Fusion polypeptide and its use

Abstract

The present invention relates to a fusion polypeptide, which comprises a carrier protein and a polypeptide of interest, wherein the carrier protein has a plurality of helical domains connected by a loop, 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 present invention also relates to a polynucleotide and a vector encoding the fusion polypeptide, and a host cell comprising the polynucleotide and / or the vector. Further, the present invention also relates to the use of the fusion polypeptide of the present invention in treating cancer and / or activating immune cells.

Description

Technical Field

[0001] The present invention relates to fusion polypeptides. In particular, the present invention relates to modified immunomodulatory molecules and their use in treating cancer. Background Art

[0002] Modulating the immune system through cytokines, such as activating immune cells, is a strategy for cancer immunotherapy. However, certain sites on cytokines have an impact on their functions, such as immunomodulatory effects.

[0003] For example, interleukin-2 (IL-2) can regulate the function of immune cells. After binding to the receptors IL2Rα (CD25), IL2Rβ (CD122), and IL2Rγ (CD132), IL-2 can activate downstream signaling pathways, including JAK1 and JAK3 kinases and transcription factor STAT5, to stimulate the activation and proliferation of immune cells (such as T cells and natural killer (NK) cells). IL-2 can both signal transduction through the CD25 / CD122 / CD132 trimer and activate signaling through the CD122 / CD132 dimer. After CD25 binds to IL-2, it changes the conformation of IL-2, increasing its affinity for the CD122 / CD132 dimer by 100 times (KD value increases from 1nM to 10pM). CD122 / CD132 dimers are mainly expressed on the surface of CD8+ memory T cells and NK cells, while CD25 / CD122 / CD132 trimers are expressed in large quantities 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 according to the activation of different immune cell types.

[0004] IL-2 is also a potential drug for cancer immunotherapy that has attracted much attention. Recombinant IL-2 (aldesleukin) was approved by the U.S. Food and Drug Administration (FDA) in 1998 for the treatment of metastatic melanoma and renal cancer. It is the only IL-2-based drug approved so far. However, only 10% of patients respond and it has significant side effects. This is mainly due to the dual nature of IL-2, which requires a larger dose to stimulate immune activation, but higher doses can cause side effects such as capillary leak syndrome.

[0005] Interleukin-15 (IL-15) can also activate immune responses and plays an important role in the differentiation and proliferation of T cells and NK cells, as well as the development of dendritic cells. The working mechanism of IL-15 is similar to that of IL-2. It activates downstream signaling pathways (JAK1 / JAK3 and STAT3 / STAT5) by binding to CD122 / CD132 receptor dimers, but its α receptor is different from IL-2 and is its unique IL-15Rα (CD215) receptor. It is generally believed that after IL-15 binds to the IL-15Rα receptor on the cell membrane, it is presented to the CD122 / CD132 receptor with high affinity in "trans" (trans, cell-cell contact) or "cis" (cis, on the same cell). It has also been found that IL-15 can bind to CD122 / CD132 with moderate affinity and function without binding to IL-15Rα.

[0006] Recent literature reports (Alexandra Berger et al. 2019J for ImmunoTherapy for Cancer) show that IL-15 alone shows more durable anti-tumor activity in mouse tumor models compared with the IL-15 / IL-15Rα receptor extracellular domain complex. The applicant believes that this may be because although the complex can quickly and effectively stimulate immunity, it will also cause immune cell exhaustion more quickly and reduce the durability of the reaction. Those skilled in the art also know that strong immune stimulation may bring safety issues.

[0007] In addition, cytokines such as IL-2 and IL-15 have a short half-life in vivo and require continuous injection.

[0008] Therefore, there is still a need to develop engineered recombinant cytokines such as IL-2 and IL-15 that can specifically activate immunity and have a prolonged half-life, improved activity, a more sustained ability to activate immunity, and / or higher safety, thereby improving their clinical application and commercial transformation value. Summary of the invention

[0009] In a first aspect, the present invention provides a fusion polypeptide comprising 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 loops of the carrier protein, and the carrier protein shields a site of interest on the polypeptide of interest, thereby blocking the accessibility of the site.

[0010] In some embodiments, the polypeptide of interest is derived from a cytokine of the four α-helical bundle cytokine family, which comprises four α-helical bundles, from N-terminus to C-terminus, namely helical bundle 1 (H1), helical bundle 2 (H2), helical bundle 3 (H3) and helical bundle 4 (H4).

[0011] In some embodiments, the polypeptide of interest is a cytokine of the family of circularly rearranged four α-helical bundle cytokines, which comprises four α-helical bundles from N-terminus to C-terminus, H2, H3, H4 and H1; H3, H4, H1 and H2; or H4, H1, H2 and H3.

[0012] In some embodiments, the amino acid corresponding to the N-terminus of the cytokine that is not rearranged in the circulation is connected to the amino acid corresponding to the C-terminus of the cytokine that is not rearranged in the circulation through a linker. In some embodiments, the linker is a GS linker or a polyglycine linker with a length of 1-10 amino acids.

[0013] In some embodiments, the polypeptide of interest is selected from circulating rearranged IL-2 and circulating rearranged IL-15.

[0014] In some embodiments, the cyclic rearranged IL-2 comprises four α-helical bundles, H3, H4, H1 and H2, or H4, H1, H2 and H3, from N-terminus to C-terminus. In some embodiments, the cyclic rearranged IL-2 comprises an amino acid sequence of SEQ ID NO: 2, 3, 4 or 5. In some embodiments, the site of interest is a CD25 binding site.

[0015] In some embodiments, the circularly rearranged IL-15 comprises four α-helical bundles, H3, H4, H1 and H2, from N-terminus to C-terminus. In some embodiments, the circularly rearranged IL-15 comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the site of interest is a CD215 binding site.

[0016] In some embodiments, the carrier protein is albumin, preferably human serum albumin (HSA). In some embodiments, the loop is selected from the loops located at 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 and A561-T566, the positions being numbered with reference to SEQ ID NO: 16. In some embodiments, the polypeptide of interest is a circularly rearranged IL-2, and the loop is selected from the loops located at D56-L66, V293-L305 and A362-D365 of HSA. In some embodiments, the insertion site of the polypeptide of interest is selected from D56, A300, C361 and A362 of HSA.

[0017] In some embodiments, the present invention provides a fusion polypeptide comprising a carrier protein HSA and a circularly rearranged IL-2, wherein the circularly rearranged IL-2 comprises four α-helical bundles, H3, H4, H1 and H2, or H4, H1, H2 and H3, from N-terminus to C-terminus, and wherein the circularly rearranged IL-2 is inserted into a loop of HSA, the loop being selected from the loops located at 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, the positions of which refer to SEQ ID NO:16 is numbered, and the HSA shields the CD25 binding site of the circulating rearranged IL-2, thereby blocking the accessibility of the site. In some embodiments, the circulating rearranged IL-2 comprises the amino acid sequence of SEQ ID NO:2, 3, 4 or 5. In some embodiments, the loop is selected from the loops located at D56-L66, V293-L305 and A362-D365 of HSA. In some embodiments, the insertion site of the circulating rearranged IL-2 is selected from D56, A300, C361 and A362 of HSA.

[0018] In some embodiments, the present invention provides a fusion polypeptide comprising a carrier protein HSA and a circularly rearranged IL-15, wherein the circularly rearranged IL-15 comprises four α-helical bundles, H3, H4, H1 and H2, from N-terminus to C-terminus, and wherein the circularly rearranged IL-15 is inserted into a loop of HSA, the loop being selected from the loops at 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, the positions of which are referred to in SEQ ID NO:16, the HSA shields the CD215 binding site of the circulating rearranged IL-15, thereby blocking the accessibility of the site. In some embodiments, the circulating rearranged IL-15 comprises the amino acid sequence of SEQ ID NO:7.

[0019] In some embodiments, the present invention provides a fusion polypeptide comprising an amino acid sequence of one of SEQ ID NOs: 8-11.

[0020] The present invention also provides a pharmaceutical composition comprising the fusion polypeptide of the present invention.

[0021] In a second aspect, the present invention also provides a method for treating cancer or activating immune cells or increasing the proliferation of immune cells (such as T cells or NK cells), comprising administering an effective amount of the fusion polypeptide of the present invention or the pharmaceutical composition of the present invention to a subject in need.

