CN113912734A - Fusion polypeptides and uses thereof - Google Patents

Fusion polypeptides and uses thereof Download PDF

Info

Publication number
CN113912734A
CN113912734A CN202010650886.XA CN202010650886A CN113912734A CN 113912734 A CN113912734 A CN 113912734A CN 202010650886 A CN202010650886 A CN 202010650886A CN 113912734 A CN113912734 A CN 113912734A
Authority
CN
China
Prior art keywords
leu
glu
lys
ala
val
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202010650886.XA
Other languages
Chinese (zh)
Inventor
沈健
周家宏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing University
Nanjing Normal University
Original Assignee
Nanjing Normal University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanjing Normal University filed Critical Nanjing Normal University
Priority to CN202010650886.XA priority Critical patent/CN113912734A/en
Priority to US18/004,634 priority patent/US20230322882A1/en
Priority to PCT/CN2021/105187 priority patent/WO2022007885A1/en
Priority to EP21838465.9A priority patent/EP4186928A1/en
Publication of CN113912734A publication Critical patent/CN113912734A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Abstract

The present invention relates to a fusion polypeptide comprising a carrier protein and a polypeptide of interest, wherein the carrier protein has a plurality of helical domains connected by loops, the polypeptide of interest is inserted into the loops of the carrier protein, and the carrier protein masks a site of interest on the polypeptide of interest, thereby blocking the accessibility of the site. The invention also relates to polynucleotides and vectors encoding the fusion polypeptides, and host cells comprising the polynucleotides and/or vectors. Further, the present invention also relates to the use of the fusion polypeptide of the present invention for treating cancer and/or activating immune cells.

Description

Fusion polypeptides and uses thereof
Technical Field
The present invention relates to fusion polypeptides. In particular, the invention relates to modified immune modulatory molecules and their use in the treatment of cancer.
Background
Modulation of the immune system by cytokines, such as activation of immune cells, is one strategy for cancer immunotherapy. However, certain sites on cytokines have an effect on their function (e.g., immunomodulatory effects).
For example, interleukin-2 (IL-2) may modulate the function of immune cells. IL-2, when bound to the receptors IL2R α (CD25), IL2R β (CD122) and IL2R γ (CD132), activates downstream signaling pathways, including JAK1 and JAK3 kinases and the transcription factor STAT5, to stimulate activation and proliferation of immune cells, such as T cells and Natural Killer (NK) cells. IL-2 can either signal through the CD25/CD122/CD132 trimer, or through the CD122/CD132 dimer activation signal channel. Binding of CD25 to IL-2 alters the conformation of IL-2, increasing its affinity for the CD122/CD132 dimer by a factor of 100 (KD increased from 1nM to 10 pM). The CD122/CD132 dimer is mainly expressed on the surfaces of CD8+ memory T cells and NK cells, while the CD25/CD122/CD132 trimer is abundantly expressed on the surface of regulatory T cells (Tregs) playing a role in immunosuppression. Thus, IL-2 acts to stimulate or inhibit the activation of the immune system, depending on the type of immune cell that is activated.
IL-2 is also a potential drug for cancer immunotherapy of great interest. Recombinant IL-2 (aldesleukin) was approved by the U.S. food and drug health administration (FDA) for the treatment of metastatic melanoma and renal cancer in 1998, the only IL-2-based drug approved to date, however, only 10% of patients respond and have significant side effects, primarily due to the reversible nature of IL-2, requiring large doses to stimulate immune activation, but high doses can cause side effects such as capillary leak syndrome.
Interleukin-15 (IL-15) also activates immune responses, and plays an important role in the differentiation and proliferation of T cells and NK cells, and the development of dendritic cells. IL-15 works similarly to IL-2 by binding to the CD122/CD132 receptor dimer to activate the downstream signaling pathway (JAK1/JAK3 and STAT3/STAT5), but its alpha receptor is distinct from IL-2 and is its own IL-15R alpha (CD215) receptor. It is believed that IL-15 is presented with high affinity to the CD122/CD132 receptor either "trans" (cell-cell contact) or "cis" (cis, on the same cell) after binding to the IL-15 Ra receptor on the cell membrane, and it has also been found that IL-15 can bind to and function with moderate affinity to CD122/CD132 without binding to IL-15 Ra.
Recent literature reports (Alexandra Berger et al 2019J for ImmunoTherapy for Cancer) that IL-15 alone shows more durable antitumor activity in mouse tumor models than the IL-15/IL-15 Ra receptor extracellular domain complex. The applicant believes that this may be because the complex, although capable of stimulating immunity rapidly and strongly, will also cause immune cell depletion more rapidly, reducing the durability of the response. It is also known to those skilled in the art that strong immune stimulation may pose safety problems.
In addition, cytokines, such as IL-2 and IL-15, have short half-lives in vivo and require continuous injection.
Accordingly, there is still a need to develop engineered recombinant cytokines such as IL-2 and IL-15 that are capable of specifically activating immunity and have a prolonged half-life, have improved activity, have a more durable ability to activate immunity, and/or have higher safety, thereby increasing their clinical utility and commercial transformation value.
