 34).
  • the interaction between the fusion polypeptide or polypeptide dimer of the present invention and the IL-2 receptor was measured using a high-throughput molecular interaction instrument based on the principle of surface plasmon resonance imaging (SPRi).
  • CD25+CD122 CD25+CD122
  • CD122+CD132 CD122+CD132
  • CD25+CD122+CD132 CD25+CD122+CD132.
  • B16F10 melanoma cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium + 10% (v/v) fetal bovine serum (Fetal Bovine Serum, FBS).
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS Fetal Bovine Serum
  • mice C57BL/6 mice were adaptively fed for 1 week, injected subcutaneously with B16F10 melanoma cells (1x10 6 each), and the mice were fed for 7 days to allow tumor growth.
  • the tumor-implanted mice were divided into six groups (9 mice in each group), and the fusion polypeptide or polypeptide dimer molecule described in the table and blank control (PBS) were injected into the tail vein respectively.
  • the fusion polypeptide or polypeptide dimer molecule was injected at a dose of 2 mg/kg body weight, once every nine days for a total of three injections (D0, D9 and D18), and the body weight and tumor size of each mouse were measured every three days ( length and width) to calculate tumor volume.
  • the surviving mice were sacrificed 27 days after the first injection, the tumors were photographed, and the tumors were embedded in wax for pathological sectioning for subsequent immunohistochemical experiments.
  • the immunostaining blocking solution was blocked at room temperature for 15 minutes. After blocking, the blocking solution was removed without washing, and anti-CD8 primary antibody (dilution ratio 1:2000) was added to incubate, and incubated at room temperature for 1 h;
  • nuclei were counterstained with hematoxylin for 2 min, tap water returned to blue, dehydrated with gradient concentration ethanol, cleared with xylene, and sealed with neutral gum.
  • Average Optical Density (IOD/area) Cumulative Optical Density Value/Measured Area of Stained Area.
  • the fusion polypeptide or polypeptide dimer molecule of the present invention has good in vivo tolerance potential, which proves the safety of the fusion polypeptide or polypeptide dimer molecule of the present invention.
  • CD8 was observed by immunohistochemical staining of tumor tissue sections to detect the infiltration of CD8+ T cells (killer T cells) in the tumor.
  • CD8+ T cells killer T cells
  • Figure 17A the degree of infiltration of CD8+ T cells induced by these molecules showed a high correlation with their anti-tumor activity (see Figures 16A and B), and the higher the degree of CD8+ T cell infiltration, the greater the tumor growth.
  • Figure 17B is the immunohistochemical staining photos of some representative tumor sections to more intuitively show the infiltration of CD8+ T cells in tumor tissues under the intervention of these different molecules, in which blue is the nucleus, and brown is the expression of CD8 Signal, it can be clearly observed that ZJ-15 molecule induces a large number of CD8+ T cells into tumor tissue for killing and inhibition.

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Abstract

本发明提供了一种融合多肽，其包含载体蛋白和感兴趣的多肽，其中所述载体蛋白具有由环连接的多个螺旋结构域，所述感兴趣的多肽插入所述载体蛋白的环中，所述载体蛋白遮蔽所述感兴趣的多肽上的感兴趣的位点，从而阻断所述位点的可及性。本发明还提供了多肽二聚体，其包含第一多肽和第二多肽，其中所述第一多肽包含第一二聚化结构域和感兴趣的多肽，所述第二多肽包含第二二聚化结构域和结合结构域，所述结合结构域能够与所述感兴趣的多肽上的感兴趣的位点结合。本发明还提供了编码所述融合多肽和多肽二聚体的多核苷酸和载体，以及包含所述多核苷酸和/或载体的宿主细胞。进一步，本发明还提供了本发明的融合多肽和多肽二聚体在治疗癌症和/或激活免疫细胞中的用途。

Description

融合多肽和多肽二聚体及其用途 技术领域
本发明涉及融合多肽和多肽二聚体。具体地，本发明涉及经修饰的免疫调节分子及其在治疗癌症中的用途。
背景技术
通过细胞因子调节免疫系统，例如激活免疫细胞，是癌症免疫治疗的一种策略。然而，细胞因子上的某些位点对其功能(如免疫调节作用)有影响。
例如，白介素-2(IL-2)可以调控免疫细胞的功能。IL-2与受体IL2Rα(CD25)，IL2Rβ(CD122)和IL2Rγ(CD132)结合后，能够激活下游信号通路，包括JAK1与JAK3激酶以及转录因子STAT5，以刺激免疫细胞(比如T细胞与天然杀伤(NK)细胞)的活化与增殖。IL-2既可以通过CD25/CD122/CD132三聚体进行信号转导，也可以通过CD122/CD132二聚体激活信号通络。CD25与IL-2结合后会改变IL-2的构象，使其对CD122/CD132二聚体的亲和力提高100倍(KD值从1nM提高到10pM)。CD122/CD132二聚体主要表达在CD8+记忆T细胞以及NK细胞表面，而CD25/CD122/CD132三聚体则大量表达在起免疫抑制作用的调节T细胞(Treg)表面。因此，IL-2的作用是，根据激活不同的免疫细胞类型，来刺激或者抑制免疫系统的活化。
IL-2同时也是一类备受关注的癌症免疫疗法的潜在药物。重组的IL-2(阿地白介素)于1998年被美国食品药品卫生监督管理局(FDA)批准用于治疗转移性黑色素瘤和肾癌，是迄今为止唯一被批准的基于IL-2的药物，然而只有10％的患者有响应，且其副作用大，这主要是因为IL-2的双面性，需要较大的剂量才能刺激免疫激活，但剂量较高时又会引起如毛细血管渗漏综合征等副作用。
白介素-15(IL-15)也可以激活免疫反应，对于T细胞与NK细胞的分化与增殖、以及树突细胞的发育有着重要作用。IL-15与IL-2的工作机理有类似之处，通过结合CD122/CD132受体二聚体来激活下游信号通路(JAK1/JAK3与STAT3/STAT5)，但其α受体与IL-2不同，是其特有的IL-15Rα(CD215)受体。一般认为IL-15在结合细胞膜上的IL-15Rα受体后，被“反式”(trans，细胞-细胞接触)或者“顺式”(cis，同一个细胞上)以高亲和力递呈到CD122/CD132受体上，也有发现IL-15在不结合IL-15Rα的情况下，也可以以中等亲和力结合CD122/CD132并发挥功能。
最近的文献报导(Alexandra Berger等2019 J for ImmunoTherapy for Cancer)，单独的IL-15与IL-15/IL-15Rα受体胞外区复合物相比，在小鼠肿瘤模型中显示出了更耐久的抗肿瘤活性。申请人认为，这可能是因为复合物虽然能快速强效的刺激免疫，但也会更快的引起免疫细胞耗竭，降低反应耐久性。本领域技术人员也知晓，强烈的免疫刺激可能 会带来安全性的问题。
此外，细胞因子，如IL-2和IL-15的体内半衰期短，需要持续注射。
因此，仍然需要开发经工程化的重组细胞因子如IL-2和IL-15，其能够特异性激活免疫并具有延长的半衰期，具有改进的活性，具有更持久的激活免疫的能力，和/或具有更高的安全性，从而提高其临床应用与商业转化价值。
发明内容
在第一方面，本发明提供一种融合多肽，其包含载体蛋白和感兴趣的多肽，其中所述载体蛋白具有由环连接的多个螺旋结构域，所述感兴趣的多肽插入所述载体蛋白的环中，所述载体蛋白遮蔽所述感兴趣的多肽上的感兴趣的位点，从而阻断所述位点的可及性。
在一些实施方案中，所述感兴趣的多肽衍生自四α-螺旋束细胞因子家族的细胞因子，所述细胞因子包含从N端到C端依次为螺旋束1(H1)、螺旋束2(H2)、螺旋束3(H3)和螺旋束4(H4)的四个α-螺旋束。
在一些实施方案中，所述感兴趣的多肽是循环重排的四α-螺旋束细胞因子家族的细胞因子，其包含从N端到C端依次为H2、H3、H4和H1；H3、H4、H1和H2；或H4、H1、H2和H3的四个α-螺旋束。
在一些实施方案中，所述循环重排的细胞因子中对应于未经循环重排的细胞因子的N端的氨基酸通过接头与对应于未经循环重排的细胞因子的C端的氨基酸连接。在一些实施方案中，所述接头是长度为1-10个氨基酸的GS接头或多聚甘氨酸接头。
在一些实施方案中，所述感兴趣的多肽选自循环重排的IL-2和循环重排的IL-15。
在一些实施方案中，所述循环重排的IL-2包含从N端到C端依次为H3、H4、H1和H2，或H4、H1、H2和H3的四个α-螺旋束。在一些实施方案中，所述循环重排的IL-2包含SEQ ID NO:2、3、4或5的氨基酸序列。在一些实施方案中，所述感兴趣的位点是CD25结合位点。