[0022] The present invention also provides the use of the fusion polypeptide or pharmaceutical composition of the present invention in preparing a drug for treating cancer and in preparing a drug for activating immune cells or increasing the proliferation of immune cells (such as T cells or NK cells).

[0023] In some embodiments, the fusion polypeptide or pharmaceutical composition of the present invention is also provided for treating cancer or activating immune cells or increasing the proliferation of immune cells (eg, T cells or NK cells).

[0024] In a third aspect, the present invention also provides a polynucleotide and a vector encoding the fusion polypeptide of the present invention, and a host cell comprising the polynucleotide and the vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1A schematic diagram showing 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 natural IL-2, and "loop 2" corresponds to N50-P54 of natural IL-2. Unless otherwise indicated, the positions of IL-2 referred to in this article are numbered with reference to the IL-2 precursor sequence of SEQ ID NO: 1 (UniProt P60568), wherein amino acid residues 1-21 are signal peptide sequences, and the sequence of natural IL-2 is amino acid residues 22-153 of SEQ ID NO: 1.

[0026] Figure 2 A schematic diagram showing the three-dimensional structure of wild-type IL-15 bound to its receptor (based on PDB number 4GS7), wherein the "opened loop" corresponds to S102-A105 of natural IL-15. Unless otherwise indicated, the positions of IL-15 referred to herein are numbered with reference to the IL-15 precursor sequence of SEQ ID NO: 6 (UniProt P40933), wherein residues 1-48 are signal peptides, and the sequence of natural IL-15 is amino acid residues 49-162 of SEQ ID NO: 6.

[0027] Figure 3 Schematic diagram showing the three-dimensional structure of HSA.

[0028] Figures 4A-4C , 5A-5C, 6A-6C and 7A-7C show the expression of the fusion polypeptide of the present invention and the structural model of HSA shielding the CD25 binding site on IL-2.

[0029] Figure 8-11 In vitro activity assay showing the fusion polypeptide of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0030] Although the present invention will be described in conjunction with the embodiments listed below, it should be understood that they are not intended to limit the present invention to those embodiments. On the contrary, the present invention is intended to cover all substitutions, modifications and equivalent forms that may be included within the scope of the present invention as defined by the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to the methods or materials described herein can be used to implement the present invention. The present invention is not limited to the methods and materials described. If one or more of the cited documents, patents and similar materials are different from or contradictory to the present application, including but not limited to defined terms, term usage, described technology, etc., the present application shall prevail. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference as a whole.

[0031] I. Definition

[0032] As used herein, the term "peptide" refers to a chain of at least two amino acids connected by peptide bonds. The term "polypeptide" is used interchangeably with the term "protein" herein to refer to a chain containing ten or more amino acid residues. All peptide and polypeptide formulas or sequences herein are written from left to right, indicating the direction from the amino terminus to the carboxyl terminus.

[0033] As used herein, the term "dihedral angle in a peptide chain" refers to the angle at which two adjacent peptide bond planes can rotate about the alpha carbon atom between the two peptide bond planes.

[0034] 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 non-natural amino acids in proteins. The single-letter and three-letter nomenclature for naturally occurring amino acids in proteins adopts the names commonly used in the art and can be 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).

[0035]

[0036]

[0037] As used herein, the term "polynucleotide" or "nucleic acid molecule" includes DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of DNA or RNA produced using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, preferably double-stranded DNA. Nucleotide analogs or derivatives (e.g., inosine or thiophosphate nucleotides) can be used in the synthesis of the nucleic acid. Such nucleotides can be used, for example, to prepare nucleic acids with altered base pairing ability or increased nuclease resistance.

[0038] As used herein, the term "coding" refers to a polynucleotide that directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or other start codons such as GTG and TTG and ends with stop codons such as TAA, TAG and TGA. The coding sequence can be a DNA, cDNA or recombinant nucleotide sequence.

[0039] As used herein, the term "hybridization" means that nucleotide sequences at least about 90%, preferably at least about 95%, more preferably at least about 96%, and more preferably at least 98% homologous to each other typically remain hybridized to each other under given stringent hybridization and washing conditions.

[0040] Those skilled in the art are aware of various conditions for hybridization, such as stringent hybridization conditions and highly stringent hybridization conditions. See, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, NY; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, John Wiley & Sons, NY.

[0041] As used herein, "percent amino acid identity" or "percent amino acid sequence identity" refers to comparing the amino acids of two polypeptides, when optimally aligned, the two polypeptides have about a specified percentage of identical amino acids. For example, "95% amino acid identity" means comparing the amino acids of two polypeptides, when optimally aligned, the two polypeptides have 95% of the same amino acids.

[0042] For the present invention, to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the purpose of optimal comparison (e.g., a gap may be introduced in the first amino acid or nucleic acid sequence to optimally align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions (i.e., overlapping positions) × 100). Preferably, the two sequences are of the same length. Those skilled in the art will appreciate that different computer programs may be used to determine the identity between two sequences.

[0043] As used herein, the term "conservative substitution", also referred to as substitution by a "homologous" amino acid residue, refers to substitutions in which the amino acid residue is replaced by an amino acid residue having a similar side chain, for example, amino acids with basic side chains (e.g., lysine, arginine and histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), non-charged polar side chain amino acids (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chain amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chain amino acids (e.g., threonine, valine, isoleucine) and aromatic side chain amino acids (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0044] Conservative amino acid substitutions generally have the least effect on the activity of the resulting protein. Such substitutions are described below. Conservative substitutions are replacements of an amino acid with an amino acid that is similar in size, hydrophobicity, charge, polarity, steric characteristics, aromaticity, etc. Such substitutions are generally conservative when it is desired to fine-tune the properties of a protein.

[0045] As used herein, "homologous" amino acid residues refer to amino acid residues with similar chemical properties, such as hydrophobicity, charge, polarity, steric characteristics, aromatic characteristics, etc. Examples of amino acids homologous to each other include positively charged lysine, arginine, histidine, negatively charged glutamic acid, aspartic acid, hydrophobic glycine, alanine, valine, leucine, 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.

[0046] Examples of conservative substitutions of amino acids 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 for Ile, Ile or Val for Leu, Arg or Gln for Lys, Leu or Ile for Met, Met, Leu or Tyr for Phe, Thr for Ser, Ser for Thr, Tyr for Trp, Trp or Phe for Tyr, and Ile or Leu for Val.

[0047] As used herein, the term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0048] As used herein, the term "cytokine" refers to a class of small molecule proteins with a wide range of biological activities that are synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) upon stimulation. Cytokines generally regulate cell growth and differentiation, as well as immune responses, by binding to corresponding receptors.

[0049] As used herein, the term "four α-helical bundle cytokine family" refers to cytokines that contain four α-helical bundles in the tertiary structure. Natural (wild type) cytokines of the four α-helical bundle cytokine family contain four α-helical bundles, which are helical bundle 1 (H1), helical bundle 2 (H2), helical bundle 3 (H3) and helical bundle 4 (H4) from N-terminus to C-terminus. Cytokines of the four α-helical 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.

[0050] As used herein, the term "cyclic rearrangement" refers to changing the order of arrangement of the four α-helical bundles in the primary structure of the cytokine of the four α-helical bundle cytokine family. For example, the cyclic rearranged cytokine contains four α-helical bundles from the N-terminus to the C-terminus, H2, H3, H4, H1 and H2; or H4, H1, H2 and H3. Cyclic rearrangement involves designing a polypeptide as follows: the N-terminus and the C-terminus of the polypeptide (the initial polypeptide, such as the wild-type four α-helical bundle cytokine) are fused (directly connected or connected through a linker) to form a circular molecule, and the circular molecule is opened (cut or broken) between H1 and H2, between H2 and H3, or between H3 and H4 to form a new linear polypeptide, which has a different N-terminus and C-terminus from the initial polypeptide. Cyclic rearrangement retains the sequence, structure and function of the polypeptide (except for the optional linker), while generating new C-termini and N-termini at different positions. Cyclic rearrangement also includes any process that produces the cyclic rearranged linear molecules described herein. Typically, cyclically rearranged polypeptides are expressed directly as linear molecules without actually undergoing the steps of cyclization and opening.