Disclosure of Invention
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 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 masks a site of interest on the polypeptide of interest, thereby blocking the accessibility of the site.
In some embodiments, the polypeptide of interest is derived from a cytokine of the family of four α -helix bundle cytokines comprising, in order from N-terminus to C-terminus, four α -helix bundles of helix bundle 1(H1), helix bundle 2(H2), helix bundle 3(H3), and helix bundle 4 (H4).
In some embodiments, the polypeptide of interest is a cytokine of the circularly rearranged family of four α -helical bundle cytokines comprising H2, H3, H4, and H1 in order from N-terminus to C-terminus; h3, H4, H1 and H2; or four alpha-helical bundles of H4, H1, H2 and H3.
In some embodiments, the amino acid corresponding to the N-terminus of the cytokine that is not cyclically rearranged in the cyclically rearranged cytokine is linked to the amino acid corresponding to the C-terminus of the cytokine that is not cyclically rearranged by a linker. In some embodiments, the linker is a GS linker or polyglycine linker of 1-10 amino acids in length.
In some embodiments, the polypeptide of interest is selected from the group consisting of a cyclically rearranged IL-2 and a cyclically rearranged IL-15.
In some embodiments, the cyclically rearranged IL-2 comprises four α -helix bundles of H3, H4, H1, and H2, or H4, H1, H2, and H3, in order from N-terminus to C-terminus. In some embodiments, the cyclically rearranged IL-2 comprises the amino acid sequence of SEQ ID NO 2, 3, 4 or 5. In some embodiments, the site of interest is the CD25 binding site.
In some embodiments, the cyclically rearranged IL-15 comprises four α -helical bundles of H3, H4, H1, and H2 in order from N-terminus to C-terminus. In some embodiments, the cyclically 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.
In some embodiments, the carrier protein is albumin, preferably Human Serum Albumin (HSA). In some embodiments, the loop is selected from the group consisting of 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, positions numbered with reference to SEQ ID NO: 16. In some embodiments, the polypeptide of interest is a cyclically rearranged IL-2, and the loop is selected from the group consisting of loops D56-L66, V293-L305, and A362-D365 located on HSA. In some embodiments, the insertion site for the polypeptide of interest is selected from D56, a300, C361, and a362 of HSA.
In some embodiments, the invention provides a fusion polypeptide comprising a carrier protein HSA and a cyclically rearranged IL-2, wherein the cyclically rearranged IL-2 comprises, in order from N-terminus to C-terminus, H3, H4, H1 and H2, or four alpha-helix bundles of H4, H1, H2 and H3, and wherein the circularly rearranged IL-2 is inserted into a loop of HSA, the rings are selected from the group consisting of rings 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 are referenced to SEQ ID NO:16, and said HSA masks the CD25 binding site of said circularly rearranged IL-2, thereby blocking the accessibility of said site. In some embodiments, the cyclically 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 group consisting of loops D56-L66, V293-L305, and A362-D365 located on HSA. In some embodiments, the insertion site for the circularly rearranged IL-2 is selected from D56, a300, C361, and a362 of HSA.
In some embodiments, the invention provides a fusion polypeptide comprising a carrier protein HSA and a cyclically rearranged IL-15, wherein the cyclically rearranged IL-15 comprises four alpha-helical bundles of H3, H4, H1 and H2 in that order from N-terminus to C-terminus, and wherein the cyclically rearranged IL-15 is inserted in a loop of HSA selected from the group consisting of loops located 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 are referenced to SEQ ID NO:16, and said HSA masks the CD215 binding site of said circularly rearranged IL-15, thereby blocking the accessibility of said site. In some embodiments, the cyclically rearranged IL-15 comprises the amino acid sequence of SEQ ID NO 7.
In some embodiments, the present invention provides a fusion polypeptide comprising the amino acid sequence of one of SEQ ID NOs 8-11.
The invention also provides pharmaceutical compositions comprising the fusion polypeptides of the invention.
In a second aspect, the invention also provides a method of treating cancer or activating immune cells or increasing the proliferation of immune cells (e.g., T cells or NK cells) comprising administering to a subject in need thereof an effective amount of a fusion polypeptide of the invention or a pharmaceutical composition of the invention.
The invention also provides the use of a fusion polypeptide or a pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of cancer and in the manufacture of a medicament for activating immune cells or increasing the proliferation of immune cells (e.g., T cells or NK cells).
In some embodiments, there is also provided a fusion polypeptide or a pharmaceutical composition of the invention for use in treating cancer or activating immune cells or increasing proliferation of immune cells (e.g., T cells or NK cells).
In a third aspect, the invention also provides polynucleotides and vectors encoding the fusion polypeptides of the invention, and host cells comprising the polynucleotides and vectors.