在一些实施方案中，所述循环重排的IL-15包含从N端到C端依次为H3、H4、H1和H2的四个α-螺旋束。在一些实施方案中，所述循环重排的IL-15包含SEQ ID NO:7的氨基酸序列。在一些实施方案中，所述感兴趣的位点是CD215结合位点。
在一些实施方案中，所述载体蛋白是白蛋白，优选人血清白蛋白(HSA)。在一些实施方案中，所述环选自位于D56-L66、A92-P96、D129-E131、Q170-A172、K281-L283、V293-L305、E311-S312、E321-A322、A362-D365、L398-E400、K439-R445、E465-D471、P537-E542和A561-T566的环，所述位置参照SEQ ID NO:16进行编号。在一些实施方案中，所述感兴趣的多肽是循环重排的IL-2，所述环选自位于HSA的D56-L66、V293-L305和A362-D365的环。在一些实施方案中，所述感兴趣的多肽的插入位点选自HSA的D56、A300、C361和A362。
在一些实施方案中，本发明提供一种融合多肽，其包含载体蛋白HSA和循环重排 的IL-2，其中所述循环重排的IL-2包含从N端到C端依次为H3、H4、H1和H2，或H4、H1、H2和H3的四个α-螺旋束，且其中所述循环重排的IL-2插入HSA的环中，所述环选自位于D56-L66、A92-P96、D129-E131、Q170-A172、K281-L283、V293-L305、E311-S312、E321-A322、A362-D365、L398-E400、K439-R445、E465-D471、P537-E542、A561-T566的环，所述位置参照SEQ ID NO:16进行编号，所述HSA遮蔽所述循环重排的IL-2的CD25结合位点，从而阻断所述位点的可及性。在一些实施方案中，所述循环重排的IL-2包含SEQ ID NO:2、3、4或5的氨基酸序列。在一些实施方案中，所述环选自位于HSA的D56-L66、V293-L305和A362-D365的环。在一些实施方案中，所述循环重排的IL-2的插入位点选自HSA的D56、A300、C361和A362。
在一些实施方案中，本发明提供一种融合多肽，其包含载体蛋白HSA和循环重排的IL-15，其中所述循环重排的IL-15包含从N端到C端依次为H3、H4、H1和H2的四个α-螺旋束，且其中所述循环重排的IL-15插入HSA的环中，所述环选自位于D56-L66、A92-P96、D129-E131、Q170-A172、K281-L283、V293-L305、E311-SS312、E321-A322、A362-D365、L398-E400、K439-R445、E465-D471、P537-E542和A561-T566的环，所述位置参照SEQ ID NO:16进行编号，所述HSA遮蔽所述所述循环重排的IL-15的CD215结合位点，从而阻断所述位点的可及性。在一些实施方案中，所述循环重排的IL-15包含SEQ ID NO:7的氨基酸序列。
在一些实施方案中，本发明提供一种融合多肽，其包含SEQ ID NO:8-11之一的氨基酸序列。
本发明还提供包含本发明的融合多肽的药物组合物。
在第二方面，本发明提供一种多肽二聚体，其包含第一多肽和第二多肽，
其中所述第一多肽包含第一二聚化结构域和感兴趣的多肽，其中所述感兴趣的多肽位于所述第一二聚化结构域的第一端，
其中所述第二多肽包含第二二聚化结构域和结合结构域，其中所述结合结构域位于所述第二二聚化结构域的第一端，
其中所述第一多肽与所述第二多肽通过所述第一二聚化结构域和第二二聚化结构域形成二聚体，且所述第一二聚化结构域的第一端与所述第二二聚化结构域的第一端邻近，所述结合结构域能够与所述感兴趣的多肽上的感兴趣的位点结合。
在一些实施方案中，所述感兴趣的多肽衍生自四α-螺旋束细胞因子家族的细胞因子，所述细胞因子包含从N端到C端依次为螺旋束1(H1)、螺旋束2(H2)、螺旋束3(H3)和螺旋束4(H4)的四个α-螺旋束。在一些实施方案中，所述感兴趣的多肽是循环重排的四α-螺旋束细胞因子家族的细胞因子，其包含从N端到C端依次为H2、H3、H4和H1；H3、H4、H1和H2；或H4、H1、H2和H3的四个α-螺旋束。
在一些实施方案中，所述循环重排的细胞因子中对应于天然细胞因子的N端的氨基酸通过接头与对应于天然细胞因子的C端的氨基酸连接。在一些实施方案中，所述接头是长度为1-10个氨基酸的GS接头或多聚甘氨酸接头。
在一些实施方案中，所述感兴趣的多肽是循环重排的IL-2或IL-15。
在一些实施方案中，所述循环重排的IL-2包含从N端到C端依次为H3、H4、H1和H2，或H4、H1、H2和H3的四个α-螺旋束。在一些实施方案中，所述循环重排的IL-2包含SEQ ID NO:21的氨基酸序列。在一些实施方案中，所述感兴趣的位点是CD25结合位点，所述结合结构域是CD25的胞外结构域。
在一些实施方案中，所述循环重排的IL-15包含从N端到C端依次为H3、H4、H1和H2的四个α-螺旋束。在一些实施方案中，所述循环重排的IL-15包含SEQ ID NO:22的氨基酸序列。在一些实施方案中，所述感兴趣的位点是CD215结合位点，所述结合结构域是CD215的胞外结构域。
在一些实施方案中，所述第一和第二二聚化结构域包含免疫球蛋白(Ig)的重链恒定区CH2和CH3。在一些实施方案中，所述Ig是人Ig，例如人IgG1。在一些实施方案中，所述第一二聚化结构域与第二二聚化结构域形成人IgG1的Fc区。在一些实施方案中，所述人IgG1的Fc区是经修饰的。在一些实施方案中，所述第一二聚化结构域包含SEQ ID NO:23的氨基酸序列，且所述第二二聚化结构域包含SEQ ID NO:24的氨基酸序列；或所述第一二聚化结构域包含SEQ ID NO:24的氨基酸序列，且所述第二二聚化结构域包含SEQ ID NO:23的氨基酸序列。
在一些实施方案中，所述第一二聚化结构域的第一端是C端，且所述第二二聚化结构域的第一端是C端。
在一些实施方案中，本发明的多肽二聚体包含第一多肽和第二多肽，
其中所述第一多肽包含人IgG1的Fc区的第一链和循环重排的IL-2，所述循环重排的IL-2与所述人IgG1的Fc区的第一链的C端连接，
其中所述第二多肽包含人IgG1的Fc区的第二链和CD25胞外结构域，所述CD25胞外结构域与人IgG1的Fc区的第二链的C端连接，且
其中所述循环重排的IL-2包含从N端到C端依次为H3、H4、H1和H2；或H4、H1、H2和H3的四个α-螺旋束。
在一些实施方案中，所述循环重排的IL-2包含SEQ ID NO:21的氨基酸序列。
在一些实施方案中，本发明的多肽二聚体包含第一多肽和第二多肽，
其中所述第一多肽包含人IgG1的Fc区的第一链和循环重排的IL-15，所述循环重排的IL-15与所述人IgG1的Fc区的第一链的C端连接，
其中所述第二多肽包含人IgG1的Fc区的第二链和CD215胞外结构域，所述CD215胞外结构域与人IgG1的Fc区的第二链的C端连接，且
其中所述循环重排的IL-15包含从N端到C端依次为H3、H4、H1和H2的四个α-螺旋束。
在一些实施方案中，所述循环重排的IL-15包含SEQ ID NO:22的氨基酸序列。
在一些实施方案中，所述第一链包含SEQ ID NO:23的氨基酸序列，且所述第二链包含SEQ ID NO:24的氨基酸序列；或所述第一链包含SEQ ID NO:24的氨基酸序列， 且所述第二链包含SEQ ID NO:23的氨基酸序列。
本发明还提供包含本发明的多肽二聚体的药物组合物。
在第三方面，本发明还提供一种治疗癌症或激活免疫细胞或增加免疫细胞(例如T细胞或NK细胞)的增殖的方法，包括给有需要的对象施用有效量的本发明的融合多肽、多肽二聚体或本发明的药物组合物。
本发明还提供本发明的融合多肽、多肽二聚体或药物组合物在制备用于治疗癌症的药物中的用途以及在制备用于激活免疫细胞或增加免疫细胞(例如T细胞或NK细胞)的增殖的药物中的用途。
在一些实施方案中，还提供本发明的融合多肽、多肽二聚体或药物组合物，其用于治疗癌症或激活免疫细胞或增加免疫细胞(例如T细胞或NK细胞)的增殖。
在第三方面，本发明还提供编码本发明的融合多肽或多肽二聚体的多核苷酸和载体，以及包含所述多核苷酸和载体的宿主细胞。
附图说明
图1显示野生型IL-2与其受体结合的三维结构示意图(基于PDB号2ERJ)，其中“环1”对应于天然IL-2的S95-L100，“环2”对应于天然IL-2的N50-P54。除非另有说明，本文中涉及IL-2的位置都参照SEQ ID NO:1(UniProt P60568)的IL-2前体序列编号，其中氨基酸残基1-21是信号肽序列，天然IL-2的序列是SEQ ID NO:1的氨基酸残基22-153。
图2显示野生型IL-15与其受体结合的三维结构示意图(基于PDB号4GS7)，其中“被打开的环”对应于天然IL-15的S102-A105。除非另有说明，本文中涉及IL-15的位置都参照SEQ ID NO:6(UniProt P40933)的IL-15前体序列编号，其中残基1-48是信号肽，天然IL-15的序列是SEQ ID NO:6的氨基酸残基49-162。
图3显示HSA的三维结构示意图。
图4-7显示本发明的融合多肽的表达以及HSA遮蔽IL-2上的CD25结合位点的结构模型。
图8和9显示本发明的多肽二聚体的结构模型及其表达，循环重排的IL-2的氨基酸序列如SEQ ID NO:21所示。
图10-13显示本发明的融合多肽的体外活性检测。
图14和15显示本发明的多肽二聚体的体外活性检测结果，循环重排的IL-2的氨基酸序列如SEQ ID NO:21所示。
图16显示给荷瘤小鼠注射本发明的融合多肽或多肽二聚体的实验结果，A：第一次注射27天后的肿瘤；B：肿瘤体积随时间变化的曲线；和C：小鼠体重随时间变化曲线。
图17显示来自不同组的小鼠的肿瘤的免疫组化染色结果，A：平均光密度；和B：代表性成像结果。
发明详述
尽管将结合以下所列举的实施方案描述本发明，但是应当理解，它们并不旨在将本发明限制于那些实施方案。相反，本发明旨在覆盖可包括在如权利要求所限定的本发明的范围内的所有替代、修改和等同形式。本领域技术人员将认识到许多与本文描述的方法或材料相似或等同的方法和材料可用于实施本发明。本发明不限于所描述的方法和材料。如果所引用的文献、专利和类似材料中的一个或多个与本申请不同或相矛盾，包括但不限于所定义的术语、术语用法、所描述的技术等，则以本申请为准。除非另有定义，否则本文使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常理解的相同含义。尽管与本文描述的那些类似或等同的方法和材料可以用于本发明的实践或测试中，但是下面描述了合适的方法和材料。本文提及的所有出版物、专利申请、专利和其他参考文献通过引用整体并入本文。
一、定义
如本文所用，术语“肽”表示通过肽键连接的至少两个氨基酸的链。术语“多肽”在本文中可以与术语“蛋白质”可互换使用，是指含有十个或更多个氨基酸残基的链。本文中的所有肽和多肽化学式或序列均是从左至右书写的，表示从氨基末端至羧基末端的方向。
如本文所用，术语“肽链中的二面角”是指相邻的两个肽键平面可以绕这两个肽键平面之间的α碳原子旋转的角度。
在肽的语境中，术语“氨基酸”、“残基”和“氨基酸残基”可以互换使用，包括蛋白质中天然存在的氨基酸和非天然氨基酸。蛋白质中天然存在的氨基酸的单字母和三字母命名采用本领域惯用名，可见于Sambrook,et al.(Molecular Cloning:A Laboratory Manual,2nd,ed.Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1989)。
Figure PCTCN2021105187-appb-000001
Figure PCTCN2021105187-appb-000002
如本文所用，术语“多核苷酸”或者“核酸分子”包括DNA分子(例如cDNA或基因组DNA)和RNA分子(例如mRNA)及使用核苷酸类似物产生的DNA或RNA的类似物。所述核酸分子可以是单链或双链的，优选双链DNA。所述核酸的合成可以使用核苷酸类似物或衍生物(例如肌苷或硫代磷酸核苷酸)。这种核苷酸可以用于，例如，制备具有改变的碱基配对能力或者增加的核酸酶抗性的核酸。
如本文所用，术语“编码”是指多核苷酸直接指定其蛋白质产物的氨基酸序列。编码序列的边界一般由开放读框确定，所述开放读框通常以ATG起始密码子或另外的起始密码子如GTG和TTG开始，以终止密码子如TAA、TAG和TGA结束。所述编码序列可以是DNA、cDNA或重组核苷酸序列。