[0051] For polypeptides, "linker" or "linker sequence" refers to an amino acid sequence connected to the N and / or C terminus of a polypeptide by a covalent bond. Linkers can be used to connect the N and C termini of the same polypeptide (such as in a cyclic rearrangement), or they can agree to connect the N and C termini of different polypeptides to form a fusion polypeptide. Linkers also refer to polynucleotides encoding the amino acid sequence of the linker. Generally, 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 whether it is immunogenic.

[0052] As used herein, a "wild-type" polypeptide refers to a polypeptide that occurs in nature.

[0053] As used herein, the term "modification" refers to modifications to 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 change the sequence of a polynucleotide or polypeptide, such as methylation of a polynucleotide, glycosylation of a polypeptide, etc. In this article, modifications also include cyclic rearrangements as described above.

[0054] As used herein, the term "open site" refers to the position in the cyclic molecule where the peptide bond is eliminated to form new amino and carboxyl termini during the cyclic rearrangement process, or the corresponding position in the encoding polynucleotide of the polypeptide. The open site is specified by the position of a pair of amino acids between the amino and carboxyl termini of the wild-type polypeptide, and these amino acids become the new amino and carboxyl termini of the cyclic rearranged polypeptide. For example, in IL-2 (97 / 96), the new N-terminus corresponds to the residue at position 97 of natural IL-2, and the new C-terminus corresponds to the residue at position 96 of natural IL-2; in IL-15 (105 / 102), the new N-terminus corresponds to the residue at position 105 of natural IL-15, and the new C-terminus corresponds to the residue at position 102 of natural IL-15, and the residues at positions 103 and 104 are removed.

[0055] As used herein, the term "receptor" is understood to be a protein present on the cell surface that binds to a ligand, and also encompasses soluble receptors that are not present on the cell surface, which have corresponding cell surface receptors or are associated with corresponding cell surface receptors. Cell surface receptors are generally composed of different domains or subunits with different functions, such as an extracellular domain, a transmembrane domain, and an intracellular effector domain, wherein the extracellular domain comprises a region that interacts with a ligand, the transmembrane domain anchors the receptor in the cell membrane, and the intracellular effector domain responds to ligand binding to produce a cell signal (signal transduction). Soluble receptors are generally composed of one or more extracellular domains that are proteolytically cleaved from the membrane anchoring region.

[0056] As used herein, the term "variant" refers to a polypeptide that is different from a reference polypeptide but retains the necessary properties. The primary amino acid sequence of a typical variant of a polypeptide is different from that of a reference polypeptide. Typically, the differences are limited so that the sequences of the reference polypeptide and the variant are very similar overall and identical in many regions. The amino acid sequences of the variant and the reference polypeptide may differ by one or more modifications (e.g., substitutions, additions and / or deletions). The substituted or inserted amino acid residue may or may not be one encoded by the genetic code. Variants of polypeptides may be naturally occurring, such as allelic variants; they may also be artificially produced variants. In addition, the term "variant" used herein includes variants of cyclic rearrangements of polypeptides.

[0057] As used herein, the term "carrier protein" refers to a protein that is fused to a molecule of interest (e.g., a polypeptide or a hapten) and has the functions of helping the delivery of the protein of interest, extending its half-life, and making the hapten immunogenic. The carrier protein itself does not have 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, i.e., has a "helix-loop-helix" structure, for example, albumin such as human serum albumin (HSA), albumin binding protein, etc.

[0058] As used herein, the "insertion site" of a polypeptide of interest in a carrier protein refers to the position of the residue 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.

[0059] As used herein, a carrier protein "shields" a site of interest means that the carrier protein sterically hinders the site of interest from binding to its receptor, ie, the carrier protein sterically clashes with the receptor.

[0060] As used herein, "site accessibility" refers to the ability of a site to contact and bind to its binding partner. When the accessibility of a site is blocked, it cannot contact and bind to its binding partner.

[0061] As used herein, the term "signal peptide" refers to a short peptide that directs a newly synthesized protein to the secretory pathway. Typically, the signal peptide is located at the N-terminus of the newly synthesized protein and has a length of, for example, 5-30 amino acid residues. The signal peptide can be removed during protein processing so that the mature protein does not contain the signal peptide.

[0062] As used herein, the term "treatment" refers to an approach to obtaining beneficial or desired results, including but not limited to eradication or amelioration of the underlying disease being treated. Likewise, a therapeutic benefit is obtained by the elimination or amelioration of one or more physiological symptoms associated with the underlying disease; thus, an improvement is observed in a subject, although the subject may still be suffering from the underlying disease.

[0063] As used herein, the terms "therapeutically effective amount" and "therapeutically effective dose" refer to an amount of an active ingredient that, when administered in single or repeated doses, achieves a detectable beneficial effect, including, but not limited to, an effect on any symptom, aspect, measured parameter or characteristic of a disease or condition.

[0064] As used herein, the term "dose" refers to an amount administered to a subject once (unit dose) or twice or more within a defined time interval. For example, a dose can refer to an amount administered (e.g., by one administration, or twice or more administration) within one day, two days, one week, two weeks, three weeks, or one or more months.

[0065] As used herein, the term "half-life" refers to the time consumed for the in vivo concentration of a target molecule to decrease by 50%. If the target molecule remains in the body for a longer time in a biological matrix (blood, serum, plasma, tissue) compared to an appropriate control, its half-life will increase. Compared to an appropriate control, the half-life can increase by 10%, 20%, 30%, 40%, 50% or more.

[0066] Those skilled in the art are familiar with methods for pharmacokinetic analysis and determination of ligand half-life. Detailed information can be found in Kenneth, A et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetic analysis: A Practical Approach (1996). Reference can also be made to "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2.sup.nd Rev. ex edition (1982).

[0067] 2. Cyclic rearrangement of peptides

[0068] Circular rearrangement refers to changing the arrangement order of the four α-helical bundles in the primary structure of the cytokine of the four α-helical bundle cytokine family. For example, the circularly rearranged four α-helical bundle cytokine contains four α-helical bundles from the N-terminus to the C-terminus, which are H2, H3, H4 and H1; H3, H4, H1 and H2; or H4, H1, H2 and H3. Circular rearrangement involves designing a polypeptide as follows: the N-terminus and the C-terminus of the polypeptide (the initial polypeptide, such as the wild-type four α-helical bundle cytokine) are fused (directly connected or connected through a linker) to form a circular molecule, and the circular molecule is opened (cut or broken) between H1 and H2, between H2 and H3, or between H3 and H4 to form a new linear polypeptide, which has an N-terminus and a C-terminus different from those of the initial polypeptide.

[0069] Importantly, the cyclically rearranged polypeptides retain the biological activity of the original polypeptide while providing optimized ends for fusion with other polypeptides. If the new ends interrupt critical regions of the original polypeptide, activity may be lost. Similarly, if ligation of the original ends destroys activity, the cyclically rearranged polypeptides will not retain biological activity. Therefore, there are two requirements for generating active cyclically rearranged proteins: 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 where new ends can be formed without destroying regions that are critical for its folding and biological activity.

[0070] Therefore, in general, the candidate polypeptides undergoing cyclic rearrangement have their N and C termini very close in the native folded state (native protein), for example, the distance between the N and C termini of the native protein is less than or equal to

[0071] The location of the new termini is geometrically, structurally, and functionally advantageous (relative to the native termini) for fusion to a desired polypeptide fusion partner and reduces the length of the required linker.

[0072] Figure 1 The structure of IL-2 is shown in , where loops 1 and 2 are examples of locations where new termini are formed. Figure 2 The structure of IL-15 is shown, where the "opened loop" is an example of where the new terminus is formed.

[0073] In some embodiments, in order to cyclically rearrange IL-2, the recombinant construct is engineered, the natural N-terminus and C-terminus of IL-2 are connected by a linker, and the cyclic molecule is opened between amino acid residues A93-R103 or N50-L56 to form a linear molecule with new N and C-termini. In some embodiments, 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, i.e. IL-2 (R103 / A93). In some embodiments, the cyclically rearranged IL-2 is IL-2 (L56 / N50), IL-2 (L56 / K55) or IL-2 (N53 / K52).

[0074] In some embodiments, the circulating rearranged IL-2 comprises the amino acid sequence of SEQ ID NO: 2, 3, 4 or 5.