Drawings
FIG. 1 shows a schematic three-dimensional structure of wild-type IL-2 binding to its receptor (based on PDB number 2ERJ), wherein "Loop 1" corresponds to S95-L100 of native IL-2 and "Loop 2" corresponds to N50-P54 of native IL-2. Unless otherwise indicated, positions referred to herein for IL-2 are numbered with reference to the IL-2 precursor sequence of SEQ ID NO:1(UniProt P60568) wherein amino acid residues 1-21 are the signal peptide sequence and the sequence of native IL-2 is amino acid residues 22-153 of SEQ ID NO: 1.
FIG. 2 shows a schematic three-dimensional structure of wild-type IL-15 binding to its receptor (based on PDB No. 4GS7), wherein the "opened loop" corresponds to S102-A105 of native IL-15. Unless otherwise indicated, positions referred to herein for IL-15 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 native IL-15 is amino acid residues 49-162 of SEQ ID NO: 6.
Fig. 3 shows a schematic three-dimensional structure of HSA.
FIGS. 4A-4C, 5A-5C, 6A-6C, and 7A-7C show the expression of fusion polypeptides of the invention and a structural model of HSA shielding the CD25 binding site on IL-2.
FIGS. 8-11 show in vitro activity assays for fusion polypeptides of the invention.
Detailed Description
While the invention will be described in conjunction with the embodiments enumerated below, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents, which may be included within the scope of the invention as defined by the appended claims. One skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of 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, usage of terms, described techniques, etc., the present application controls. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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 by reference in their entirety.
A, define
As used herein, the term "peptide" means a chain of at least two amino acids linked by peptide bonds. The term "polypeptide" is used interchangeably herein with the term "protein" and refers to a chain containing ten or more amino acid residues. All peptide and polypeptide chemical formulas or sequences herein are written from left to right, representing the direction from the amino terminus to the carboxy terminus.
As used herein, the term "dihedral angle in a peptide chain" refers to the angle at which two adjacent planes of peptide bonds can rotate about the alpha carbon atom between the two planes of peptide bonds.
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 one-letter and three-letter designations of the naturally occurring amino acids in proteins are used by conventional names 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, N.Y., 1989).
Figure RE-RE-GDA0002730424380000041
Figure RE-RE-GDA0002730424380000051
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 the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, preferably double-stranded DNA. The synthesis of the nucleic acid may use nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acids having altered base-pairing abilities or increased nuclease resistance.
As used herein, the term "encoding" 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 another start codon, such as GTG and TTG, and ends with a stop codon, such as TAA, TAG and TGA. The coding sequence may be a DNA, cDNA or recombinant nucleotide sequence.
As used herein, the term "hybridize" is a process in which nucleotide sequences that are 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 the conditions of a given stringent hybridization and wash.
Those skilled in the art are aware of various conditions for hybridization, such as stringent hybridization conditions and highly stringent hybridization conditions. See, e.g., Sambrook et al, 1989, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Press, n.y.; and Ausubel et al, (eds.),1995, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y..
As used herein, "percent amino acid identity" or "percent amino acid sequence identity" refers to the comparison of amino acids of two polypeptides that, when optimally aligned, have approximately the specified percentage of amino acids that are identical. For example, "95% amino acid identity" refers to comparing the amino acids of two polypeptides, which are 95% identical when optimally aligned.
For purposes of the present invention, to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity is the number of identical positions/total number of positions (i.e., overlapping positions) × 100). Preferably, the two sequences are of the same length. One skilled in the art will appreciate that different computer programs can be used to determine identity between two sequences.
As used herein, the term "conservative substitution", also referred to as substitution by a "homologous" amino acid residue, refers to a substitution in which the amino acid residue is replaced with 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), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine).
Conservative amino acid substitutions generally have minimal effect on the activity of the resulting protein. Such substitutions are described below. Conservative substitutions are those that replace an amino acid with an amino acid that is similar in size, hydrophobicity, charge, polarity, steric characteristics, aromaticity, and the like. Such substitutions are generally conservative when fine-tuning of the properties of the protein is desired.
As used herein, "homologous" amino acid residues refer to amino acid residues having similar chemical properties relating to hydrophobicity, charge, polarity, steric characteristics, aromaticity characteristics, and the like. Examples of amino acids that are homologous to each other include positively charged lysine, arginine, histidine, negatively charged glutamic acid, aspartic acid, hydrophobic glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, polar serine, threonine, cysteine, methionine, tryptophan, tyrosine, asparagine, glutamine, aromatic phenylalanine, tyrosine, tryptophan, serine and threonine of chemically similar side chain groups, or glutamine and asparagine, or leucine and isoleucine.
Examples of conservative amino acid substitutions in proteins include: ser for Ala, Lys for Arg, Gln or His for Asn, Glu for Asp, Ser for Cys, Asn for Gln, Asp for Glu, Pro for Gly, Asn or Gln for His, Leu or Val for Ile, Ile or Val for Leu, Arg or Gln for Lys, Leu or Ile for 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.
As used herein, the term "expression" includes any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
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 (e.g., 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 the corresponding receptors.