如本文所用，术语“杂交”是在给定的严格杂交和洗涤条件下，彼此至少大约90％、优选至少大约95％、更优选至少大约96％、更优选至少98％同源的核苷酸序列通常保持彼此杂交。
本领域技术人员知道各种用于杂交的条件，如严格杂交条件和高度严格杂交条件。参见，例如，Sambrook et al.,1989,Molecular Cloning,A Laboratory Manual,Cold Spring Harbor Press,N.Y.；和Ausubel et al.(eds.),1995,Current Protocols in Molecular Biology,John Wiley&Sons,N.Y.。
如本文所用，“氨基酸相同性百分比”或者“氨基酸序列相同性百分比”是指比较两个多肽的氨基酸，当最佳比对时，所述两个多肽具有大约指定的相同氨基酸百分比。例如，“95％的氨基酸相同性”是指比较两个多肽的氨基酸，当最佳比对时，所述两个多肽有95％的氨基酸相同。
对于本发明，为确定两个氨基酸序列或两个核酸序列的相同性百分比，以最佳比较为目的比对序列(例如在第一个氨基酸或核酸序列中可导入缺口，以与第二个氨基酸或核酸序列进行最佳比对)。然后比较在相应氨基酸位置或核苷酸位置的氨基酸残基或核苷酸。当第一个序列中的位置在第二个序列中相应位置由相同氨基酸残基或核苷酸占据时，则这些分子在这个位置是相同的。两个序列之间的相同性百分比是所述序列共有的相同位置的数量的函数(即相同性百分比＝相同位置的数量/位置(即重叠位置)的总数量×100)。优选地，这两个序列是相同长度的。本领域技术人员知晓，可以使用不同的计算机程序确定两个序列之间的相同性。
如本文所用，术语“保守取代”也称为由“同源”氨基酸残基取代，是指其中氨基酸残 基由具有相似侧链的氨基酸残基置换的取代，例如，碱性侧链的氨基酸(例如赖氨酸、精氨酸和组氨酸)、酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、非荷电极性侧链氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-分支的侧链氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)及芳香侧链氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。
保守氨基酸取代通常对所得蛋白质的活性的影响最小。这种取代在下文描述。保守取代是用大小、疏水性、电荷、极性、空间特征、芳香性等相似的氨基酸置换一个氨基酸。当希望精细调节蛋白质的特性时，这种取代通常是保守的。
如本文所用，“同源”氨基酸残基是指具有相似化学性质的氨基酸残基，所述化学性质涉及疏水性、电荷、极性、空间特征、芳香性特征等。彼此同源的氨基酸的例子包括正电荷的赖氨酸、精氨酸、组氨酸，负电荷的谷氨酸、天冬氨酸，疏水性的甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸，极性的丝氨酸、苏氨酸、半胱氨酸、甲硫氨酸、色氨酸、酪氨酸、天冬酰胺、谷氨酰胺，芳香性的苯丙氨酸、酪氨酸、色氨酸，化学相似侧链基团的丝氨酸与苏氨酸，或者谷氨酰胺和天冬酰胺，或者亮氨酸和异亮氨酸。
蛋白质中氨基酸保守取代的例子包括：Ser取代Ala，Lys取代Arg，Gln或His取代Asn，Glu取代Asp，Ser取代Cys，Asn取代Gln，Asp取代Glu，Pro取代Gly，Asn或Gln取代His，Leu或Val取代Ile，Ile或Val取代Leu，Arg或Gln取代Lys，Leu或Ile取代Met，Met、Leu或Tyr取代Phe，Thr取代Ser，Ser取代Thr，Tyr取代Trp，Trp或Phe取代Tyr，及Ile或Leu取代Val。
如本文所用，术语“表达”包括多肽生产中包含的任何步骤，包括但不限于转录、转录后修饰、翻译、翻译后修饰和分泌。
如本文所用，术语“细胞因子”是指由免疫细胞(如单核细胞、巨噬细胞、T细胞、B细胞、NK细胞等)和某些非免疫细胞(内皮细胞、表皮细胞、纤维母细胞等)经刺激而合成、分泌的一类具有广泛生物学活性的小分子蛋白质。细胞因子一般通过结合相应受体调节细胞生长和分化，以及免疫应答。
如本文所用，术语“四α-螺旋束细胞因子家族”涉及三级结构中包含四个α-螺旋束的细胞因子。天然(野生型)的四α-螺旋束细胞因子家族的细胞因子包含从N端到C端依次为螺旋束1(H1)、螺旋束2(H2)、螺旋束3(H3)和螺旋束4(H4)的四个α-螺旋束。四α-螺旋束细胞因子家族的细胞因子包括但不限于IL-2、IL-4、IL-6、IL-7、IL-9、IL-15、IL-21、G-CSF和GM-CSF。
如本文所用，术语“循环重排”是指改变四α-螺旋束细胞因子家族的细胞因子中四个α-螺旋束在一级结构中的排列顺序。例如，循环重排的细胞因子包含从N端到C端依次为H2、H3、H4和H1；H3、H4、H1和H2；或H4、H1、H2和H3的四个α-螺旋束。循环重排涉及对多肽进行如下设计：将多肽(初始多肽，如野生型四α-螺旋束细胞 因子)的N端与C端融合(直接连接或通过接头连接)形成环状分子，并在H1和H2之间、H2和H3之间或H3和H4之间打开(切割或断裂)所述环状分子，形成新的线性多肽，其具有与初始多肽不同的N端和C端。循环重排保留了多肽的序列、结构和功能(除了任选存在的接头以外)，同时在不同的位置产生了新的C末端和N末端。循环重排还包括产生本文所述的循环重排的线性分子的任何过程。通常，循环重排的多肽直接表达为的线性分子，而不真正经历环化和打开的步骤。
对于多肽，“接头”或“接头序列”是指通过共价键与多肽的N和/或C末端连的氨基酸序列。接头可以用于连接同一多肽的N和C末端(如在循环重排中)，也可以同意连接不同多肽的N末端和C末端以形成融合多肽。接头也涉及编码接头的氨基酸序列的多核苷酸。通常，接头没有特定的生物学活性。然而，可以基于接头或所得分子的某些性质来选择组成接头的氨基酸，例如柔性，亲水性，净电荷或是否具有蛋白水解敏感性，以及没有免疫原性。
如本文所用，“野生型”多肽是指天然存在的多肽。
如本文所用，术语“修饰”是指对多核苷酸或多肽序列的修饰，包括但不限于，取代、删除、插入和/或添加一或多个核苷酸或氨基酸。修饰也包括不改变多核苷酸或多肽的序列的化学修饰，如多核苷酸的甲基化、多肽的糖基化等。在本文中，修饰也包括如上所述的循环重排。
如本文所用，术语“打开位点”是指循环重排的过程中，环状分子中消除肽键以形成新的氨基和羧基末端的位置，或者所述多肽的编码多核苷酸中相应的位置。打开位点由位于野生型多肽的氨基和羧基末端之间的一对氨基酸的位置指定，这些氨基酸成为循环重排的多肽的新的氨基和羧基末端。例如，在IL-2(97/96)中，新的N末端对应于天然IL-2的位置97的残基，且新的C末端对应于天然IL-2的位置96的残基；在IL-15(105/102)中，新的N末端对应于天然IL-15的位置105的残基，且新的C末端对应于天然IL-15的位置102的残基，位置103和104的残基被去除。
如本文所用，术语“受体”应理解为与配体结合的存在于细胞表面的蛋白质，也涵盖不存在于细胞表面的可溶性受体，其具有对应的细胞表面受体或与对应的细胞表面受体相关。细胞表面受体通常由具有不同功能的不同结构域或亚基组成，例如胞外结构域、跨膜结构域和胞内效应结构域，所述胞外结构域包含与配体相互作用的区域，所述跨膜结构域将所述受体锚定在细胞膜中，而所述胞内效应结构域响应配体结合产生细胞信号(信号转导)。可溶性受体通常由从膜锚定区的蛋白水解切割的一个或多个胞外结构域组成。
如本文所用，术语“变体”是指与参考多肽不同但保留必要特性的多肽。多肽的典型变体的一级氨基酸序列与参考多肽不同。通常，差异是有限的，使得参考多肽和变体的序列总体上非常相似，并且在许多区域是相同的。变体和参考多肽的氨基酸序列可以通过一个或多个修饰(例如，取代，添加和/或缺失)而不同。取代或插入的氨基酸残基可以是或不是遗传密码编码的一个。多肽的变体可以是天然存在的，例如等位基因变体； 也可以是人工产生的变体。另外，本文所用的术语“变体”包括多肽的循环重排的变体。
如本文所用，术语“载体蛋白”是指与感兴趣的分子(例如，多肽或半抗原)融合，具有帮助感兴趣的蛋白的递送、延长其半衰期、使半抗原产生免疫原性等功能的蛋白质。载体蛋白本身不具有所述感兴趣的分子的生物学活性。本发明所用的载体蛋白具有多个由环连接的螺旋结构，即具有“螺旋-环-螺旋”结构，例如，白蛋白如人血清白蛋白(HSA)、白蛋白结合蛋白等。
如本文所用，感兴趣的多肽在载体蛋白中的“插入位点”是指感兴趣的多肽插入载体蛋白后，在一级结构中最接近所述感兴趣的多肽的N端的所述载体蛋白的残基的位置。
如本文所用，载体蛋白“遮蔽”感兴趣的位点是指载体蛋白在空间上阻碍感兴趣的位点与其受体结合，即载体蛋白与所述受体在空间上冲突(clash)。
如本文所用，“位点的可及性”是指位点与其结合伴侣接触并结合的能力。当位点的可及性被阻断，其不能与其结合伴侣接触并结合。
如本文所用，术语“免疫球蛋白(Ig)”指具有抗体活性或化学结构的，与抗体分子相似的球蛋白。天然免疫球蛋白是由两条相同的轻链和两条相同的重链通过链间二硫键连接而成的四肽链结构。
如本文所用，术语“Fc片段”或“Fc区”是免疫球蛋白的部分，木瓜蛋白酶水解Ig，可将Ig裂解为两个完全相同的Fab片段和一个Fc片段。天然抗体的Fc片段包含通过二硫键连接的两条相同的多肽。每条多肽包含两个重链恒定区(CH2和CH3，例如IgG的Fc区)，或三个重链恒定区(CH2、CH3和CH4，例如IgM和IgE的Fc区)。
如本文所用，术语“信号肽”是指引导新合成的蛋白质向分泌通路转移的短肽。通常，信号肽位于新合成的蛋白质的N末端，长度为，例如，5-30个氨基酸残基。信号肽可以在蛋白质加工的过程中被去除，从而成熟的蛋白质并不包含信号肽。
如本文所用，术语“治疗”是指获得有益或期望结果的方法，包括但不限于根除或改善所治疗的潜在疾病。同样，通过消除或改善与基础疾病相关的一种或多种生理症状，从而获得治疗益处；这样，尽管受试者仍可能患有基础疾病，但在受试者中观察到了改善。
如本文所用，术语“治疗有效量”和“治疗有效剂量”是指活性组分的量，当以单一剂量或重复剂量施用时，所述有效量可以实现可检测的有益效果，包括但不限于，对疾病或病症的任何症状、方面、测量的参数或特征的效果。
如本文所用，术语“剂量”是指一次(单位剂量)或在限定的时间间隔内两次或多次施用给对象的量。例如，剂量可以指在一天、两天、一周、两周、三周或一个或多个月内施用(例如，通过一次施用，或两次或更多次施用)的量。
如本文所用，术语“半衰期”是指目标分子的体内浓度降低50％所消耗的时间。如果与适当的对照相比，目标分子在生物基质(血液，血清，血浆，组织)中体内存留更长的时间，则其半衰期将增加。与适当的对照相比，半衰期可以增加10％、20％、30％、 40％、50％或更多。
本领域技术人员熟悉药物代谢动力学分析和确定配体半衰期的方法。可以在Kenneth,A et al.,Chemical Stability of Pharmaceuticals:A Handbook for Pharmacists和Peters et al.,Pharmacokinetc analysis:A Practical Approach(1996)中找到详细信息。还可以参考"Pharmacokinetics",M Gibaldi&D Perron,published by Marcel Dekker,2.sup.nd Rev.ex edition(1982)。
二、多肽的循环重排
循环重排是指改变四α-螺旋束细胞因子家族的细胞因子中四个α-螺旋束在一级结构中的排列顺序。例如，循环重排的四α-螺旋束细胞因子包含从N端到C端依次为H2、H3、H4和H1；H3、H4、H1和H2；或H4、H1、H2和H3的四个α-螺旋束。循环重排涉及对多肽进行如下设计：将多肽(初始多肽，如野生型四α-螺旋束细胞因子)的N端与C端融合(直接连接或通过接头连接)形成环状分子，并在H1和H2之间、H2和H3之间或H3和H4之间打开(切割或断裂)所述环状分子，形成新的线性多肽，其具有与初始多肽不同的N端和C端。
重要的是，循环重排的多肽在保留初始多肽的生物学活性的同时，提供用于与其它多肽融合的经优化的末端。如果新末端中断了初始多肽的关键区域，则可能会失去活性。同样，如果连接原始末端会破坏活性，则循环重排的多肽也不能保留生物学活性。因此，对于产生有活性的循环重排的蛋白质有两个要求：1)连接初始多肽的末端不破坏其生物学活性；2)初始多肽中必须存在至少一个“打开位点”，在所述位点可以形成新的末端而不会破坏对于其折叠和生物学活性至关重要的区域。