[0075] In some embodiments, in order to perform a circular rearrangement of IL-15, a recombinant construct is engineered by connecting the native N-terminus and C-terminus of IL-15 via a linker to form a circular molecule, and opening the circular molecule between amino acid residues S102-A105 to form a linear molecule with new N- and C-termini. For example, a new C-terminus is formed at amino acid residues S102, G103, or D104, and a new N-terminus is formed at G103, D104, A105, or S106. In some embodiments, 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).

[0076] In a preferred embodiment, the length of the linker used to connect the N and C termini of the initial polypeptide is related to the distance between the N and C termini in the original protein. In some embodiments, the length of the linker used to connect the N and C termini of the initial polypeptide is 1-10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In some embodiments, the length of the linker is greater than 10 amino acids. Preferably, a 3 amino acid linker such as GSG is used to connect the N and C termini of natural IL-2; a four amino acid linker such as GGGG (SEQ ID NO: 17) is used to connect the N and C termini of natural IL-15.

[0077] Those skilled in the art will recognize that other modifications may be made to the original polypeptide, for example, amino acid substitutions may be made. In some embodiments, the residue T23 of native IL-2 (i.e., T2 of SEQ ID NO: 1) is substituted with A. In some embodiments, the residue C145 of native IL-2 (C124 of SEQ ID NO: 1) is substituted with S.

[0078] In some embodiments, the circulating rearranged IL-2 comprises the amino acid sequence of SEQ ID NO: 2, 3, 4 or 5.

[0079] In some embodiments, the circulating rearranged IL-15 comprises the amino acid sequence of SEQ ID NO:7.

[0080] 3. Fusion peptide

[0081] The fusion polypeptide of the present invention comprises a carrier protein and a polypeptide of interest, wherein the carrier protein has a plurality of helical domains connected by loops, the polypeptide of interest is inserted into the loops 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.

[0082] The inventors have discovered that when a polypeptide of interest is inserted into a loop of a carrier protein, by selecting a suitable insertion site, adjusting the linker between the polypeptide of interest and the carrier protein (e.g., its sequence and / or length), deleting one or more amino acids in the inserted loop, deleting one or more amino acids at the N and / or C terminus of the polypeptide of interest, or any combination thereof, the relative position of the carrier protein and the polypeptide of interest can be controlled using the dihedral angle in the peptide chain so that the carrier protein shields the site of interest.

[0083] In some embodiments, the polypeptide of interest is derived from a cytokine of the four α-helical bundle cytokine family, including but not limited to IL-2, IL-4, IL6, IL-7, IL-9, IL15 and IL21. Preferably, the polypeptide of interest is a cytokine of the four α-helical bundle cytokine family that is cyclically rearranged. In a preferred embodiment, the polypeptide of interest is a cyclically rearranged IL-2 or IL-15.

[0084] Natural IL-2 has binding sites for CD25, CD122, and CD132 (respectively called IL-2 receptor α, β, and γ). IL-2 can bind to the CD25 / CD122 / CD132 trimer, thereby activating T regulatory cells (Treg) expressing the trimer and inhibiting the immune response, and can also bind to the CD122 / CD132 dimer, thereby activating immune cells expressing the dimer (such as CD8+ memory T cells and NK cells) and stimulating their proliferation. After CD25 binds to IL-2, it changes the conformation of IL-2, increasing its affinity for the CD122 / CD132 dimer.

[0085] Therefore, in some embodiments, the polypeptide of interest is circulating rearranged IL-2, the site of interest is the CD25 binding site, and the fusion polypeptide has activity comparable to or improved upon that of native IL-2, such as the activity of activating the JAK1 / JAK3 and STAT3 / STAT5 signaling pathways.

[0086] IL-15 can also activate immune responses and plays an important role in the differentiation and proliferation of T cells and NK cells, as well as the development of dendritic cells. The working mechanism of IL-15 is similar to that of IL-2. It activates downstream signaling pathways (JAK1 / JAK3 and STAT3 / STAT5) by binding to CD122 / CD132 receptor dimers, but its α receptor is different from IL-2, which is its unique IL-15Rα (CD215). After IL-15 binds to IL-15Rα, it binds to CD122 / CD132 with high affinity, while IL-15 binds to CD122 / CD132 with moderate affinity without binding to IL-15Rα.

[0087] Therefore, in some embodiments, the polypeptide of interest is a circulating rearranged IL-15, the site of interest is a CD215 binding site, and the fusion polypeptide has an activity comparable to or improved upon that of native IL-15, such as the activity of activating the JAK1 / JAK3 and STAT3 / STAT5 signaling pathways.

[0088] Preferably, the carrier protein suitable for the present invention is capable of binding to the neonatal Fc receptor (FcRn) (such as albumin), or the carrier protein is bound to a protein capable of binding to FcRn (such as albumin binding protein).

[0089] Human serum albumin (HSA) is one of the most stable and most abundant proteins in human serum (35-50 g / L), accounting for half of the proteins in serum. Its main functions are to transport substances (such as hormones, fatty acids, etc.), maintain pH and osmotic pressure, etc. HSA in this article includes wild-type HSA and modified HSA.

[0090] HSA is a fully α-helical protein with a molecular weight of 66.5 kDa. It 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 then be recycled to the cell surface and released back into the blood, thereby preventing HSA from entering the lysosome and being degraded. FcRn mainly binds to DIII and part of DI.

[0091] The precursor sequence of HSA is shown in SEQ ID NO: 16 (Uniprot P02768), and all positions related to HSA herein are numbered with reference to SEQ ID NO: 16. Residues 1-24 in the precursor sequence of HSA are signal peptides, and native HSA contains residues 25-609 of SEQ ID NO:16.

[0092] In some embodiments, the carrier protein is HSA. Preferably, the insertion of the polypeptide of interest does not affect the binding of HSA to FcRn. In some embodiments, the polypeptide of interest is inserted into a loop of HSA, the loop being selected from the loops located at 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, preferably, the loops located at D56-L66, V293-L305 and A362-D365. In some embodiments, the insertion site of the polypeptide of interest is amino acid residue D56, A300, C361 or A362 of HSA.

[0093] The relative position between the carrier protein and the polypeptide of interest can be controlled by deleting one or more amino acids in the loops of the carrier protein, such as HSA, or without deleting amino acids in the loops of the carrier protein.

[0094] HSA can also be modified to improve its properties. For example, the free cysteine ​​in wild-type HSA can be replaced with other amino acids, such as serine. In some embodiments, the HSA comprises the amino acid substitution C58S.

[0095] In some embodiments, the carrier protein has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16 and has the same or similar structure as wild-type HSA.

[0096] In some embodiments, the N-terminus and C-terminus of the polypeptide of interest are connected to the carrier protein through a joint. In some embodiments, the N-terminus of the polypeptide of interest is connected to the carrier protein through a joint, and the C-terminus of the polypeptide of interest is directly connected to the carrier protein. In some embodiments, the N-terminus of the polypeptide of interest is directly connected to the carrier protein, and the C-terminus of the polypeptide of interest is connected to the carrier protein through a joint. In some embodiments, the N-terminus and C-terminus of the polypeptide of interest are directly connected to the carrier protein. In some embodiments, the joint 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. In some embodiments, the joint is selected from a spiral joint, or a flexible joint (such as a GS joint and a polyglycine joint).

[0097] In some embodiments, the fusion polypeptide of the present invention comprises a carrier protein HSA and a circularly rearranged IL-2, wherein the circularly rearranged IL-2 comprises four α-helical bundles, H3, H4, H1 and H2, or H4, H1, H2 and H3, from N-terminus to C-terminus, and wherein the circularly rearranged IL-2 is inserted into a loop of HSA, the loop being selected from the loops at 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 and A561-T566, the positions of which are referred to in SEQ ID NO: In some embodiments, the circulating rearranged IL-2 comprises an amino acid sequence of SEQ ID NO: 2, 3, 4 or 5. In some embodiments, the loop is selected from the loops located at D56-L66, V293-L305 and A362-D365 of HSA. In some embodiments, the insertion site of the circulating rearranged IL-2 is selected from D56, A300, C361 and A362 of HSA.