As used herein, the term "family of four α -helical bundle cytokines" relates to cytokines that comprise four α -helical bundles in the tertiary structure. The natural (wild-type) cytokine of the four α -helix bundle cytokine family comprises four α -helix bundles in order from N-terminus to C-terminus of helix bundle 1(H1), helix bundle 2(H2), helix bundle 3(H3) and helix bundle 4 (H4). 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.
As used herein, the term "cyclic rearrangement" refers to changing the order of the arrangement of the four α -helix bundles in the primary structure in the cytokines of the four α -helix bundle cytokine family. For example, a circularly rearranged cytokine comprises H2, H3, H4, and H1 in order from N-terminus to C-terminus; h3, H4, H1 and H2; or four alpha-helical bundles of H4, H1, H2 and H3. The cyclic rearrangement involves designing polypeptides as follows: the N-terminus of a polypeptide (the original polypeptide, such as a wild-type tetra α -helical bundle cytokine) is fused (directly linked or linked through a linker) to the C-terminus to form a circular molecule, and the circular molecule is opened (cleaved or cleaved) between H1 and H2, between H2 and H3, or between H3 and H4 to form a new linear polypeptide having a different N-terminus and C-terminus than the original polypeptide. The cyclic rearrangement preserves the sequence, structure and function of the polypeptide (except for the optional linker) while creating new C-and N-termini at different positions. Cyclic rearrangements also include any process that produces cyclic rearranged linear molecules described herein. Typically, cyclically rearranged polypeptides are directly expressed as linear molecules without actually undergoing the cyclization and opening steps.
For a polypeptide, a "linker" or "linker sequence" refers to an amino acid sequence that is covalently linked to the N-and/or C-terminus of a polypeptide. Linkers can be used to join the N-and C-termini of the same polypeptide (e.g., in a cyclic rearrangement), or can agree to join the N-and C-termini of different polypeptides to form a fusion polypeptide. A linker also relates to a polynucleotide encoding the amino acid sequence of the linker. Generally, the linker has no specific biological activity. However, the amino acids that make up the linker may be selected based on certain properties of the linker or the resulting molecule, such as flexibility, hydrophilicity, net charge or whether proteolytically sensitive, and lack of immunogenicity.
As used herein, a "wild-type" polypeptide refers to a naturally occurring polypeptide.
As used herein, the term "modification" refers to a modification of a polynucleotide or polypeptide sequence, including, but not limited to, substitution, deletion, insertion, and/or addition of one or more nucleotides or amino acids. Modifications also include chemical modifications that do not alter the sequence of the polynucleotide or polypeptide, such as methylation of polynucleotides, glycosylation of polypeptides, and the like. In this context, modifications also include cyclic rearrangements as described above.
As used herein, the term "open site" refers to a location in a cyclic molecule where peptide bonds are eliminated during cyclic rearrangement to form new amino and carboxy termini, or the corresponding location in the encoding polynucleotide of the polypeptide. The opening site is designated by the position of a pair of amino acids located between the amino and carboxy termini of the wild-type polypeptide, which become the new amino and carboxy termini of the cyclically rearranged polypeptide. For example, in IL-2(97/96), the new N-terminus corresponds to the residue at position 97 of native IL-2 and the new C-terminus corresponds to the residue at position 96 of native IL-2; in IL-15 (105/102), the new N-terminus corresponds to the residue at position 105 of native IL-15 and the new C-terminus corresponds to the residue at position 102 of native IL-15, with the residues at positions 103 and 104 removed.
As used herein, the term "receptor" is to be understood as a cell surface-presented protein that binds to a ligand, and also encompasses soluble receptors not present on a cell surface that have or are associated with a corresponding cell surface receptor. Cell surface receptors are typically composed of different domains or subunits with different functions, such as an extracellular domain comprising a region that interacts with a ligand, a transmembrane domain that anchors the receptor in the cell membrane, and an intracellular effector domain that generates a cellular signal (signal transduction) in response to ligand binding. Soluble receptors are typically composed of one or more extracellular domains that are proteolytically cleaved from the membrane anchoring region.
As used herein, the term "variant" refers to a polypeptide that differs from a reference polypeptide but retains the necessary properties. A typical variant of a polypeptide differs in primary amino acid sequence from the reference polypeptide. Typically, the differences are limited such that the sequences of the reference polypeptide and the variant are very similar overall and are identical in many regions. The amino acid sequences of the variant and reference polypeptides 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 the polypeptide may be naturally occurring, e.g., allelic variants; or may be artificially generated variants. In addition, the term "variant" as used herein includes cyclically rearranged variants of a polypeptide.
As used herein, the term "carrier protein" refers to a protein fused to a molecule of interest (e.g., a polypeptide or hapten) that functions to facilitate delivery of the protein of interest, increase its half-life, make the hapten immunogenic, and the like. The carrier protein itself does not have the biological activity of the molecule of interest. The carrier proteins used in the present invention have multiple helix structures connected by loops, i.e., have a "helix-loop-helix" structure, e.g., albumins such as Human Serum Albumin (HSA), albumin binding proteins, and the like.