因此，一般而言，进行循环重排的候选多肽在天然折叠状态(原始蛋白质)中，原始的N和C末端非常接近，例如，原始蛋白质的N和C末端距离小于或等于
Figure PCTCN2021105187-appb-000003
对于与所需的多肽融合伴侣融合并减少所需接头的长度而言，新末端的位置在几何、结构和功能上(相对于天然末端)是有利的。
图1中示出了IL-2的结构，其中环1和2是形成新末端的位置的示例。图2示出了IL-15的结构，其中“被打开的环”是形成新末端的位置的示例。
在一些实施方案中，为了对IL-2进行循环重排，对重组构建体进行工程化设计，将IL-2的天然N末端和C末端通过接头连接，并在氨基酸残基A93-R103或N50-L56之间打开所述环状分子形成具有新的N和C端的线性分子。在一些方案中，在氨基酸残基R103形成新的N末端，并在A93形成新的C末端，氨基酸残基Q94-P102被删除，即IL-2(R103/A93)。在一些方案中，所述循环重排的IL-2是IL-2(L56/N50)、IL-2(L56/K55)或IL-2(N53/K52)。在一些实施方案中，在N97形成新的N端，并在K96形成新的C端，即IL-2(N97/K96)。
在一些实施方案中，所述循环重排的IL-2包含SEQ ID NO:2、3、4或5的氨基酸序列。
在一些实施方案中，为了对IL-15进行循环重排，对重组构建体进行工程化，将IL-15的天然N末端和C末端通过接头连接形成环状分子，并在氨基酸残基S102-A105之间打开所述环状分子形成具有新的N和C端的线性分子。例如，在氨基酸残基S102、G103或D104形成新的C端，并在G103、D104、A105或S106形成新的N末端。在一些实施方案中，所述循环重排的IL-15是IL-15(G103/S102)、IL-15(D104/S102)、IL-15(A105/S102)、IL-15(S106/S102)、IL-15(S106/G103)、IL-15(A105/G103)、IL-15(D104/G103)、IL-15(A105/D104)、IL-15(S106/D104)、IL-15(S106/A105)。
在优选的实施方案中，用于连接初始多肽的N和C末端的接头的长度与原始蛋白质中N和C末端的距离相关。在一些实施方案中，用于连接初始多肽的N和C末端的接头的长度为1-10个氨基酸，例如1、2、3、4、5、6、7、8、9或10个氨基酸。在一些实施方案中，所述接头的长度大于10个氨基酸。优选地，使用3个氨基酸的接头如GSG连接天然IL-2的N和C末端；使用四个氨基酸的接头如GGGG(SEQ ID NO:17)连接天然IL-15的N和C末端。
本领域技术人员会认识到，可以对初始多肽进行其他修饰，例如，可以进行氨基酸取代。在一些实施方案中，将天然IL-2的残基T23(即SEQ ID NO:1的T2)取代为A。在一些实施方案中，将天然IL-2的残基C145(SEQ ID NO:1的C124)取代为S。
在一些实施方案中，所述循环重排的IL-2包含SEQ ID NO:2、3、4或5的氨基酸序列。
在一些实施方案中，循环重排的IL-15包含SEQ ID NO:7的氨基酸序列。
在一些实施方案中，循环重排的IL-2包含SEQ ID NO:23所示的氨基酸序列。
在一些实施方案中，循环重排的IL-15包含SEQ ID NO:24所示的氨基酸序列。
三、融合多肽
本发明的融合多肽包含载体蛋白和感兴趣的多肽，其中所述载体蛋白具有由环连接的多个螺旋结构域，所述感兴趣的多肽插入所述载体蛋白的环中，所述载体蛋白遮蔽所述感兴趣的多肽上的感兴趣的位点，从而阻断所述位点的可及性。
发明人发现，将感兴趣的多肽插入载体蛋白的环中时，通过选择合适的插入位点，调整感兴趣的多肽与载体蛋白之间接头(例如其序列和/或长度)，删除所插入的环中的一或多个氨基酸，在感兴趣的多肽的N和/或C端删除一或多个氨基酸，或其任意组合，可以利用肽链中的二面角控制载体蛋白和感兴趣的多肽的相对位置，使得所述载体蛋白遮蔽所述感兴趣的位点。
在一些实施方案中，所述感兴趣的多肽衍生自四α-螺旋束细胞因子家族的细胞因子，包括但不限于IL-2、IL-4、IL6、IL-7、IL-9、IL15和IL21。优选地，所述感兴趣的多肽是循环重排的四α-螺旋束细胞因子家族的细胞因子。在优选的实施方案中，所述感兴趣的多肽是循环重排的IL-2或IL-15。
天然IL-2上具有CD25、CD122和CD132(分别称为IL-2受体α、β和γ)结合位点。 IL-2既可以与CD25/CD122/CD132三聚体结合，从而激活表达所述三聚体的T调节细胞(Treg)而抑制免疫应答，也可以与CD122/CD132二聚体结合，从而激活表达所述二聚体的免疫细胞(如CD8+记忆T细胞和NK细胞)并刺激其增殖。CD25与IL-2结合后会改变IL-2的构象，使其对CD122/CD132二聚体的亲和力提高。
因此，在一些实施方案中，所述感兴趣的多肽是循环重排的IL-2，所述感兴趣的位点是CD25结合位点，所述融合多肽具有与天然IL-2相当或改进的活性，例如激活JAK1/JAK3与STAT3/STAT5信号通路的活性。
IL-15也可以激活免疫反应，对于T细胞与NK细胞的分化与增殖、以及树突细胞的发育有着重要作用。IL-15与IL-2的工作机理有类似之处，通过结合CD122/CD132受体二聚体来激活下游信号通路(JAK1/JAK3与STAT3/STAT5)，但其α受体与IL-2不同，是其特有的IL-15Rα(CD215)。IL-15与IL-15Rα结合后以高亲和力与CD122/CD132结合，而IL-15在不与IL-15Rα结合的情况下，以中等亲和力与CD122/CD132结合。
因此，在一些实施方案中，所述感兴趣的多肽是循环重排的IL-15，所述感兴趣的位点是CD215结合位点，所述融合多肽具有与天然IL-15相当或改进的活性，例如激活JAK1/JAK3与STAT3/STAT5信号通路的活性。
优选地，适用于本发明的载体蛋白能够与新生儿Fc受体(FcRn)结合(如白蛋白)，或者所述载体蛋白与能够结合FcRn的蛋白质结合(如白蛋白结合蛋白)。
人血清白蛋白(HSA)是人血清中最稳定以及含量最高的蛋白质之一(35-50g/L)，占血清中蛋白质的一半，其主要作用是运输物质(如激素、脂肪酸等)，维持pH与渗透压等。本文中的HSA包括野生型HSA和经修饰的HSA。
HSA是一个全α-螺旋蛋白，其分子量为66.5kDa，由三个类似的结构域(DI、DII、DIII)组成一个“心形”的结构(图3)。HSA在酸性条件下(pH<6.5)可以与人FcRn结合，随后回收至细胞表面并释放回血液中，从而避免HSA进入溶酶体被降解。FcRn主要与DIII以及部分DI结合。
HSA的前体序列如SEQ ID NO:16(Uniprot P02768)所示，本文中所有涉及HSA的位置参照SEQ ID NO:16进行编号。HSA的前体序列中残基1-24是信号肽，天然HSA包含SEQ ID NO:16的残基25-609。
在一些实施方案中，所述载体蛋白是HSA。优选地，所述感兴趣的多肽的插入不影响HSA与FcRn的结合。在一些实施方案中，所述感兴趣的多肽插入HSA的环中，所述环选自位于D56-L66、A92-P96、D129-E131、Q170-A172、K281-L283、V293-L305、E311-S312、E321-A322、A362-D365、L398-E400、K439-R445、E465-D471、P537-E542、A561-T566的环，优选地，位于D56-L66、V293-L305和A362-D365的环。在一些实施方案中，所述感兴趣的多肽的插入位点是HSA的氨基酸残基D56、A300、C361或A362。
可以通过删除载体蛋白如HSA的环中的一或多个氨基酸来控制载体蛋白和感兴趣的多肽之间的相对位置，也可以不删除载体蛋白的环中的氨基酸。
也可以对HSA进行修饰以改进其性质。例如，可以将野生型HSA中的游离的半胱 氨酸取代为其他氨基酸，如丝氨酸。在一些实施方案，所述HSA包含氨基酸取代C58S。
在一些实施方案中，所述载体蛋白与SEQ ID NO:16具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性，且具有与野生型HSA相同或相似的结构。
在一些实施方案中，所述感兴趣的多肽的N端和C端通过接头与所述载体蛋白连接。在一些实施方案中，所述感兴趣的多肽的N端通过接头与所述载体蛋白连接，所述所述感兴趣的多肽的C端直接与所述载体蛋白连接。在一些实施方案中，所述感兴趣的多肽的N端直接与所述载体蛋白连接，所述所述感兴趣的多肽的C端通过接头与所述载体蛋白连接。在一些实施方案中，所述感兴趣的多肽的N端和C端直接与所述载体蛋白连接。在一些实施方案中，所述接头具有1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个氨基酸。在一些实施方案中，所述接头选自螺旋接头，或柔性接头(如GS接头和多聚甘氨酸接头)。
在一些实施方案中，本发明的融合多肽包含载体蛋白HSA和循环重排的IL-2，其中所述循环重排的IL-2包含从N端到C端依次为H3、H4、H1和H2，或H4、H1、H2和H3的四个α-螺旋束，且其中所述循环重排的IL-2插入HSA的环中，所述环选自位于D56-L66、A92-P96、D129-E131、Q170-A172、K281-L283、V293-L305、E311-S312、E321-A322、A362-D365、L398-E400、K439-R445、E465-D471、P537-E542和A561-T566的环，所述位置参照SEQ ID NO:16进行编号，所述HSA遮蔽所述循环重排的IL-2的CD25结合位点，从而阻断所述位点的可及性。在一些实施方案中，所述循环重排的IL-2包含SEQ ID NO:2、3、4或5的氨基酸序列。在一些实施方案中，所述环选自位于HSA的D56-L66、V293-L305和A362-D365的环。在一些实施方案中，所述循环重排的IL-2的插入位点选自HSA的D56、A300、C361和A362。
在一些实施方案中，所述循环重排的IL-2包含SEQ ID NO:2的氨基酸序列。在一些实施方案中，所述环是位于HSA的A362-D365的环，优选地，所述插入位点为HSA的C361。在一些实施方案中，所述循环重排的IL-2的N端通过接头EAAAKAEAAA(SEQ ID NO:19)与HSA的C361连接，C端通过接头GS与HSA的D365连接，HSA的残基362-364被删除。
在一些实施方案中，所述循环重排的IL-2包含SEQ ID NO:4的氨基酸序列。在一些实施方案中，所述环是位于HSA的D56-L66的环，优选地，所述插入位点为HSA的D56。在一些实施方案中，所述循环重排的IL-2的N端通过接头AAAAAK(SEQ ID NO:20)与HSA的D56连接，C端直接与HSA的E57连接。
在一些实施方案中，所述循环重排的IL-2包含SEQ ID NO:5的氨基酸序列。在一些实施方案中，所述环是位于HSA的V293-L305的环，优选地，所述插入位点为HSA的A300。在一些实施方案中，所述循环重排的IL-2的N端直接与HSA的A300连接，C端通过接头G与HSA的D301连接。
在一些实施方案中，所述循环重排的IL-2由氨基酸序列SEQ ID NO:4组成。在一些实施方案中，所述环是位于HSA的A362-D365的环，优选地，所述插入位点为HSA 的A362。在一些实施方案中，所述循环重排的IL-2的N端直接与HSA的A362连接，C端直接与HSA的A363连接。
在一些实施方案中，本发明提供一种融合多肽，其包含载体蛋白HSA和循环重排的IL-15，其中所述循环重排的IL-15包含从N端到C端依次为H3、H4、H1和H2的四个α-螺旋束，且其中所述循环重排的IL-15插入HSA的环中，所述环选自位于D56-L66、A92-P96、D129-E131、Q170-A172、K281-L283、V293-L305、E311-SS312、E321-A322、A362-D365、L398-E400、K439-R445、E465-D471、P537-E542和A561-T566的环，所述位置参照SEQ ID NO:16进行编号，所述HSA遮蔽所述所述循环重排的IL-15的CD215结合位点，从而阻断所述位点的可及性。在一些实施方案中，所述循环重排的IL-15包含SEQ ID NO:7的氨基酸序列。
在一些实施方案中，本发明的融合多肽包含SEQ ID NO:8-11之一的氨基酸序列。在一些实施方案中，本发明的融合多肽包含与SEQ ID NO:8-11之一的氨基酸序列具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性的氨基酸序列，其中所述融合多肽包含天然的CD25结合位点，其具有与天然IL-2相当或改进的活性，例如激活JAK1/JAK3与STAT3/STAT5信号通路的活性。