[0098] In some embodiments, the circulating rearranged IL-2 comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the loop is located at A362-D365 of HSA, preferably, the insertion site is C361 of HSA. In some embodiments, the N-terminus of the circulating rearranged IL-2 is connected to C361 of HSA via the linker EAAAKAEAAA (SEQ ID NO: 19), the C-terminus is connected to D365 of HSA via the linker GS, and residues 362-364 of HSA are deleted.

[0099] In some embodiments, the circulating rearranged IL-2 comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the loop is located at D56-L66 of HSA, preferably, the insertion site is D56 of HSA. In some embodiments, the N-terminus of the circulating rearranged IL-2 is connected to D56 of HSA through the linker AAAAK (SEQ ID NO: 20), and the C-terminus is directly connected to E57 of HSA.

[0100] In some embodiments, the circulating rearranged IL-2 comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the loop is located at V293-L305 of HSA, preferably, the insertion site is A300 of HSA. In some embodiments, the N-terminus of the circulating rearranged IL-2 is directly connected to A300 of HSA, and the C-terminus is connected to D301 of HSA via linker G.

[0101] In some embodiments, the circulating rearranged IL-2 consists of the amino acid sequence SEQ ID NO: 4. In some embodiments, the loop is located at A362-D365 of HSA, preferably, the insertion site is A362 of HSA. In some embodiments, the N-terminus of the circulating rearranged IL-2 is directly linked to A362 of HSA, and the C-terminus is directly linked to A363 of HSA.

[0102] In some embodiments, the present invention provides a fusion polypeptide comprising a carrier protein HSA and a circularly rearranged IL-15, wherein the circularly rearranged IL-15 comprises four α-helical bundles, H3, H4, H1 and H2, from N-terminus to C-terminus, and wherein the circularly rearranged IL-15 is inserted into a loop of HSA, the loop being selected from the loops at 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, the positions of which are referred to in SEQ ID NO:16, the HSA shields the CD215 binding site of the circulating rearranged IL-15, thereby blocking the accessibility of the site. In some embodiments, the circulating rearranged IL-15 comprises the amino acid sequence of SEQ ID NO:7.

[0103] In some embodiments, the fusion polypeptide of the present invention comprises an amino acid sequence of one of SEQ ID NOs: 8 to 11. In some embodiments, the fusion polypeptide of the present invention comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with an amino acid sequence of one of SEQ ID NOs: 8 to 11, wherein the fusion polypeptide comprises a native CD25 binding site, and has an activity comparable to or improved on that of native IL-2, such as the activity of activating JAK1 / JAK3 and STAT3 / STAT5 signaling pathways.

[0104] In some embodiments, the fusion polypeptide of the present invention has a more persistent immunostimulatory effect. In some embodiments, the fusion polypeptide of the present invention has a higher safety.

[0105] 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%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more compared to native IL-2. 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%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more compared to native IL-15.

[0106] IV. Expression of Fusion Peptides

[0107] In order to express the fusion polypeptide of the present invention, the present invention also provides a polynucleotide encoding the fusion polypeptide of the present invention.

[0108] Nucleic acid molecules of all or part of the nucleic acid sequence 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 sequence.

[0109] The polynucleotides of the present invention can be amplified using cDNA, mRNA or genomic DNA as templates and suitable oligonucleotide primers according to standard PCR amplification techniques. The nucleic acids amplified in this way can be cloned into suitable vectors and characterized by DNA sequence analysis.

[0110] The polynucleotides of the present invention can be prepared by standard synthetic techniques, for example, using an automated DNA synthesizer.

[0111] The present invention also relates to complementary strands of nucleic acid molecules described herein. Nucleic acid molecules complementary to other nucleotide sequences are molecules that are fully complementary to the nucleotide sequences so that they can hybridize with other nucleotide sequences to form stable duplexes. Of course, the polynucleotides of the present invention do not include polynucleotides that hybridize only to poly A sequences (such as the 3' terminal poly (A) of mRNA) or to a complementary poly T (or U) residue.

[0112] The present invention also provides a vector comprising the polynucleotide of the present invention, preferably an expression vector. Further, the present invention also provides a host cell comprising the polynucleotide of the present invention or a vector (preferably, an expression vector). In some embodiments, the polynucleotide of the present invention is integrated into the genome of the host cell. In some embodiments, the polynucleotide of the present invention is not integrated into the genome of the host cell.

[0113] In some embodiments, a single expression vector is used to express the first polypeptide and the second polypeptide of the fusion polypeptide of the present invention. In some embodiments, different expression vectors are used to express the first polypeptide and the second polypeptide of the fusion polypeptide of the present invention.

[0114] The selection of expression vector is related to the host cell for expressing the fusion polypeptide. Host cells that can be used to express the fusion polypeptide of the present invention include but are not limited to bacteria (including Escherichia coli), yeast, insect cells and mammalian cells, such as COS, CHO, HeLa and 293-6E cells. Expression vectors suitable for various host cells are known in the art. For example, expression vectors suitable for bacteria include, but are not limited to, pET vectors (such as pET-28a, pET-30a, pET-32a and pET-40a, etc.), pEX vectors (such as pEX-1), pGH112, pUC118 and pEZZ18; expression vectors suitable 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; expression vectors suitable for mammalian cells include, but are not limited to, pCMVInt, pGL4.23, pX334, pX458, pBiFC-CC155, pDP4rs, pDC312, and pcDNA.

[0115] In some embodiments, the host cell is a 293-6E cell. In some embodiments, the expression vector is pcDNA3.4.

[0116] The inclusion of a signal peptide in a precursor polypeptide will help the secretion and / or processing of the expressed polypeptide. Therefore, in some embodiments, the polynucleotide of the present invention also comprises a sequence encoding a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of METDTLLLWVLLLWVPGSTG (SEQ ID NO: 18).

[0117] The polynucleotide or vector of the present invention can be transferred (transfected) into the selected host cell by methods known in the art, such as calcium chloride transformation and calcium phosphate treatment for Escherichia coli, electroporation, lipofectamine treatment or PEI treatment for mammalian cells. Cells transformed by the vector can be screened based on antibiotic resistance genes (e.g., amp, gpt, neo and hyg genes) in the vector.

[0118] After expression, the recombinant fusion protein 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, etc. Substantially pure compositions having a purity of at least about 90 to 95% are preferred, and 98 to 99% or higher purity is most preferred for pharmaceutical use.

[0119] To facilitate purification, the fusion polypeptide of the present invention may further comprise a tag sequence, including but not limited to a His6 tag, a FLAG tag, and the like.

[0120] In some embodiments, the fusion polypeptide of the present invention comprises an amino acid sequence of one of SEQ ID NOs: 12 to 15. In some embodiments, the fusion polypeptide of the present invention comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with an amino acid sequence of one of SEQ ID NOs: 12 to 15, wherein the fusion polypeptide comprises a native CD25 binding site, and has an activity comparable to or improved on that of native IL-2, such as the activity of activating JAK1 / JAK3 and STAT3 / STAT5 signaling pathways.

[0121] 5. Pharmaceutical Composition

[0122] The present invention provides a pharmaceutical composition comprising a fusion polypeptide of the present invention. In one embodiment, 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.

[0123] Pharmaceutically acceptable carriers include, but are not limited to, solvents, emulsifiers, buffers, stabilizers, etc. The solvents include water, aqueous solutions, and non-aqueous solvents (eg, vegetable oils).

[0124] The pharmaceutical composition of the present invention can be administered by any suitable route, including subcutaneous, intramuscular, intraarticular, intravenous, intradermal, intraperitoneal, intranasal, intracranial, parenteral administration. Preferably, the pharmaceutical composition of the present invention is administered intravenously. It should be understood that the route of administration can vary with the therapeutic agent, the condition and age of the recipient, and the disease being treated.

[0125] The pharmaceutical composition of the present invention may be in the form of a solution or a lyophilized preparation.

[0126] In some embodiments, the pharmaceutical composition of the present invention is provided in the form of a lyophilized powder to be rehydrated before administration. The pharmaceutical composition of the present invention can also be provided in a liquid form, which can be directly administered to a patient. In some embodiments, the composition is provided in a pre-filled syringe in a liquid form.