As used herein, an "insertion site" of a polypeptide of interest in a carrier protein refers to the position of a residue of the carrier protein in the primary structure that is closest to the N-terminus of the polypeptide of interest after the polypeptide of interest is inserted into the carrier protein.
As used herein, a carrier protein "masks" a site of interest by the carrier protein meaning that the carrier protein sterically hinders the binding of the site of interest to its receptor, i.e., the carrier protein sterically interferes with the receptor (clasps).
As used herein, "accessibility of a site" refers to the ability of the site to contact and bind to its binding partner. When the site's accessibility is blocked, it cannot contact and bind to its binding partner.
As used herein, the term "signal peptide" refers to a short peptide that directs the transfer of a newly synthesized protein to the secretory pathway. Typically, the signal peptide is located at the N-terminus of the newly synthesized protein and is, for example, 5-30 amino acid residues in length. The signal peptide may be removed during protein processing so that the mature protein does not contain the signal peptide.
As used herein, the term "treatment" refers to a method of obtaining beneficial or desired results, including but not limited to eradication or amelioration of the underlying disease being treated. Likewise, therapeutic benefit is achieved by eliminating or ameliorating one or more physiological symptoms associated with the underlying disease; thus, although the subject may still have the underlying disease, an improvement is observed in the subject.
As used herein, the terms "therapeutically effective amount" and "therapeutically effective dose" refer to an amount of active ingredient that, when administered in a single dose or repeated doses, can achieve a detectable beneficial effect, including, but not limited to, an effect on any symptom, aspect, measured parameter or characteristic of a disease or disorder.
The term "dose" as used herein 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 two or more administrations) within one day, two days, one week, two weeks, three weeks, or one or more months.
As used herein, the term "half-life" refers to the time taken for the in vivo concentration of a target molecule to decrease by 50%. The half-life of the target molecule will be increased if it remains in vivo in the biological matrix (blood, serum, plasma, tissue) for a longer time than in the appropriate control. The half-life may be increased by 10%, 20%, 30%, 40%, 50% or more compared to an appropriate control.
Those skilled in the art are familiar with pharmacokinetic analysis and methods for determining the half-life of a ligand. Details can be found in Kenneth, A et al, Chemical Stability of Pharmaceuticals, A Handbook for Pharmacists and Peters et al, pharmaceutical analysis, A Practical Approach (1996). Reference may also be made to "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker,2.sup.nd Rev. ex edition (1982).
Second, cyclic rearrangement of polypeptides
By cyclic rearrangement is meant altering the order of the four α -helix bundles in the primary structure in the cytokines of the four α -helix bundle cytokine family. For example, a circularly rearranged four α -helical bundle cytokine comprises H2, H3, H4, and H1 in order from N-terminus to C-terminus; h3, H4, H1 and H2; or four alpha-helical bundles of H4, H1, H2 and H3. The cyclic rearrangement involves designing polypeptides as follows: the N-terminus of the polypeptide (the original polypeptide, such as wild-type tetra α -helical bundle cytokine) is fused (directly linked or linked through a linker) to the C-terminus to form a circular molecule, and the circular molecule is opened (cleaved or cleaved) between H1 and H2, between H2 and H3, or between H3 and H4 to form a new linear polypeptide having a different N-terminus and C-terminus than the original polypeptide.
Importantly, the cyclically rearranged polypeptide provides optimized termini for fusion with other polypeptides while retaining the biological activity of the original polypeptide. If the new end breaks a critical region of the original polypeptide, activity may be lost. Similarly, a cyclically rearranged polypeptide will not retain biological activity if ligation of the original ends would destroy activity. Thus, there are two requirements for proteins that produce active cyclic rearrangements: 1) the ends of the original polypeptide are ligated without destroying its biological activity; 2) at least one "opening site" must be present in the original polypeptide at which a new end can be formed without disrupting the regions critical to its folding and biological activity.
Thus, in general, a candidate polypeptide undergoing a cyclic rearrangement will have its original N-and C-termini in close proximity in the native folded state (original protein), e.g., the N-and C-termini of the original protein will be less than or equal to the distance between the N-and C-termini
Figure RE-RE-GDA0002730424380000091
The location of the new end is geometrically, structurally and functionally (relative to the native end) advantageous for fusion with a desired polypeptide fusion partner and for reducing the length of the desired linker.
The structure of IL-2 is shown in FIG. 1, where loops 1 and 2 are examples of locations where new ends are formed. FIG. 2 shows the structure of IL-15, where "opened loop" is an example of a location where a new tip is formed.
In some embodiments, recombinant constructs are engineered for cyclic rearrangement of IL-2 by linking the native N-and C-termini of IL-2 via a linker and opening the circular molecule between amino acid residues A93-R103 or N50-L56 to form a linear molecule with new N and C termini. In some embodiments, amino acid residue Q94-P102 is deleted, i.e., IL-2(R103/A93), at amino acid residue R103 forming a new N-terminus and A93 forming a new C-terminus. In some embodiments, the cyclically rearranged IL-2 is IL-2(L56/N50), IL-2 (L56/K55), or IL-2 (N53/K52).