在一些实施方案中，本发明的融合多肽具有更持久的免疫刺激作用。在一些实施方案中，本发明的融合多肽具有更高的安全性。
在一些实施方案中，与天然IL-2相比，本发明的融合多肽的半衰期增加10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、150％、200％、300％、400％、500％、600％、700％、800％、900％、1000％或更多。在一些实施方案中，与天然IL-15相比，本发明的融合多肽的半衰期增加10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、150％、200％、300％、400％、500％、600％、700％、800％、900％、1000％或更多。
四、多肽二聚体
本发明的多肽二聚体包含第一多肽和第二多肽，
其中所述第一多肽包含第一二聚化结构域和感兴趣的多肽，其中所述感兴趣的多肽位于所述第一二聚化结构域的第一端，
其中所述第二多肽包含第二二聚化结构域和结合结构域，其中所述结合结构域位于所述第二二聚化结构域的第一端，
其中所述第一多肽与所述第二多肽通过所述第一二聚化结构域和第二二聚化结构域形成二聚体，且所述第一二聚化结构域的第一端与所述第二二聚化结构域的第一端邻近，所述结合结构域能够与所述感兴趣的多肽上的感兴趣的位点结合。
在一些实施方案中，所述感兴趣的多肽衍生自四α-螺旋束细胞因子家族的细胞因子，包括但不限于IL-2、IL-4、IL6、IL-7、IL-9、IL15和IL21。优选地，所述感兴趣的多肽是循环重排的四α-螺旋束细胞因子家族的细胞因子。在优选的实施方案中，所述感 兴趣的多肽是循环重排的IL-2或IL-15，所述循环重排如前文所述。
天然IL-2上具有CD25、CD122和CD132(分别称为IL-2受体α、β和γ)结合位点。IL-2既可以与CD25/CD122/CD132三聚体结合，从而激活表达所述三聚体的T调节细胞(Treg)而抑制免疫应答，也可以与CD122/CD132二聚体结合，从而激活表达所述二聚体的免疫细胞(如CD8+记忆T细胞和NK细胞)并刺激其增殖。CD25与IL-2结合后会改变IL-2的构象，使其对CD122/CD132二聚体的亲和力提高。
因此，在一些实施方案中，所述感兴趣的多肽是循环重排的IL-2，所述感兴趣的位点是CD25结合位点，所述结合结构域是CD25的胞外结构域，所述多肽二聚体具有与天然IL-2相当或改进的活性，例如激活JAK1/JAK3与STAT3/STAT5信号通路的活性。
IL-15也可以激活免疫反应，对于T细胞与NK细胞的分化与增殖、以及树突细胞的发育有着重要作用。IL-15与IL-2的工作机理有类似之处，通过结合CD122/CD132受体二聚体来激活下游信号通路(JAK1/JAK3与STAT3/STAT5)，但其α受体与IL-2不同，是其特有的IL-15Rα(CD215)。IL-15与IL-15Rα结合后以高亲和力与CD122/CD132结合，而IL-15在不与IL-15Rα结合的情况下，以中等亲和力与CD122/CD132结合。
因此，在一些实施方案中，所述感兴趣的多肽是循环重排的IL-15，所述感兴趣的位点是CD215结合位点，所述结合结构域是CD215的胞外结构域，所述多肽二聚体具有与天然IL-15相当或改进的活性，例如激活JAK1/JAK3与STAT3/STAT5信号通路的活性。
在本发明的多肽二聚体中，所述感兴趣的位点和所述结合结构域位于不同的(第一和第二)多肽中。所述第一和第二二聚化结构域形成二聚体，而使得所述所述感兴趣的位点和所述结合结构域接近。所述第一与第二二聚化结构域通过，例如但不限于，共价连接、氢键、静电作用和/或范德华力，优选共价连接，形成二聚体。
在一些实施方案中，所述第一和第二二聚化结构域通过二硫键形成二聚体。在一些实施方案中，所述第一和第二二聚化结构域包含免疫球蛋白(Ig)，例如人Ig(如人IgG1)的重链恒定区CH2和CH3。在一些实施方案中，所述第一二聚化结构域与第二二聚化结构域形成人IgG1的Fc区。
可以在Fc段中引入不同的突变以实现不同的功能，例如，增加Fc与FcRn在酸性条件下的亲和力；降低或者增加Fc与不同Fcγ受体以及C1q的亲和力，以减弱或者增强抗体依赖性细胞毒性(ADCC)、抗体依赖性细胞吞噬作用(ADCP)、补体依赖性细胞毒性(CDC)等。在一些实施方案中，本发明的多肽二聚体中的Fc区包含Fc沉默突变，例如L234A+L235A+P329G，以降低ADCC、ADCP和CDC，其中Fc区的氨基酸位置编号遵循IMGT EU编号规则(http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGH Gnber.html)。
在一些实施方案中，所述Fc区是同源二聚体，即所述第一二聚化结构域与所述第二二聚化结构域的序列相同。在一些实施方案中，所述Fc区是异源二聚体，即所述第一二聚化结构域与所述第二二聚化结构域的序列不同。在一些实施方案中，对所述Fc 区进行杵臼结构修饰。例如，在一条多肽链引入突变T366Y(第一代杵臼结构)或者S354C+T366W(第二代杵臼结构)，形成杵(knob)链；在另一条多肽链Y407T(第一代杵臼结构)或者Y349C+T366S+L368A+Y407V(第二代杵臼结构)，形成臼(hole)链，编号规则同上。在一些实施方案中，所述第一多肽包含杵链，所述第二多肽包含臼链。在一些实施方案中，所述第一多肽包含臼链，所述第二多肽包含杵链。
在一些实施方案中，所述杵链包含SEQ ID NO:23或与SEQ ID NO:23具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性的氨基酸序列。在一些实施方案中，所述臼链包含SEQ ID NO:24或与SEQ ID NO:24具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性的氨基酸序列。
在一些实施方案中，所述第一二聚化结构域包含SEQ ID NO:23或与SEQ ID NO:23具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性的氨基酸序列，且所述第二二聚化结构域包含SEQ ID NO:24或与SEQ ID NO:24具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性的氨基酸序列；或所述第一二聚化结构域包含SEQ ID NO:24或与SEQ ID NO:24具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性的氨基酸序列，且所述第二二聚化结构域包含SEQ ID NO:23或与SEQ ID NO:23具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性的氨基酸序列。
在涉及免疫球蛋白Fc区的一些实施方案中，所述感兴趣的多肽位于第一二聚化结构域(即Fc区的第一链)的C端，且所述结合结构域位于所述第二二聚化结构域(即Fc区的第二链)的C端。在涉及免疫球蛋白Fc区的一些实施方案中，所述感兴趣的多肽位于第一二聚化结构域(即Fc区的第一链)的N端，且所述结合结构域位于所述第二二聚化结构域(即Fc区的第二链)的N端。
在一些实施方案中，所述感兴趣的多肽通过接头与所述第一二聚化结构域连接，且所述结合结构域通过接头与所述第二二聚化结构域连接。在一些实施方案中，所述接头是柔性接头。在一些实施方案中，所述接头具有1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个氨基酸。在一些实施方案中，所述接头具有1-20、2-18、3-16、5-15、6-12或8-10个氨基酸。在一些实施方案中，所述接头具有氨基酸序列GGGGSGGGGS(SEQ ID NO:30)。
在一些实施方案中，本发明的多肽二聚体包含第一多肽和第二多肽，其中所述第一多肽包含人IgG1的Fc区的第一链和循环重排的IL-2，所述循环重排的IL-2与所述人IgG1的Fc区的第一链的C端连接，其中所述第二多肽包含人IgG1的Fc区的第二链和CD25胞外结构域，所述CD25胞外结构域与人IgG1的Fc区的第二链的C端连接，且其中所述循环重排的IL-2如前文所述，例如所述循环重排的IL-2由氨基酸序列SEQ ID NO:21组成。在一些实施方案中，所述第一链由SEQ ID NO:23的氨基酸序列组成，且所述第二链由SEQ ID NO:24的氨基酸序列组成；或所述第一链由SEQ ID NO:24的氨基酸序列组成，且所述第二链由SEQ ID NO:23的氨基酸序列组成。
在一些实施方案中，本发明的多肽二聚体包含第一多肽和第二多肽，其中所述第一多肽包含人IgG1的Fc区的第一链和循环重排的IL-15，所述循环重排的IL-15与所述人IgG1的Fc区的第一链的C端连接，其中所述第二多肽包含人IgG1的Fc区的第二链和CD215胞外结构域，所述CD215胞外结构域与人IgG1的Fc区的第二链的C端连接，且其中所述循环重排的IL-15如前文所述，例如所述循环重排的IL-15包含氨基酸序列SEQ ID NO:22。在一些实施方案中，所述第一链包含SEQ ID NO:23的氨基酸序列，且所述第二链包含SEQ ID NO:24的氨基酸序列；或所述第一链包含SEQ ID NO:24的氨基酸序列，且所述第二链包含SEQ ID NO:23的氨基酸序列。
本领域技术人员会认识到，可以在不降低其生物学活性的情况下对循环重排的多肽的氨基酸序列进行修饰。此类修饰是本领域技术人员众所周知的，并且包括添加残基，例如在氨基末端添加的甲硫氨酸以提供起始位点，或在任一末端添加额外的氨基酸以保护蛋白质免受外肽酶的破坏。
本领域的技术人员将认识到可以进行其他修改。例如，可以进行氨基酸取代，如保守取代，而不影响蛋白质的活性。或者，可以缩短或完全消除分子的非必需区域。因此，在分子本身不参与分子活性的区域中，它们可以被消除或被较短的片段取代，这些较短的片段仅用于维持分子的活性成分之间的正确空间关系。
在一些实施方案中，所述循环重排的IL-2包含与SEQ ID NO:21具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性的氨基酸序列组成，其中第一多肽包含天然的CD25结合位点，所述多肽二聚体具有与天然IL-2相当或改进的活性。
在一些实施方案中，所述循环重排的IL-15包含与SEQ ID NO:22具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性的氨基酸序列，其中第一多肽包含天然的CD215结合位点，所述多肽二聚体具有与天然IL-15相当或改进的活性。
在一些实施方案中，所述第一多肽包含SEQ ID NO:25的氨基酸序列，所述第二多肽包含SEQ ID NO:26的氨基酸序列。在一些实施方案中，所述第一多肽包含与SEQ ID NO:25具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性的氨基酸序列，所述第二多肽包含与SEQ ID NO:26具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性的氨基酸序列，其中所述第一多肽包含天然的CD25结合位点，且所述多肽二聚体具有与天然IL-2相当或改进的活性。
在一些实施方案中，所述第一多肽包含SEQ ID NO:27的氨基酸序列，所述第二多肽包含SEQ ID NO:28的氨基酸序列。在一些实施方案中，所述第一多肽包含与SEQ ID NO:27具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性的氨基酸序列，所述第二多肽包含与SEQ ID NO:28具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性的氨基酸序列，其中所述第一多肽包含天然的CD25结合位点，且所述多肽二聚体具有与天然IL-2相当或改进的活性。
在一些实施方案中，与天然IL-2相比，本发明的多肽二聚体的半衰期增加10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、150％、200％或更多。