[0127] In some embodiments, the compositions of the present invention are encapsulated in liposomes. In some embodiments, the liposomes can be coated with a flexible water-soluble polymer that avoids uptake by organs of the mononuclear phagocyte system, primarily the liver and spleen. Suitable hydrophilic polymers for coating liposomes include, but are not limited to, PEG, polyvinyl pyrrolidone, polyvinyl methyl ether, polymethyl oxazoline, polyethyl oxazoline, polyhydroxypropyl oxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethylcellulose hydroxyethylamide, hydrophilic polyvinyl alcohol, and the like.

[0128] The pharmaceutical composition of the present invention can be administered once or multiple times depending on the dosage and frequency required and tolerated by the patient. In any case, the pharmaceutical composition administered should provide a sufficient amount of the protein of the present invention to effectively treat the patient.

[0129] 6. Therapeutic Application

[0130] The present invention also provides methods of using the fusion polypeptides of the present invention to treat diseases, such as diseases involving immunosuppression.

[0131] In some embodiments, a method for treating cancer is provided, comprising administering a therapeutically effective amount of a fusion polypeptide or pharmaceutical composition of the present invention to a subject in need thereof. The cancers include, but are not limited to, lung cancer, liver cancer, kidney cancer, head and neck cancer, colorectal cancer, gastric cancer, nasopharyngeal carcinoma, glioma, melanoma, and osteosarcoma.

[0132] In some embodiments, a method for activating immune cells or increasing the proliferation of immune cells is provided, comprising administering an effective amount of the fusion polypeptide of the present invention or the pharmaceutical composition of the present invention to a subject in need thereof. In some embodiments, the immune cells are T cells or NK cells.

[0133] In some embodiments, the use of the fusion polypeptide or pharmaceutical composition of the present invention in the preparation of a medicament for treating cancer is provided. The cancer includes but is not limited to lung cancer, liver cancer, kidney cancer, head and neck cancer, colorectal cancer, gastric cancer, nasopharyngeal carcinoma, glioma, melanoma and osteosarcoma.

[0134] In some embodiments, the use of the fusion polypeptide or pharmaceutical composition of the present invention in the preparation of a medicament for activating immune cells or increasing the proliferation of immune cells is provided. In some embodiments, the immune cells are T cells or NK cells. Example

[0135] Through the following examples, those skilled in the art will understand the present invention more clearly. It should be understood that the examples are only for illustration and are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the gene cloning operation can be specifically referred to Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0136] Example 1. Construction of expression vector

[0137] The construction of the expression vector was completed by Nanjing GenScript Biotechnology Co., Ltd., including: synthesizing a nucleic acid encoding the amino acid sequence of SEQ ID NO: 12-15, wherein the nucleic acid additionally comprises a nucleotide sequence encoding the signal peptide of SEQ ID NO: 18; constructing the nucleic acid into the mammalian cell expression vector pcDNA3.4 by molecular cloning methods, and amplifying and purifying the plasmid.

[0138] Example 2: Expression of fusion polypeptide

[0139] In this example, 293-6E cells were transfected with nucleic acid encoding the fusion polypeptide for eukaryotic expression. The expression and purification of the fusion polypeptide were performed by Nanjing GenScript Biotechnology Co., Ltd. The process is briefly described as follows:

[0140] -293-6E cells in serum-free FreeStyle TM The cells were cultured in Erlenmeyer flasks (Corning Inc., Acton, MA) in 293 Expression medium (Thermo Fisher Scientific, Carlsbad, CA, USA) in a shaking incubator (VWR Scientific, Chester, PA) at 37°C and 5% CO2.

[0141] - One day before transfection, dilute cells to the appropriate density.

[0142] -On the day of transfection, the plasmid mixture (the mass ratio of the plasmid encoding the first polypeptide to the second polypeptide is 1:1) and the transfection reagent (such as polyetherimide, Polyetherimide, PEI) are mixed in a suitable ratio (such as a mass ratio of 1:3) in the culture medium, and then added to the cell culture medium for transfection to achieve secretory expression of the target protein.

[0143] - After six days, the cell survival rate is about 50%-80%. The supernatant is obtained by centrifugation, which contains the target protein (the expressed polypeptide does not contain a signal peptide).

[0144] -The supernatant was filtered through a 0.22 μm filter and loaded onto the HisTrap at 3 ml / min. TM After washing and elution in a FF Crud 5 ml (GE, catalog number 17-5286-01) column, the purified proteins were pooled and stored in phosphate buffered saline (PBS pH 7.2) by buffer exchange.

[0145] The molecular weight, purity and sequence coverage of the purified protein were evaluated by SDS-PAGE, immunoblotting (results not shown), high performance liquid chromatography coupled with molecular sieve separation (SEC-HPLC) and liquid chromatography mass spectrometry (LC-MS). The primary antibody used in immunoblotting 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).

[0146] The results are as follows Figures 4A-4C , 5A-5C, 6A-6C and 7A-7C, Figures 4A-4C : Opening in loop 1 of IL-2 to form a circularly rearranged IL-2, with the insertion site at C361 of HSA (SEQ ID NO: 12); Figures 5A-5C : Opening in loop 2 of IL-2 to form a circularly rearranged IL-2, with the insertion site at D56 of HSA (SEQ ID NO: 13); Figures 6A-6C : Opening in loop 2 of IL-2 to form a circularly rearranged IL-2 with an insertion site at A300 of HSA (SEQ ID NO: 14); and Figures 7A-7C : Loop 1 of IL-2 is opened to form a circularly rearranged IL-2, with the insertion site at A362 of HSA (SEQ ID NO: 15). Figure 4A , 5A , 6A and 7A show the structural model of HSA shielding the CD25 binding site on IL-2, Figure 4B ,5B , 6B and 7B show the SDS-PAGE results of reduced and non-reduced fusion polypeptides, while Figure 4C , 5C , 6C and 7C show the results of molecular sieve analysis.

[0147] like Figures 4A-4C As shown in Figures 5A-5C, 6A-6C and 7A-7C, the four fusion polypeptides all achieved high expression levels, and SDS-PAGE and SEC-HPLC showed high purity and high uniformity; high sequence coverage was detected by LC-MS, confirming the integrity and correctness of the purified fusion polypeptides.

[0148] Example 3: Detection of fusion polypeptide activity

[0149] In this example, HEK-Blue TM IL-2 cell line (InvivoGen), the activity of these fusion proteins in activating downstream signaling pathways was detected at the cellular level. TM The IL-2 cell line was 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 receptor molecules, human JAK3 kinase and transcription factor STAT5. In addition, the secreted embryonic alkaline phosphatase (SEAP) reporter gene regulated by STAT5 was also transferred. When this cell line is stimulated by IL-2, it activates the downstream signaling pathway to activate STAT5 and upregulate the secretion and expression of SEAP.

[0150] The above test was completed by Nanjing Topology Biotechnology Co., Ltd. The process is briefly described as follows:

[0151] 1. Experimental Preparation

[0152] 1.1. Prepare QUANTI-Blue solution: Thaw QB reagent and QB buffer at room temperature, add to 98 mL of sterile water, mix thoroughly, dispense into 10 mL / tube, and store at -20℃ away from light.

[0153] 1.2. Serum inactivation: Take 45 mL of serum (FBS) into a 50 mL centrifuge tube, heat-inactivate in a 56°C water bath for 30 min (mix every 10 min), and store at 4°C.

[0154] 1.3. Culture medium

[0155] Growth medium: DMEM + 10% FBS + 1% PS + 100μg / mL Normocin + 1μg / ml puromycin + 1X HEK-Blue TMCLR Selection; Test medium: DMEM+10% FBS (inactivated)+1% PS+100 μg / mL Normocin; Freezing medium: DMEM+20% FBS+10% DMSO.

[0156] 2. Cell culture, passaging and storage

[0157] 2.1. Place the cells in growth medium and culture and subculture in a 37°C, 5% CO2 incubator;

[0158] 2.2. When the cell confluence reaches 70-80% and the cells are in good condition, discard the supernatant and resuspend the cells in growth medium preheated in a 37°C water bath. After counting the cells, centrifuge at 200×g for 5 min. Discard the supernatant and resuspend the cells in freezing medium precooled at 4°C and adjust the cell density to 5-7×10 6 cells / mL, 1 mL per tube, placed in a programmed cooling box, placed at -80°C overnight and then stored in liquid nitrogen for a long term.