In some embodiments, the cyclically rearranged IL-2 comprises the amino acid sequence of SEQ ID NO 2, 3, 4 or 5.
In some embodiments, to cyclically rearrange IL-15, recombinant constructs are engineered, the natural N-and C-termini of IL-15 are joined by a linker to form a circular molecule, and the circular molecule is opened 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 residue S102, G103 or D104, and a new N-terminus is formed at G103, D104, A105 or S106. In some embodiments, the cyclically rearranged IL-15 is IL-15(G103/S102), IL-15(D104/S102), IL-15 (A105/S102), IL-15(S106/G103), IL-15(A105/G103), IL-15 (D104/G103), IL-15(A105/D104), IL-15 (S106/A105).
In a preferred embodiment, the length of the linker used to link the N and C termini of the original polypeptide is related to the distance of the N and C termini in the original protein. In some embodiments, the linker used to link the N and C termini of the initial polypeptide is 1-10 amino acids in length, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, the linker is greater than 10 amino acids in length. Preferably, a3 amino acid linker such as GSG is used to link the N and C termini of native IL-2; the N-and C-termini of native IL-15 are linked using a four amino acid linker such as GGGG (SEQ ID NO: 17).
One 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.
In some embodiments, the cyclically rearranged IL-2 comprises the amino acid sequence of SEQ ID NO 2, 3, 4 or 5.
In some embodiments, the cyclically rearranged IL-15 comprises the amino acid sequence of SEQ ID NO 7.
Fusion polypeptide
The fusion polypeptide of the present invention comprises a carrier protein having multiple helical domains connected by loops and a polypeptide of interest inserted into the loops of the carrier protein, the carrier protein masking a site of interest on the polypeptide of interest, thereby blocking the accessibility of the site.
The inventors have found that when a polypeptide of interest is inserted into a loop of a carrier protein, the relative position of the carrier protein and the polypeptide of interest can be controlled by dihedral angles in the peptide chain such that the carrier protein masks the site of interest by selecting an appropriate insertion site, adjusting the linker (e.g., its sequence and/or length) between the polypeptide of interest and the carrier protein, 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.
In some embodiments, the polypeptide of interest is derived from a cytokine from the four α -helical bundle cytokine family, including but not limited to IL-2, IL-4, IL6, IL-7, IL-9, IL15, and IL 21. Preferably, the polypeptide of interest is a cytokine of the circularly rearranged four α -helical bundle cytokine family. In a preferred embodiment, the polypeptide of interest is a cyclically rearranged IL-2 or IL-15.
Native IL-2 has binding sites for CD25, CD122, and CD132 (referred to as IL-2 receptors α, β, and γ, respectively). IL-2 can bind to both the CD25/CD122/CD132 trimer, thereby activating T regulatory cells (Tregs) expressing the trimer to suppress the immune response, and to the CD122/CD132 dimer, thereby activating immune cells (e.g., CD8+ memory T cells and NK cells) expressing the dimer and stimulating their proliferation. Binding of CD25 to IL-2 alters the conformation of IL-2, resulting in increased affinity for the CD122/CD132 dimer.
Thus, in some embodiments, the polypeptide of interest is a cyclically rearranged IL-2, the site of interest is a CD25 binding site, and the fusion polypeptide has activity comparable to or improved over native IL-2, such as activity that activates JAK1/JAK3 and STAT3/STAT5 signaling pathways.
IL-15 also activates immune responses, and has important roles in the differentiation and proliferation of T cells and NK cells, and the development of dendritic cells. IL-15 works similarly to IL-2 by binding to the CD122/CD132 receptor dimer to activate the downstream signaling pathway (JAK1/JAK3 and STAT3/STAT5), but its alpha receptor is distinct from IL-2 and is its own IL-15R alpha (CD 215). IL-15 binds to IL-15 Ra with high affinity to CD122/CD132, whereas IL-15 binds to CD122/CD132 with moderate affinity without binding to IL-15 Ra.
Thus, in some embodiments, the polypeptide of interest is a cyclically rearranged IL-15, the site of interest is a CD215 binding site, and the fusion polypeptide has activity comparable to or improved over native IL-15, such as activity that activates JAK1/JAK3 and STAT3/STAT5 signaling pathways.
Preferably, a carrier protein suitable for use in the present invention is capable of binding to neonatal Fc receptor (FcRn) (e.g. albumin), or the carrier protein binds to a protein capable of binding FcRn (e.g. albumin binding protein).
Human Serum Albumin (HSA) is one of the most stable and high-content proteins (35-50g/L) in human serum, and accounts for half of the proteins in serum, and has the main functions of transporting substances (such as hormones, fatty acids and the like) and maintaining pH, osmotic pressure and the like. HSA herein includes wild-type HSA and modified HSA.
HSA is an all α -helical protein with a molecular weight of 66.5kDa, consisting of three similar domains (DI, DII, DIII) in a "heart-shaped" structure (fig. 3). HSA can bind to human FcRn under acidic conditions (pH <6.5) and is subsequently recovered to the cell surface and released back into the blood, thereby avoiding degradation of HSA into the lysosome. FcRn binds mainly to DIII as well as part of DI.