在一些实施方案中，与天然IL-15相比，本发明的多肽二聚体的半衰期增加10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、150％、200％或更多。
五、融合多肽和多肽二聚体的表达
为表达本发明的融合多肽，本发明还提供编码本发明的融合多肽的多核苷酸。
为表达本发明的多肽二聚体，本发明还提供编码本发明的多肽二聚体的多核苷酸。在一些实施方案中，所述第一多肽和第二多肽由单一的多核苷酸编码。在一些实施方案中，所述第一多肽和第二多肽由不同的多核苷酸编码。
本发明的全部或部分核酸序列的核酸分子可以通过聚合酶链反应(PCR)分离，所述PCR使用基于所述序列中包含的序列信息设计合成的寡核苷酸引物。
本发明的多核苷酸可以使用cDNA、mRNA或者基因组DNA作为模板及合适的寡核苷酸引物根据标准PCR扩增技术进行扩增。如此扩增的核酸可以克隆进合适的载体中，并通过DNA序列分析进行表征。
本发明的多核苷酸可以通过标准的合成技术制备，例如使用自动化DNA合成仪制备。
本发明还涉及本文描述的核酸分子的互补链。与其它核苷酸序列互补的核酸分子是与该核苷酸序列充分互补的分子，使得其可以与其他核苷酸序列杂交，从而形成稳定双链体。当然，本发明的多核苷酸不包括仅与poly A序列(如mRNA的3’末端poly(A))或者与互补的一段poly T(或U)残基杂交的多核苷酸。
本发明还提供包含本发明的多核苷酸的载体，优选表达载体。进一步，本发明还提供包含本发明的多核苷酸或载体(优选地，表达载体)的宿主细胞。在一些实施方案中，本发明的多核苷酸整合入宿主细胞的基因组。在一些实施方案中，本发明的多核苷酸不整合入宿主细胞的基因组。
在一些实施方案中，用单一表达载体表达本发明的融合多肽的第一多肽和第二多肽。在一些实施方案中，用不同的表达载体表达本发明的融合多肽的第一多肽和第二多肽。
表达载体的选择与用于表达融合多肽或多肽二聚体的宿主细胞有关。可以用于表达本发明的融合多肽宿主细胞包括但不限于细菌(包括大肠杆菌)、酵母、昆虫细胞以及哺乳动物细胞，例如COS、CHO、HeLa和293-6E细胞。适用于各种宿主细胞的表达载体是本领域已知的。例如，适用于细菌的表达载体包括但不限于pET载体(如pET-28a、pET-30a、pET-32a和pET-40a等)、pEX载体(如pEX-1)、pGH112、pUC118和pEZZ18；适用于酵母的表达载体包括但不限于pESP载体(如pESP-1、pESP-2、pESP-3等)、pDR196、pHiSi、p53his、pSH47和pYCP211；适用于昆虫细胞的表达载体包括但不限于pCoBlast、pIEX/Bac-3、pIEXBac-c-EGFP-4、pFastBac1-His-C、pIEXBac-c-EGFP-3、pFastBac1-GST-N和pIEXBac-c-EGFP-2；适用于哺乳动物细胞的表达载体包括但不限于pCMVInt、pGL4.23、pX334、pX458、pBiFC-CC155、pDP4rs、pDC312和pcDNA。
在一些实施方案中，所述宿主细胞是293-6E细胞。在一些实施方案中，所述表达载体是pcDNA3.4。
前体多肽中包含信号肽会帮助所表达的多肽分泌和/或加工。因此，在一些实施方案中，本发明的多核苷酸还包含编码信号肽的序列。在一些实施方案中，所述信号肽包含METDTLLLWVLLLWVPGSTG(SEQ ID NO:18)的氨基酸序列。
本发明的多核苷酸或载体可以通过本领域已知的方法转移(转染)到选定的宿主细胞中，例如用于大肠杆菌的氯化钙转化和磷酸钙处理，电穿孔，脂质转染胺处理或用于哺乳动物细胞的PEI处理。可以根据载体中抗生素抗性基因(例如amp，gpt，neo和hyg基因)筛选由载体转化的细胞。
表达后，重组融合蛋白可以根据本领域的标准程序进行纯化，包括硫酸铵沉淀，亲和色谱，使用离子或疏水树脂的柱色谱，凝胶电泳等。优选纯度至少为约90至95％的基本上纯的组合物，最优选用于药物用途的纯度为98至99％或更高。
为了便于纯化，本发明的融合多肽还可以包含标签序列，包括但不限于His6标签、FLAG标签等。
在一些实施方案中，本发明的融合多肽包含SEQ ID NO:12-15之一的氨基酸序列。在一些实施方案中，本发明的融合多肽包含与SEQ ID NO:12-15之一的氨基酸序列具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性的氨基酸序列，其中所述融合多肽包含天然的CD25结合位点，其具有与天然IL-2相当或改进的活性，例如激活JAK1/JAK3与STAT3/STAT5信号通路的活性。
在一些实施方案中，本发明的多核苷酸或载体如表达载体编码SEQ ID NO:31的氨基酸序列，和SEQ ID NO:32的氨基酸序列。在一些实施方案中，本发明的多核苷酸或载体如表达载体编码与SEQ ID NO:31具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性的氨基酸序和与SEQ ID NO:32具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性的氨基酸序列，其中所表达的第一多肽包含天然的CD25结合位点，且所述多肽二聚体具有与天然IL-2相当或改进的活性，例如激活JAK1/JAK3与STAT3/STAT5信号通路的活性。
在一些实施方案中，本发明的多核苷酸或载体如表达载体编码SEQ ID NO:33的氨基酸序列和SEQ ID NO:34的氨基酸序列。在一些实施方案中，本发明的多核苷酸或载体如表达载体编码与SEQ ID NO:33具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性的氨基酸序列和与SEQ ID NO:34具有至少70％、80％、90％、95％、96％、97％、98％或99％的序列相同性的氨基酸序列，其中所表达的第一多肽包含天然的CD25结合位点，且所述多肽二聚体具有与天然IL-2相当或改进的活性，例如激活JAK1/JAK3与STAT3/STAT5信号通路的活性。
六、药物组合物
本发明提供了包含本发明的融合多肽的药物组合物。在一实施方案中，所述药物组 合物包含本发明的融合多肽和至少一种药学上可接受的载体。可以根据已知方法将本发明的融合多肽与药学上可接受的载体组合来制备所述药物组合物。
本发明还提供了包含本发明的多肽二聚体的药物组合物。在一实施方案中，所述药物组合物包含本发明的多肽二聚体和至少一种药学上可接受的载体。可以根据已知方法将本发明的多肽二聚体与药学上可接受的载体组合来制备所述药物组合物。
药学上可接受的载体包括但不限于溶剂、乳化剂、缓冲剂、稳定剂等。所述溶剂包括水、水性溶液、非水性溶剂(例如植物油)。
本发明的药物组合物可以通过任何合适的途径施用，包括皮下、肌内、关节内、静脉内、皮内、腹腔内、鼻内、颅内、胃肠外施用。优选地，本发明的药物组合物是静脉内施用的。应理解，施用途径可以随治疗剂、接受者的状况和年龄以及所治疗的疾病而变化。
本发明的药物组合物可以是溶液形式或冻干制剂。
在一些实施方案中，本发明药物组合物以冻干粉末形式提供，以在给药前复水。本发明的药物组合物也可以以液体形式提供，其可以直接施用于患者。在一些实施方案中，将组合物以液体形式提供在预填充的注射器中。
在一些实施方案中，本发明的组合物被封装在脂质体中。在一些实施方案中，可以用柔性水溶性聚合物包被脂质体，该聚合物避免被单核吞噬细胞系统的器官摄取，主要是肝脏和脾脏。适用于包被脂质体的亲水性聚合物包括但不限于PEG、聚乙烯基吡咯烷酮、聚乙烯基甲基醚、聚甲基恶唑啉、聚乙基恶唑啉、聚羟丙基恶唑啉、聚羟丙基甲基丙烯酰胺、聚甲基丙烯酰胺、聚二甲基丙烯酰胺、聚羟丙基甲基丙烯酸酯、聚氢氧乙基丙烯酸酯、羟甲基纤维素羟乙基酰胺、亲水性聚乙烯醇等。
可以根据患者所要求和耐受的剂量和频率来一次或多次施用本发明的药物组合物。无论如何，所施用的药物组合物应提供足够量的本发明的蛋白质以有效治疗患者。
六、治疗应用
本发明还提供使用本发明的融合多肽治疗疾病，如涉及免疫抑制的疾病的方法。
在一些实施方案中，提供一种治疗癌症的方法，包括给有需要的对象施用治疗有效量的本发明的融合多肽或药物组合物。所述癌症包括但不限于肺癌、肝癌、肾癌、头颈癌、结肠直肠癌、胃癌、鼻咽癌、神经胶质瘤、黑色素瘤和骨肉瘤。
在一些实施方案中，提供一种激活免疫细胞或增加免疫细胞的增殖的方法，包括给有需要的对象施用有效量的本发明的融合多肽或本发明的药物组合物。在一些实施方案中，所述免疫细胞是T细胞或NK细胞。
在一些实施方案中，提供本发明的融合多肽或药物组合物在制备用于治疗癌症的药物中的用途。所述癌症包括但不限于肺癌、肝癌、肾癌、头颈癌、结肠直肠癌、胃癌、鼻咽癌、神经胶质瘤、黑色素瘤和骨肉瘤。
在一些实施方案中，提供本发明的融合多肽或药物组合物在制备用于激活免疫细胞 或增加免疫细胞的增殖的药物中的用途。在一些实施方案中，所述免疫细胞是T细胞或NK细胞。
本发明还提供使用本发明的多肽二聚体治疗疾病，如涉及免疫抑制的疾病的方法。
在一些实施方案中，提供一种治疗癌症的方法，包括给有需要的对象施用治疗有效量的本发明的多肽二聚体或药物组合物。所述癌症包括但不限于肺癌、肝癌、肾癌、头颈癌、结肠直肠癌、胃癌、鼻咽癌、神经胶质瘤、黑色素瘤和骨肉瘤。
在一些实施方案中，提供一种激活免疫细胞或增加免疫细胞的增殖的方法，包括给有需要的对象施用有效量的本发明的多肽二聚体或本发明的药物组合物。在一些实施方案中，所述免疫细胞是T细胞或NK细胞。
在一些实施方案中，提供本发明的多肽二聚体或药物组合物在制备用于治疗癌症的药物中的用途。所述癌症包括但不限于肺癌、肝癌、肾癌、头颈癌、结肠直肠癌、胃癌、鼻咽癌、神经胶质瘤、黑色素瘤和骨肉瘤。
在一些实施方案中，提供本发明的多肽二聚体或药物组合物在制备用于激活免疫细胞或增加免疫细胞的增殖的药物中的用途。在一些实施方案中，所述免疫细胞是T细胞或NK细胞。
实施方案
P1、一种多肽二聚体，其包含第一多肽和第二多肽，
其中所述第一多肽包含第一二聚化结构域和感兴趣的多肽，其中所述感兴趣的多肽位于所述第一二聚化结构域的第一端，
其中所述第二多肽包含第二二聚化结构域和结合结构域，其中所述结合结构域位于所述第二二聚化结构域的第一端，
其中所述第一多肽与所述第二多肽通过所述第一二聚化结构域和第二二聚化结构域形成二聚体，且所述第一二聚化结构域的第一端与所述第二二聚化结构域的第一端邻近，所述结合结构域能够与所述感兴趣的多肽上的感兴趣的位点结合。
P2、实施方案P1的多肽二聚体，其中所述感兴趣的多肽衍生自四α-螺旋束细胞因子家族的细胞因子，所述细胞因子包含从N端到C端依次为螺旋束1(H1)、螺旋束2(H2)、螺旋束3(H3)和螺旋束4(H4)的四个α-螺旋束。
P3、实施方案P2的多肽二聚体，其中所述感兴趣的多肽是循环重排的四α-螺旋束细胞因子家族的细胞因子，其包含从N端到C端依次为H2、H3、H4和H1；H3、H4、H1和H2；或H4、H1、H2和H3的四个α-螺旋束。
P4、实施方案P3的多肽二聚体，其中所述循环重排的细胞因子中对应于天然细胞因子的N端的氨基酸通过接头与对应于天然细胞因子的C端的氨基酸连接。
P5、实施方案P4的多肽二聚体，其中所述接头是长度为1-10个氨基酸的GS接头或多聚甘氨酸接头。
P6、实施方案P1-P5任一项的多肽二聚体，其中所述感兴趣的多肽是循环重排的IL-2 或IL-15。
P7、实施方案P6的多肽二聚体，其中所述循环重排的IL-2包含从N端到C端依次为H3、H4、H1和H2，或H4、H1、H2和H3的四个α-螺旋束。
P8、实施方案P7的多肽二聚体，其中所述循环重排的IL-2包含SEQ ID NO:21的氨基酸序列。
P9、实施方案P7或P8的多肽二聚体，其中所述感兴趣的位点是CD25结合位点，所述结合结构域是CD25的胞外结构域。
P10、实施方案P6的多肽二聚体，其中所述循环重排的IL-15包含从N端到C端依次为H3、H4、H1和H2的四个α-螺旋束。
P11、实施方案P10的多肽二聚体，其中所述循环重排的IL-15包含SEQ ID NO:4的氨基酸序列。
P12、实施方案P10或11的多肽二聚体，其中所述感兴趣的位点是CD215结合位点，所述结合结构域是CD215的胞外结构域。
P13、实施方案P1-P12任一项的多肽二聚体，其中所述第一和第二二聚化结构域包含免疫球蛋白(Ig)的重链恒定区CH2和CH3。
P14、实施方案P13的多肽二聚体，其中所述Ig是人Ig。
P15、实施方案P14的多肽二聚体，其中所述Ig是人IgG1。
P16、实施方案P1-P15任一项的多肽二聚体，其中所述第一二聚化结构域与第二二聚化结构域形成人IgG1的Fc区。