[0159] 3. HEK-Blue IL-2 System Validation

[0160] 3.1. Preparation of HEK-Blue IL-2 cell suspension: Rinse the cells twice with pre-warmed DPBS and resuspend them in pre-warmed DPBS to form a single cell suspension. After counting the cells, centrifuge at 200 × g for 5 min. Resuspend them in pre-warmed test medium at a cell density of 2.8 × 10 5 cells / mL;

[0161] 3.2. Human IL-2 protein (positive control, AcroBiosystems, catalog number IL2-H4113) and HSA (negative control) were prepared as follows: starting concentration 650 pM, 5-fold dilution, 8 gradients.

[0162] 3.3. Add 20 μL of samples with a series of concentration gradients and 180 μL of cell suspension to each well of the culture plate (three replicates for each sample), and culture in a 37°C, 5% CO2 incubator for 20-24 hours.

[0163] 3.4. Add the supernatant of each culture to a 96-well plate, 20 μL / well, and add 180 μL / well of QUANTI-Blue solution that has been thawed and restored to room temperature. Incubate at 37°C for 1-3 hours, read the absorbance OD 620-655nm with a microplate reader, and analyze the data.

[0164] 4. Activity detection of fusion polypeptide.

[0165] The activity of the fusion polypeptide prepared in step 2 is detected by the method in step 3, wherein each fusion polypeptide is also prepared into a series of gradient dilution samples according to the method in step 3.2.

[0166] The results are as follows Figure 8-11 As shown, Figure 8 : Opening in loop 1 of IL-2 to form a circularly rearranged IL-2, with the insertion site at C361 of HSA (SEQ ID NO: 12); Fig. 9 : Opening in loop 2 of IL-2 to form a circularly rearranged IL-2, with the insertion site at D56 of HSA (SEQ ID NO: 13); Fig.10 : Opening in loop 2 of IL-2 to form a circularly rearranged IL-2 with an insertion site at A300 of HSA (SEQ ID NO: 14); and Fig.11 : Loop 1 of IL-2 is opened to form a circularly rearranged IL-2, with the insertion site at A362 of HSA (SEQ ID NO: 15).

[0167] Comparison of the 50% maximal effect concentration (EC50) of each fusion polypeptide tested with the EC50 of human IL-2 is shown in Tables 1-4 below.

[0168] Table 1. Comparison of the fusion polypeptide of SEQ ID NO: 12 and human IL-2

[0169] Human IL-2 Fusion polypeptide of SEQ ID NO:12 EC50(pM) 5.123 1.750

[0170] Table 2. Comparison of the fusion polypeptide of SEQ ID NO: 13 and human IL-2

[0171] Human IL-2 Fusion polypeptide of SEQ ID NO:13 EC50(pM) 7.934 6.356

[0172] Table 3. Comparison of the fusion polypeptide of SEQ ID NO: 14 and human IL-2

[0173] Human IL-2 Fusion polypeptide of SEQ ID NO: 14 EC50(pM) 2.583 5.859

[0174] Table 4. Comparison of the fusion polypeptide of SEQ ID NO: 15 and human IL-2