The precursor sequence of HSA is shown in SEQ ID NO:16(Uniprot P02768), and all positions referred to herein for HSA are numbered with reference to SEQ ID NO: 16. Residues 1-24 of the precursor sequence of HSA are signal peptides, and native HSA comprises residues 25-609 of SEQ ID NO 16.
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 the loop of HSA selected from the group consisting of 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 loops located at D56-L66, V293-L305 and A362-D365. In some embodiments, the insertion site for the polypeptide of interest is amino acid residue D56, a300, C361, or a362 of HSA.
The relative position between the carrier protein and the polypeptide of interest may be controlled by deleting one or more amino acids in the loop of the carrier protein, such as HSA, or may not be deleted.
HSA may also be modified to improve its properties. For example, the free cysteine in wild-type HSA may be substituted with another amino acid, such as serine. In some embodiments, the HSA comprises the amino acid substitution C58S.
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.
In some embodiments, the N-terminus and C-terminus of the polypeptide of interest are linked to the carrier protein by linkers. In some embodiments, the N-terminus of the polypeptide of interest is linked to the carrier protein via a linker and the C-terminus of the polypeptide of interest is directly linked to the carrier protein. In some embodiments, the N-terminus of the polypeptide of interest is directly linked to the carrier protein and the C-terminus of the polypeptide of interest is linked to the carrier protein by a linker. In some embodiments, the N-terminus and C-terminus of the polypeptide of interest are directly linked to the carrier protein. In some embodiments, the linker has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. In some embodiments, the linker is selected from a helical linker, or a flexible linker (such as a GS linker and a polyglycine linker).
In some embodiments, the fusion polypeptide of the invention comprises a carrier protein HSA and a cyclically rearranged IL-2, wherein the cyclically rearranged IL-2 comprises, in order from N-terminus to C-terminus, H3, H4, H1 and H2, or four alpha-helix bundles of H4, H1, H2 and H3, and wherein the circularly rearranged IL-2 is inserted into a loop of HSA, the rings are selected from the group consisting of rings 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 are referenced to SEQ ID NO:16, and said HSA masks the CD25 binding site of said circularly rearranged IL-2, thereby blocking the accessibility of said site. In some embodiments, the cyclically 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 group consisting of loops D56-L66, V293-L305, and A362-D365 located on HSA. In some embodiments, the insertion site for the circularly rearranged IL-2 is selected from D56, a300, C361, and a362 of HSA.
In some embodiments, the cyclically rearranged IL-2 comprises the amino acid sequence of SEQ ID NO 2. In some embodiments, the loop is that located at a362-D365 of HSA, preferably the insertion site is C361 of HSA. In some embodiments, the N-terminus of the cyclically rearranged IL-2 is linked to C361 of HSA via linker EAAAKAEAAA (SEQ ID NO:19), the C-terminus is linked to D365 of HSA via linker GS, and residues 362-364 of HSA are deleted.
In some embodiments, the cyclically rearranged IL-2 comprises the amino acid sequence of SEQ ID NO 4. In some embodiments, the loop is at D56-L66 of HSA, preferably the insertion site is D56 of HSA. In some embodiments, the N-terminus of the cyclically rearranged IL-2 is linked to D56 of HSA via linker AAAAAK (SEQ ID NO: 20) and the C-terminus is directly linked to E57 of HSA.
In some embodiments, the cyclically rearranged IL-2 comprises the amino acid sequence of SEQ ID NO 5. In some embodiments, the loop is that of V293-L305 of HSA, preferably the insertion site is a300 of HSA. In some embodiments, the N-terminus of the cyclically rearranged IL-2 is directly linked to a300 of HSA and the C-terminus is linked to D301 of HSA via linker G.
In some embodiments, the cyclically rearranged IL-2 consists of the amino acid sequence SEQ ID NO 4. In some embodiments, the loop is that located at a362-D365 of HSA, preferably the insertion site is a362 of HSA. In some embodiments, the N-terminus of the cyclically rearranged IL-2 is directly linked to a362 of HSA and the C-terminus is directly linked to a363 of HSA.
In some embodiments, the invention provides a fusion polypeptide comprising a carrier protein HSA and a cyclically rearranged IL-15, wherein the cyclically rearranged IL-15 comprises four alpha-helical bundles of H3, H4, H1 and H2 in that order from N-terminus to C-terminus, and wherein the cyclically rearranged IL-15 is inserted in a loop of HSA selected from the group consisting of loops located 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 are referenced to SEQ ID NO:16, and said HSA masks the CD215 binding site of said circularly rearranged IL-15, thereby blocking the accessibility of said site. In some embodiments, the cyclically rearranged IL-15 comprises the amino acid sequence of SEQ ID NO 7.