P17、实施方案P1-P16任一项的多肽二聚体，其中所述第一二聚化结构域包含SEQ ID NO:21的氨基酸序列，且所述第二二聚化结构域包含SEQ ID NO:22的氨基酸序列；或所述第一二聚化结构域包含SEQ ID NO:22的氨基酸序列，且所述第二二聚化结构域包含SEQ ID NO:21的氨基酸序列。
P18、实施方案P13-P17任一项的多肽二聚体，其中所述第一二聚化结构域的第一端是C端，且所述第二二聚化结构域的第一端是C端。
P19、一种多肽二聚体，其包含第一多肽和第二多肽，
其中所述第一多肽包含人IgG1的Fc区的第一链和循环重排的IL-2，所述循环重排的IL-2与所述人IgG1的Fc区的第一链的C端连接，
其中所述第二多肽包含人IgG1的Fc区的第二链和CD25胞外结构域，所述CD25胞外结构域与人IgG1的Fc区的第二链的C端连接，且
其中所述循环重排的IL-2包含从N端到C端依次为H3、H4、H1和H2；或H4、H1、H2和H3的四个α-螺旋束。
P20、实施方案P19的多肽二聚体，其中所述循环重排的IL-2包含SEQ ID NO:2的氨基酸序列。
P21、一种多肽二聚体，其包含第一多肽和第二多肽，
其中所述第一多肽包含人IgG1的Fc区的第一链和循环重排的IL-15，所述循环重 排的IL-15与所述人IgG1的Fc区的第一链的C端连接，
其中所述第二多肽包含人IgG1的Fc区的第二链和CD215胞外结构域，所述CD215胞外结构域与人IgG1的Fc区的第二链的C端连接，且
其中所述循环重排的IL-15包含从N端到C端依次为H3、H4、H1和H2的四个α-螺旋束。
P22、实施方案P21的多肽二聚体，其中所述循环重排的IL-15包含SEQ ID NO:22的氨基酸序列。
P23、实施方案P19-P22任一项的多肽二聚体，其中所述第一链包含SEQ ID NO:23的氨基酸序列，且所述第二链包含SEQ ID NO:24的氨基酸序列；或所述第一链包含SEQ ID NO:24的氨基酸序列，且所述第二链包含SEQ ID NO:23的氨基酸序列。
P24、一种药物组合物，包含实施方案P1-P23任一项的多肽二聚体。
P25、实施方案P1-P23任一项的多肽二聚体在制备用于治疗癌症的药物中的用途。
P26、实施方案P1-P23任一项的多肽二聚体在制备用于激活免疫细胞或增加免疫细胞的增殖的药物中的用途。
P27、实施方案P26的用途，其中所述免疫细胞是T细胞或NK细胞。
P28、一种或多种分离的多核苷酸，其编码实施方案P1-P23任一项的多肽二聚体。
P29、一或多种表达载体，其包含实施方案P28的多核苷酸。
P30、一种宿主细胞，其包含实施方案P28的多核苷酸或实施方案P29的载体。
实施例
通过以下实施例，本领域技术人员会更清楚地理解本发明。应理解，实施例只是用于说明，而非限制本发明的范围。如无特别说明，本发明中使用的实验方法均为常规方法，基因克隆操作具体可参见Sambrook,et al.(Molecular Cloning:A Laboratory Manual,2nd,ed.Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1989)。
实施例1、表达载体的构建
表达载体的构建由南京金斯瑞生物科技有限公司完成，包括：合成编码SEQ ID NO:12-15和31-34的氨基酸序列的核酸，所述核酸额外包含编码SEQ ID NO:18的信号肽的核苷酸序列；通过分子克隆方法将所述核酸构建到哺乳动物细胞表达载体pcDNA 3.4中，以及质粒的扩增与纯化等。
实施例2、融合多肽和多肽二聚体的表达
在本实施例中，用编码融合多肽的核酸转染293-6E细胞，进行真核表达。融合多肽的表达和纯化由南京金斯瑞生物科技有限公司完成，过程简述如下：
-293-6E细胞在无血清的FreeStyle TM 293 Expression培养基(Thermo Fisher  Scientific,Carlsbad,CA,USA)中，置于Erlenmeyer锥形瓶(Corning Inc.,Acton,MA)中，在震荡培养器(VWR Scientific,Chester,PA)上进行培养，培养条件为37℃与5％CO 2。
-在转染前一天，将细胞稀释到合适的密度。
-在转染当天，质粒混合物(编码第一多肽和第二多肽的质粒质量比1：1)与转染试剂(比如聚醚酰亚胺，Polyetherimide，PEI)以合适的比例(比如1：3质量比)在培养基中进行混合，然后加入细胞培养液中进行转染，进行目标蛋白的分泌表达。
-六天后，细胞的存活率为50％-80％左右，通过离心获取上清液，其中含有目标蛋白(所表达的多肽中不包含信号肽)。
-上清液经过0.22μm滤膜过滤后，以3毫升/分钟的速度被加载到HisTrap TM FF Crude 5ml(GE，货号17-5286-01)层析柱中，经过洗涤和洗脱后，纯化得到的蛋白被汇集在一起并通过缓冲液交换储存于磷酸盐缓冲溶液(PBS pH 7.2)中。
纯化所得蛋白的分子量、纯度与序列覆盖度，用SDS-PAGE、免疫印迹(结果未显示)、高效液相色谱与分子筛联用(SEC-HPLC)以及液相色谱质谱(LC-MS)的方法进行了评估。免疫印迹中所用的一抗为鼠源抗His标签抗体(GenScript，货号A00186)或者鼠源抗FLAG抗体(GenScript，货号A00187)，二抗为辣根过氧化物酶修饰的山羊抗鼠IgG抗体(GenScript，货号A00160)。
结果如图4-9所示，图4：在IL-2的环1中打开形成循环重排的IL-2，插入位点在HSA的C361(SEQ ID NO:12)；图5：在IL-2的环2中打开形成循环重排的IL-2，插入位点在HSA的D56(SEQ ID NO:13)；图6：在IL-2的环2中打开形成循环重排的IL-2，插入位点在HSA的A300(SEQ ID NO:14)；和图7：在IL-2的环1中打开形成循环重排的IL-2，插入位点在HSA的A362(SEQ ID NO:15)。图8：循环重排的IL-2与经杵臼结构(knob-in-hole)修饰的人IgG1 Fc区的杵(knob)链融合(SEQ ID NO:31)，CD25的胞外结构域与经杵臼结构修饰的人IgG1 Fc区的臼(hole)链融合(SEQ ID NO:32)；图9：循环重排的IL-2与经杵臼结构修饰的人IgG1 Fc区的臼链融合(SEQ ID NO:33)，CD25的胞外结构域与经杵臼结构修饰的人IgG1 Fc区的杵链融合(SEQ ID NO:34)。在图4-7中，A幅显示HSA遮蔽IL-2上的CD25结合位点的结构模型，B幅显示还原和非还原的融合多肽的SDS-PAGE结果，而C幅显示分子筛分析结果。在图8和9中，A幅显示本发明的多糖二聚体的结构模型，B幅显示还原和非还原的多肽二聚体的SDS-PAGE结果，而C幅显示分子筛分析结果。
如图4-7所示，四种融合多肽都实现了高表达水平，SDS-PAGE与SEC-HPLC显示了高纯度与高均一性；通过LC-MS检测到了高序列覆盖度，确认了经纯化的融合多肽的完整性与正确性。
如图8和9所示，两种多肽二聚体都实现了高表达水平，SDS-PAGE与SEC-HPLC显示了高纯度与高均一性；通过LC-MS检测到了高序列覆盖度，确认了经纯化的二聚 体的完整性与正确性。
实施例3、检测融合多肽和多肽二聚体的活性
本实施例利用HEK-Blue TM IL-2细胞系(InvivoGen)，在细胞水平检测了这些融合蛋白激活下游信号通路的活性。HEK-Blue TM IL-2细胞系是在人胚胎肾293细胞(HEK-293)中稳定转染了编码IL-2信号通路所需分子的基因后构建而成，这些分子包括CD25、CD122和CD132受体分子、人JAK3激酶以及转录因子STAT5，另外还转入了受STAT5调控的分泌型胚胎碱性磷酸酶(SEAP)报告基因。当此细胞系被IL-2刺激后，会激活下游信号通路活化STAT5，上调SEAP的分泌表达。
上述检测由南京拓扑信生物科技有限公司完成，过程简述如下：
1.实验准备
1.1.配制QUANTI-Blue溶液：将QB reagent和QB buffer室温解冻融化，加入98mL无菌水中，充分混匀后，分装10mL/管，-20℃避光保存。
1.2.血清灭活：取45mL血清(FBS)至50mL离心管中，在56℃水浴中热灭活30min(每10min混匀一次)，4℃暂存。
1.3.培养基
生长培养基：DMEM+10％FBS+1％PS+100μg/mL Normocin+1μg/ml puromycin+1X HEK-Blue TM CLR Selection；测试培养基：DMEM+10％FBS(灭活)+1％PS+100μg/mL Normocin；冷冻培养基：DMEM+20％FBS+10％DMSO。
2.细胞的培养、传代和保存
2.1.将细胞置于生长培养基中，在37℃，5％CO 2培养箱中培养和传代；
2.2.当细胞汇合度达到70-80％，且状态较好时，弃上清，用在37℃水浴中预热的生长培养基重悬细胞，细胞计数后200×g离心5min；弃上清，用4℃预冷的冷冻培养基重悬细胞，并调整细胞密度至5-7×10 6细胞/mL，1mL每管，放入程序降温盒中，-80℃放置过夜后液氮长期保存。
3.HEK-Blue IL-2系统验证
3.1.制备HEK-Blue IL-2细胞悬液：用预热的DPBS轻轻润洗细胞两次，并用预热的DPBS重悬形成单个细胞悬液，细胞计数后，200×g离心5min；用预热的测试培养基重悬，细胞密度为2.8×10 5细胞/mL；
3.2.人源IL-2蛋白(阳性对照，北京百普赛斯生物科技有限公司AcroBiosystems，货号IL2-H4113)和HSA(阴性对照)或人源IgG1 Fc段(阴性对照，北京百普赛斯生物科技有限公司AcroBiosystems，货号FCC-H5214)均按以下配制系列梯度稀释样品：起始浓度650pM，5倍稀释，8个梯度。
3.3.在培养板的每个孔加入20μL的系列浓度梯度的样品和180μL细胞悬液(每个 样品三个重复)，置于37℃，5％CO 2培养箱中培养20-24小时。
3.4.将各培养物的上清液加入96孔板中，20μL/孔，并加入解冻并恢复到室温的QUANTI-Blue溶液180μL/孔，37℃，温育1-3h后，用酶标仪读取吸光度OD 620-655nm，分析数据。
4.融合多肽的活性检测。
采用步骤3中的方法检测实施例2中制备的融合多肽和多肽二聚体的活性，其中各融合多肽也按照步骤3.2中的方法配制系列梯度稀释样品。
结果如图10-15所示，图10：在IL-2的环1中打开形成循环重排的IL-2，插入位点在HSA的C361(SEQ ID NO:12)；图11：在IL-2的环2中打开形成循环重排的IL-2，插入位点在HSA的D56(SEQ ID NO:13)；图12：在IL-2的环2中打开形成循环重排的IL-2，插入位点在HSA的A300(SEQ ID NO:14)；和图13：在IL-2的环1中打开形成循环重排的IL-2，插入位点在HSA的A362(SEQ ID NO:15)。图14：第一多肽包含循环重排的IL-2与经杵臼结构修饰的人IgG1 Fc区的杵链融合(SEQ ID NO:31)，第二多肽包含CD25的胞外结构域与经杵臼结构修饰的人IgG1 Fc区的臼链融合(SEQ ID NO:32)；图15：第一多肽包含循环重排的IL-2与经杵臼结构修饰的人IgG1 Fc区的臼链融合(SEQ ID NO:33)，第二多肽包含CD25的胞外结构域与经杵臼结构修饰的人IgG1 Fc区的杵链融合(SEQ ID NO:34)。
所检测各融合多肽的50％最大效应浓度(EC50)与人IL-2的EC50的比较示于下表1-6。
表1、SEQ ID NO:12的融合多肽与人IL-2的比较
  人IL-2 SEQ ID NO:12的融合多肽
EC50(pM) 5.123 1.750
表2、SEQ ID NO:13的融合多肽与人IL-2的比较
  人IL-2 SEQ ID NO:13的融合多肽
EC50(pM) 7.934 6.356
表3、SEQ ID NO:14的融合多肽与人IL-2的比较
  人IL-2 SEQ ID NO:14的融合多肽
EC50(pM) 2.583 5.859
表4、SEQ ID NO:15的融合多肽与人IL-2的比较
  人IL-2 SEQ ID NO:15的融合多肽
EC50(pM) 2.755 5.253
表5、多肽二聚体1(SEQ ID NO:31+SEQ ID NO:32)与人IL-2的比较
  人IL-2 多肽二聚体1
EC50(pM) 6.117 10.83
表6、多肽二聚体2(SEQ ID NO:33+SEQ ID NO:34)与人IL-2的比较
  人IL-2 多肽二聚体2
EC50(pM) 5.406 10.17
结果显示，本发明的融合多肽显示出于野生型IL-2相当或更高的活性，而使用HSA或Fc区片段则没有显示相应的应答(结果未显示)。