[0175] Human IL-2 Fusion polypeptide of SEQ ID NO:15 EC50(pM) 2.755 5.253

[0176] The results showed that the fusion polypeptide of the present invention exhibited an activity comparable to or higher than that of wild-type IL-2, while no corresponding response was shown using HSA (results not shown). Sequence Listing <110> Nanjing Normal University <120> Fusion polypeptide and its use <130> I2019TC4009CB <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 153 <212> PRT <213> Homo sapiens <400> 1 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser Thr Leu Thr 145 150 <210> 2 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> IL-2-1 <400> 2 Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly 1 5 10 15 Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile 20 25 30 Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser 35 40 45 Thr Leu Thr Gly Ser Gly Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln 50 55 60 Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly 65 70 75 80 Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys 85 90 95 Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu 100 105 110 Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala 115 120 125 <210> 3 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> IL-2* <400> 3 Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr 1 5 10 15 Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu 20 25 30 Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg 35 40 45 Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser 50 55 60 Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val 65 70 75 80 Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr 85 90 95 Leu Thr Gly Ser Gly Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu 100 105 110 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 115 120 125 Asn Asn 130 <210> 4 <211> 135 <212> PRT <213> Artificial Sequence <220> <223> IL-2-2 <400> 4 Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr 1 5 10 15 Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu 20 25 30 Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg 35 40 45 Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser 50 55 60 Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val 65 70 75 80 Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr 85 90 95 Leu Thr Gly Ser Gly Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu 100 105 110 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 115 120 125 Asn Asn Tyr Lys Asn Pro Lys 130 135 <210> 5 <211> 135 <212> PRT <213> Artificial Sequence <220> <223> IL-2-3 <400> 5 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 1 5 10 15 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 20 25 30 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 35 40 45 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 50 55 60 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 65 70 75 80 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 85 90 95 Ile Ser Thr Leu Thr Gly Ser Gly Pro Ala Ser Ser Ser Thr Lys Lys 100 105 110 Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu 115 120 125 Asn Gly Ile Asn Asn Tyr Lys 130 135 <210> 6 <211> 162 <212> PRT <213> Homo sapiens <400> 6 Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr 1 5 10 15 Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His 20 25 30 Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40 45 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 50 55 60 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 65 70 75 80 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 85 90 95 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 100 105 110 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 115 120 125 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 130 135 140 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 145 150 155 160 Thr Ser <210> 7 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> IL-15-1 <400> 7 Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn 1 5 10 15 Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys 20 25 30 Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val 35 40 45 His Ile Val Gln Met Phe Ile Asn Thr Ser Gly Gly Gly Gly Asn Trp 50 55 60 Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser 65 70 75 80 Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser 85 90 95 Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile 100 105 110 Ser Leu Glu Ser Gly Asp 115 <210> 8 <211> 720 <212> PRT <213> Artificial Sequence <220> <223> C361-IL2-1 <400> 8 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Ser Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 The Ser Al of Lys Gln Arg Leu Lys Cys Al Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Can His Thr Glu Cys His Gly Asp Lead to Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Ser 290,295,300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Glu Ala Ala Ala Lys Ala Glu 355 360 365 Ala Ala Ala Asp Leu I Will Be Asn I Asn I Will I Will Glu 370 375 380 Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr 385 390 395 400 Only Thr Ile Val Glu Pheu Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser 405 410 415 Ile Ile Ser Thr Leu Thr Gly Ser Gly Pro Ala Ser Ser Ser Thr Lys 420 425 430 Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile 435 440 445 Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu 450 455 460 Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu 465 470 475 480 Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu 485 490 495 Ala Gly Ser Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe 500 505 510 Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu 515 520 525 Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val 530 535 540 Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu 545 550 555 560 Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro 565,570,575 Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu 580,585,590 Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val 595,600,605 Thr Lys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser 610 615 620 Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu 625 630 635 640 Ser Glu Lys Glu Arg 645,650,655 Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys Lys Pro 660,665,670 Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala 675,680,685 Phe Val Glu Lys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala 690,695,700 Glu Glu Gly Lys Lys Leu Val Ala Ser Gln Ala Ala Leu Gly Leu 705 710 715 720 <210> 9 <211> 726 <212> PRT <213> Artificial Sequence <220> <223> D56-IL2-2 <400> 9 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Ser Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Ala Ala Ala Ala Ala Lys Leu Thr 50 55 60 Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu 65 70 75 80 Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val 85 90 95 Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu 100 105 110 Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr 115 120 125 Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe 130 135 140 Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 145 150 155 160 Gly Ser Gly Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 165 170 175 Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 180 185 190 Tyr Lys Asn Pro Lys Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His 195 200 205 Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr 210 215 220 Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn 225 230 235 240 Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu 245 250 255 Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu 260 265 270 Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro 275 280 285 Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala 290,295,300 Phe Thr Glu Cys Gln Ala Asp Lys Ala Cys Leu Leu 305 310 315 320 Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys 325 330 335 Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe 340 345 350 Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu 355 360 365 Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr 370 375 380 Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp 385 390 395 400 Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu 405 410 415 Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Wing 420 425 430 Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala 435 440 445 Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys 450 455 460 Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro 465 470 475 480 Asp Tyr Ser Val Val Val Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr 485,490,495 Thr Leu Glu Lys Cys Ala Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Ala 500 505 510 Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu 515,520,525 Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe 530 535 540 Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser 545 550 555 560 Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser 565,570,575 Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp 580,585,590 Tyr Leo Ser Will Be Leo Asn Gln Leo Cys Leo Will His Glu Lys Thr 595 600 605 Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn 610 615 620 Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro 625 630 635 640 Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr 645 650 655 Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu 660 665 670 Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val 675 680 685 Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp 690 695 700 Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser 705 710 715 720 Gln Ala Ala Leu Gly Leu 725 <210> 10 <211> 721 <212> PRT <213> Artificial Sequence <220> <223> A300-IL-2-3 <400> 10 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Ser Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 The Ser Al of Lys Gln Arg Leu Lys Cys Al Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys His Gly Asp Leads To Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asn Pro Lys Leu 290,295,300 Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu 305 310 315 320 Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu 325 330 335 Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp 340 345 350 Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu 355 360 365 Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu 370 375 380 Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu 385 390 395 400 Thr Gly Ser Gly Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln 405 410 415 Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn 420 425 430 Asn Tyr Lys Gly Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 435 440 445 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 450 455 460 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 465 470 475 480 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 485 490 495 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 500 505 510 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 515 520 525 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 530 535 540 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 545 550 555 560 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 565 570 575 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 580 585 590 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 595 600 605 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 610 615 620 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 625 630 635 640 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 645 650 655 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 660 665 670 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 675 680 685 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 690 695 700 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 705 710 715 720 Leu <210> 11 <211> 720 <212> PRT <213> Artificial Sequence <220> <223> A362-IL2-3 <400> 11 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Ser Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 The Ser Al of Lys Gln Arg Leu Lys Cys Al Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Can His Thr Glu Cys His Gly Asp Lead to Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Ser 290,295,300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr With Glu Lys Cys Ala Asn Pro Lys With Thr Arg 355 360 365 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 370 375 380 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 385 390 395 400 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 405 410 415 Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 420 425 430 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 435 440 445 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly 450 455 460 Ser Gly Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu 465 470 475 480 His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr 485 490 495 Lys Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe 500 505 510 Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu 515 520 525 Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val 530 535 540 Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu 545 550 555 560 Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro 565 570 575 Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu 580 585 590 Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val 595 600 605 Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser 610 615 620 Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu 625 630 635 640 Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg 645 650 655 Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro 660 665 670 Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala 675 680 685 Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala 690 695 700 Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 705 710 715 720 <210> 12 <211> 726 <212> PRT <213> Artificial Sequence <220> <223> C361-IL-2-1-His <400> 12 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Ser Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 The Ser Al of Lys Gln Arg Leu Lys Cys Al Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Glu Ala Ala Ala Lys Ala Glu 355 360 365 Ala Ala Ala Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu 370 375 380 Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr 385 390 395 400 Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser 405 410 415 Ile Ile Ser Thr Leu Thr Gly Ser Gly Pro Ala Ser Ser Ser Thr Lys 420 425 430 Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile 435 440 445 Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu 450 455 460 Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu 465 470 475 480 Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu 485 490 495 Ala Gly Ser Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe 500 505 510 Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu 515 520 525 Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val 530 535 540 Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu 545 550 555 560 Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro 565,570,575 Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu 580,585,590 Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val 595,600,605 Thr Lys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser 610 615 620 Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu 625 630 635 640 Ser Glu Lys Glu Arg 645,650,655 Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys Lys Pro 660,665,670 Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala 675,680,685 Phe Val Glu Lys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala 690,695,700 Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 705 710 715 720 His His His His His His 725 <210> 13 <211> 732 <212> PRT <213> Artificial Sequence <220> <223> D56-IL-2-2-His <400> 13 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Ser Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Ala Ala Ala Ala Ala Lys Leu Thr 50 55 60 Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu 65 70 75 80 Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val 85 90 95 Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu 100 105 110 Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr 115 120 125 Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe 130 135 140 Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 145 150 155 160 Gly Ser Gly Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 165 170 175 Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 180 185 190 Tyr Lys Asn Pro Lys Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His 195 200 205 Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr 210 215 220 Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn 225 230 235 240 Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu 245 250 255 Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu 260 265 270 Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg His Pro 275 280 285 Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala 290,295,300 Phe Thr Glu Cys Gln Ala Asp Lys Ala Cys Leu Leu 305 310 315 320 Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys 325 330 335 Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe 340 345 350 Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu 355 360 365 Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr 370 375 380 Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp 385 390 395 400 Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu 405 410 415 Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Wing 420 425 430 Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala 435 440 445 Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys 450 455 460 Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro 465 470 475 480 Asp Tyr Ser Val Val Val Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr 485,490,495 Thr Leu Glu Lys Cys Ala Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Ala 500 505 510 Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu 515,520,525 Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe 530 535 540 Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser 545 550 555 560 Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser 565,570,575 Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp 580 585 590 Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr 595 600 605 Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn 610 615 620 Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro 625 630 635 640 Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr 645 650 655 Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu 660 665 670 Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val 675 680 685 Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp 690 695 700 Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser 705 710 715 720 Gln Ala Ala Leu Gly Leu His His His His His His 725 730 <210> 14 <211> 727 <212> PRT <213> Artificial Sequence <220> <223> A300-IL-2-3_His <400> 14 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Ser Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 The Ser Al of Lys Gln Arg Leu Lys Cys Al Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Can His Thr Glu Cys His Gly Asp Lead to Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asn Pro Lys Leu 290 295 300 Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu 305 310 315 320 Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu 325 330 335 Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp 340 345 350 Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu 355 360 365 Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu 370 375 380 Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu 385 390 395 400 Thr Gly Ser Gly Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln 405 410 415 Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn 420 425 430 Asn Tyr Lys Gly Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 435 440 445 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 450 455 460 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 465 470 475 480 Thr Thr Thr Thr Thr Glu Lys Cys 485,490,495 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 500 505 510 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu And Lys Gln Asn Cys 515,520,525 Leu Phe Glu Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 530 535 540 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 545 550 555 560 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Lys His 565,570,575 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 580,585,590 Your Asn Gln Your Cys Will Your Glu Lys Thr Pro Will Be Asp Arg 595 600 605 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 610 615 620 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 625 630 635 640 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 645 650 655 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 660 665 670 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 675 680 685 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 690 695 700 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 705 710 715 720 Leu His His His His His His 725 <210> 15 <211> 726 <212> PRT <213> Artificial Sequence <220> <223> A362-IL-2-3_His <400> 15 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Ser Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 The Ser Al of Lys Gln Arg Leu Lys Cys Al Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys His Gly Asp Leads To Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Ser 290,295,300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Asn Pro Lys Leu Thr Arg 355 360 365 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 370 375 380 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 385 390 395 400 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 405 410 415 Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 420 425 430 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 435 440 445 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly 450 455 460 Ser Gly Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu 465 470 475 480 His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr 485 490 495 Lys Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe 500 505 510 Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu 515 520 525 Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val 530 535 540 Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu 545 550 555 560 Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro 565 570 575 Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu 580 585 590 Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val 595 600 605 Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser 610 615 620 Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu 625 630 635 640 Ser Glu Lys Glu Arg 645,650,655 Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys Lys Pro 660,665,670 Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala 675,680,685 Phe Val Glu Lys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala 690,695,700 Glu Glu Gly Lys Lys Leu Val Ala Ser Gln Ala Ala Leu Gly Leu 705 710 715 720 His Hiss Hiss Hiss 725 <210> 16 <211> 609 <212> PRT <213> Homo sapiens <400> 16 Met Lys Trp Val Thr Phe Ile Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Leu Lys Glu Cys Cys Glu 290,295,300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 405 410 415 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 595 600 605 Leu <210> 17 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 17 Gly Gly Gly Gly 1 <210> 18 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> signal peptide <400> 18 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly 20 <210> 19 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 19 Glu Ala Ala Ala Lys Ala Glu Ala Ala Ala 1 5 10 <210> 20 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 20 Ala Ala Ala Ala Ala Lys 1 5

Claims

1. A fusion polypeptide consisting of an amino acid sequence of one of SEQ ID NOs: 8-11, or consisting of an amino acid sequence of one of SEQ ID NOs: 8-11 and a tag sequence.

2. A pharmaceutical composition comprising the fusion polypeptide of claim 1.

3. Use of the fusion polypeptide of claim 1 in preparing a drug for treating cancer.

4. An isolated polynucleotide encoding the fusion polypeptide of claim 1. An expression vector comprising the polynucleotide of claim 4. A host cell comprising the polynucleotide of claim 4 or the expression vector of claim 5.

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