实施例4、检测融合多肽与IL-2受体的相互作用
使用基于表面等离子共振成像(SPRi)原理的高通量分子互作仪测量本发明的融合多肽或多肽二聚体与IL-2受体之间的相互作用。
1.将以下受体蛋白或受体蛋白的组合固定化在羧基芯片表面：
CD25；
CD122；
CD25+CD122；
CD122+CD132；和
CD25+CD122+CD132。
2.以梯度浓度的野生型IL-2蛋白溶液作为流动相，根据制造商说明进行SPRi互作实验，测量结合信号和亲和力。
3.根据步骤2的结果配制梯度浓度的本发明的融合多肽或多肽二聚体溶液作为流动相，根据制造商说明进行SPRi互作实验，测量结合信号和亲和力。
实施例5、检测融合多肽的体内活性
测量五个所挑选的融合多肽或多肽二聚体分子(见下表7)在B16F10黑色素瘤小鼠模型中激活免疫系统杀伤肿瘤的活性，并观测CD8+T细胞在肿瘤组织中的浸润情况。实验委托江苏基楚生物科技有限公司进行。
表7
分子代号 分子名称 SEQ ID NO:
ZJ-1 IL-2 环1重排-KiH-CD25 31+32
ZJ-2 HSA_C361-IL2 环1重排 12
ZJ-6 HSA_D56-IL2 环2重排 13
ZJ-12 HSA_A300-IL2 环2重排 14
ZJ-15 HSA_A362-IL-2 环2重排 15
5.1.体内模型中的抗肿瘤活性测定
在DMEM(Dulbecco's Modified Eagle's Medium)培养基+10％(体积/体积比)的胎牛血清(Fetal Bovine Serum,FBS)中培养B16F10黑色素瘤细胞。
适应性喂养C57BL/6小鼠1周，在皮下注射B16F10黑色素瘤细胞(1x10 6每只)，继续饲养小鼠7天让肿瘤生长。将植瘤的小鼠分为六组(每组9只小鼠)，分别在尾静脉中注射表中所述融合多肽或多肽二聚体分子以及空白对照(PBS)。所述融合多肽或多肽二聚体分子的注射剂量为2毫克/公斤体重，每九天注射一次，总共注射三次(D0、D9和D18)，每三天测量每个小鼠的体重以及肿瘤尺寸(长和宽)以计算肿瘤体积。第一次注射27天后牺牲存活的老鼠，取肿瘤拍照，并将肿瘤包埋在蜡中进行病理切片用于接下来的免疫组织化学实验。
5.2.免疫组织化学检测肿瘤切片中的CD8表达
a).处理切片以脱蜡水化，二甲苯3次，每次5min，无水乙醇处理2次，每次5min，95％乙醇2次，每次5min，蒸馏水2次，每次5min；
b).采用柠檬酸抗原修复法热修复后冷却至室温，PBS洗3次，每次5min；
c).免疫染色封闭液室温封闭15min，封闭后去除封闭液不洗，加抗CD 8一抗((稀释比例1:2000)孵育，室温温育1h；
d).PBS洗3次，每次5min，二抗温育1h；
e).PBS洗3次，每次5min，DAB显色，显微镜下观察确定显色终点；
f).显色完毕苏木精复染核2min，自来水返蓝，梯度浓度乙醇脱水，用二甲苯透明化，中性树胶封片。
g).使用Image ProPlus软件对免疫组化染色结果进行半定量分析：
平均光密度(IOD/area)＝累积光密度值/测量的染色区域的面积。
平均光密度的数值越大表示阳性越强。
5.3.实验结果
如图16A与B所示，在2毫克每公斤的剂量下，所选的五个融合多肽或多肽二聚体小鼠黑色素瘤模型中均表现出抗肿瘤活性，其中ZJ-15的活性最强，肿瘤几乎没有生长。与现有技术(Charych et al.,Clin Canc Res 2016)报道的数据相比，在相同的动物肿瘤模型、剂量、给药方式与频率的情况下，本发明的融合多肽或多肽二聚体分子(特别是ZJ-15与ZJ-12)显示出了显著优于野生型IL-2的体内抗肿瘤活性。
如图16C所示，以2毫克每公斤的剂量(其对于细胞因子而言是相对高的的剂量)给小鼠施用本发明的融合多肽或多肽二聚体分子，小鼠的体重仍在稳定增长。这样的结果显示本发明的融合多肽或多肽二聚体分子具有较好的体内耐受潜力，证明了本发明的融合多肽或多肽二聚分子的安全性。
通过肿瘤组织切片的免疫组织化学染色，观察其中的CD8表达情况，以检测CD8+T细胞(杀伤性T细胞)在肿瘤中的浸润。如图17A所示，这些分子诱导的CD8+T细胞的浸润程度与它们的抗肿瘤活性(参见图16A和B)显示了很高的相关性，CD8+T细胞浸润程度越高，肿瘤生长越受到抑制，与发明人所预期的本发明的融合多肽或多肽二聚体分子的工作机理一致，即本发明的融合多肽或多肽二聚体分子可以更好地激活体内本身的免疫系统以杀伤肿瘤组织。图17B是一些具有代表性的肿瘤切片的免疫组化染色照片，以更直观的显示在这些不同分子干预下CD8+T细胞在肿瘤组织中的浸润情况，其中蓝色为细胞核，棕色为CD8表达信号，可以很明显的观察到例如ZJ-15分子诱导了大量的CD8+T细胞进入肿瘤组织进行杀伤与抑制。

Claims (31)

  1. 一种融合多肽，其包含载体蛋白和感兴趣的多肽，其中所述载体蛋白具有由环连接的多个螺旋结构域，所述感兴趣的多肽插入所述载体蛋白的环中，所述载体蛋白遮蔽所述感兴趣的多肽的感兴趣的位点，从而阻断所述位点的可及性。
  2. 权利要求1的融合多肽，其中所述感兴趣的多肽衍生自四α-螺旋束细胞因子家族的细胞因子，所述细胞因子包含从N端到C端依次为螺旋束1(H1)、螺旋束2(H2)、螺旋束3(H3)和螺旋束4(H4)的四个α-螺旋束。
  3. 权利要求2的融合多肽，其中所述感兴趣的多肽是循环重排的四α-螺旋束细胞因子家族的细胞因子，其包含从N端到C端依次为H2、H3、H4和H1；H3、H4、H1和H2；或H4、H1、H2和H3的四个α-螺旋束。
  4. 权利要求3的融合多肽，其中所述循环重排的细胞因子中对应于未经循环重排的细胞因子的N端的氨基酸通过接头与对应于未经循环重排的细胞因子的C端的氨基酸连接。
  5. 权利要求4的融合多肽，其中所述接头是长度为1-10个氨基酸的GS接头或多聚甘氨酸接头。
  6. 权利要求1-5任一项的融合多肽，其中所述感兴趣的多肽选自循环重排的IL-2和循环重排的IL-15。
  7. 权利要求6的融合多肽，其中所述循环重排的IL-2包含从N端到C端依次为H3、H4、H1和H2，或H4、H1、H2和H3的四个α-螺旋束。
  8. 权利要求7的融合多肽，其中所述循环重排的IL-2包含SEQ ID NO:2、3、4或5的氨基酸序列。
  9. 权利要求7或8的融合多肽，其中所述感兴趣的位点是CD25结合位点。
  10. 权利要求6的融合多肽，其中所述循环重排的IL-15包含从N端到C端依次为H3、H4、H1和H2的四个α-螺旋束。
  11. 权利要求10的融合多肽，其中所述循环重排的IL-15包含SEQ ID NO:7的氨基酸序列。
  12. 权利要求10或11的融合多肽，其中所述感兴趣的位点是CD215结合位点。
  13. 权利要求1-12任一项的融合多肽，其中所述载体蛋白是白蛋白。
  14. 权利要求13的融合多肽，其中所述载体蛋白是人血清白蛋白(HSA)。
  15. 权利要求14的融合多肽，其中所述环选自位于D56-L66、A92-P96、D129-E131、Q170-A172、K281-L283、V293-L305、E311-S312、E321-A322、A362-D365、L398-E400、K439-R445、E465-D471、P537-E542和A561-T566的环，所述位置参照SEQ ID NO:16进行编号。
  16. 权利要求15的融合多肽，其中所述感兴趣的多肽是循环重排的IL-2，所述环选自位于HSA的D56-L66、V293-L305和A362-D365的环。
  17. 权利要求16的融合多肽，其中所述感兴趣的多肽的插入位点选自HSA的D56、A300、C361和A362。
  18. 一种融合多肽，其包含载体蛋白HSA和循环重排的IL-2，其中所述循环重排的IL-2包含从N端到C端依次为H3、H4、H1和H2，或H4、H1、H2和H3的四个α-螺旋束，且其中所述循环重排的IL-2插入HSA的环中，所述环选自位于D56-L66、A92-P96、D129-E131、Q170-A172、K281-L283、V293-L305、E311-S312、E321-A322、A362-D365、L398-E400、K439-R445、E465-D471、P537-E542和A561-T566的环，所述位置参照SEQ ID NO:16进行编号，所述HSA遮蔽所述循环重排的IL-2的CD25结合位点，从而阻断所述位点的可及性。
  19. 权利要求18的融合多肽，其中所述循环重排的IL-2包含SEQ ID NO:2、3、4或5的氨基酸序列。
  20. 权利要求18或19的融合多肽，其中所述环选自位于HSA的D56-L66、V293-L305和A362-D365的环。
  21. 权利要求18-20任一项的融合多肽，其中所述循环重排的IL-2的插入位点选自HSA的D56、A300、C361和A362。
  22. 一种融合多肽，其包含载体蛋白HSA和循环重排的IL-15，其中所述循环重排的IL-15包含从N端到C端依次为H3、H4、H1和H2的四个α-螺旋束，且其中所述循环重排的IL-15插入HSA的环中，所述环选自位于D56-L66、A92-P96、D129-E131、Q170-A172、K281-L283、V293-L305、E311-SS312、E321-A322、A362-D365、L398-E400、K439-R445、E465-D471、P537-E542和A561-T566的环，所述位置参照SEQ ID NO:16进行编号，所述HSA遮蔽所述循环重排的IL-15的CD215结合位点，从而阻断所述位点的可及性。
  23. 权利要求22的融合多肽，其中所述循环重排的IL-15包含SEQ ID NO:7的氨基酸序列。
  24. 一种融合多肽，其包含SEQ ID NO:8-11之一的氨基酸序列。
  25. 一种药物组合物，其包含权利要求1-24任一项的融合多肽。
  26. 权利要求1-24任一项的融合多肽在制备用于治疗癌症的药物中的用途。
  27. 权利要求1-24任一项的融合多肽在制备用于激活免疫细胞或增加免疫细胞的增殖的药物中的用途。
  28. 权利要求27的用途，其中所述免疫细胞是T细胞或NK细胞。
  29. 一种分离的多核苷酸，其编码权利要求1-24任一项的融合多肽。
  30. 一种表达载体，其包含权利要求29的多核苷酸。
  31. 一种宿主细胞，其包含权利要求29的多核苷酸或权利要求30的表达载体。
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019222283A1 (en) * 2018-05-14 2019-11-21 Harpoon Therapeutics, Inc. Binding moiety for conditional activation of immunoglobulin molecules
CN110724198A (zh) * 2018-07-17 2020-01-24 上海一宸医药科技有限公司 长效纤连蛋白iii型结构域融合蛋白

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019222283A1 (en) * 2018-05-14 2019-11-21 Harpoon Therapeutics, Inc. Binding moiety for conditional activation of immunoglobulin molecules
CN110724198A (zh) * 2018-07-17 2020-01-24 上海一宸医药科技有限公司 长效纤连蛋白iii型结构域融合蛋白

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Molecular Biology", 1995, JOHN WILEY & SONS
CHARYCH ET AL., CLIN CANC RES, 2016
KENNETH, A ET AL.: "Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists", 1996
M GIBALDID PERRON: "Pharmacokinetics", 1982, MARCEL DEKKER
SAMBROOK ET AL.: "Molecular Cloning, A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY

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