In some embodiments, the fusion polypeptide of the invention comprises an amino acid sequence of one of SEQ ID NOs 8-11. In some embodiments, a fusion polypeptide of the invention comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence of one of SEQ ID NOs 8-11, wherein the fusion polypeptide comprises a native CD25 binding site having comparable or improved activity as native IL-2, e.g., activity to activate JAK1/JAK3 and STAT3/STAT5 signaling pathways.
In some embodiments, the fusion polypeptides of the invention have a more durable immunostimulatory effect. In some embodiments, the fusion polypeptides of the invention have greater safety.
In some embodiments, the half-life of a fusion polypeptide 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 as compared to native IL-2. In some embodiments, the half-life of a fusion polypeptide 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 as compared to native IL-15.
Expression of fusion polypeptides
To express the fusion polypeptides of the invention, the invention also provides polynucleotides encoding the fusion polypeptides of the invention.
Nucleic acid molecules of all or part of a nucleic acid sequence of the invention can be isolated by Polymerase Chain Reaction (PCR) using synthetic oligonucleotide primers designed based on sequence information contained in the sequence.
The polynucleotides of the invention may be amplified using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid thus amplified can be cloned into a suitable vector and characterized by DNA sequence analysis.
Polynucleotides of the invention can be prepared by standard synthetic techniques, for example using an automated DNA synthesizer.
The invention also relates to the complementary strand of the nucleic acid molecules described herein. A nucleic acid molecule that is complementary to another nucleotide sequence is a molecule that is sufficiently complementary to the nucleotide sequence that it can hybridize to the other nucleotide sequence, thereby forming a stable duplex. Of course, polynucleotides of the invention do not include polynucleotides that hybridize only to poly A sequences (e.g., the 3' end of mRNA poly (A)) or to a complementary stretch of poly T (or U) residues.
The invention also provides vectors, preferably expression vectors, comprising a polynucleotide of the invention. Further, the present invention also provides a host cell comprising a polynucleotide or vector (preferably, an expression vector) of the present invention. In some embodiments, the polynucleotide of the invention is integrated into the genome of the host cell. In some embodiments, the polynucleotide of the invention does not integrate into the genome of the host cell.
In some embodiments, the first and second polypeptides of the fusion polypeptides of the invention are expressed using a single expression vector. In some embodiments, the first and second polypeptides of the fusion polypeptides of the invention are expressed using different expression vectors.
The choice of expression vector is relevant to the host cell used to express the fusion polypeptide. Host cells that can be used to express the fusion polypeptides of the invention include, but are not limited to, bacterial (including E.coli), yeast, insect cells, and mammalian cells, such as COS, CHO, HeLa, and 293-6E cells. Expression vectors suitable for use in a variety of host cells are known in the art. For example, expression vectors suitable for use in bacteria include, but are not limited to, pET vectors (e.g., pET-28a, pET-30a, pET-32a, pET-40a, and the like), pEX vectors (e.g., pEX-1), pGH112, pUC118, and pEZZ 18; expression vectors suitable for use in yeast include, but are not limited to, pESP vectors (e.g., pESP-1, pESP-2, pESP-3, etc.), pDR196, pHiSi, p53his, pSH47, and pYCP 211; 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 use in mammalian cells include, but are not limited to, pCMVInt, pGL4.23, pX334, pX458, pBiFC-CC155, pDP4rs, pDC312, and pcDNA.
In some embodiments, the host cell is a 293-6E cell. In some embodiments, the expression vector is pcdna3.4.
The inclusion of a signal peptide in the precursor polypeptide may aid in secretion and/or processing of the expressed polypeptide. Thus, in some embodiments, the polynucleotides of the invention further comprise a sequence encoding a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of METDTLLLWVLLLWVPGSTG (SEQ ID NO: 18).
The polynucleotides or vectors of the invention may be transferred (transfected) into a selected host cell by methods known in the art, such as calcium chloride transformation and calcium phosphate treatment for E.coli, electroporation, lipofectamine treatment or PEI treatment for mammalian cells. Cells transformed with the vector can be selected based on the antibiotic resistance genes (e.g., amp, gpt, neo, and hyg genes) in the vector.
After expression, the recombinant fusion protein may be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity chromatography, column chromatography using ionic or hydrophobic resins, gel electrophoresis, and the like. Substantially pure compositions having a purity of at least about 90 to 95% are preferred, with purity of 98 to 99% or higher most preferred for pharmaceutical use.
For ease of purification, the fusion polypeptides of the invention may also comprise a tag sequence, including but not limited to a His6 tag, a FLAG tag, and the like.
In some embodiments, the fusion polypeptide of the invention comprises an amino acid sequence of one of SEQ ID NOs 12-15. In some embodiments, a fusion polypeptide of the invention comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence of one of SEQ ID NOs 12-15, wherein the fusion polypeptide comprises a native CD25 binding site having comparable or improved activity as native IL-2, e.g., activity to activate JAK1/JAK3 and STAT3/STAT5 signaling pathways.
Medicine composition
The present invention provides pharmaceutical compositions comprising the fusion polypeptides of the invention. In one embodiment, the pharmaceutical composition comprises the fusion polypeptide of the invention and at least one pharmaceutically acceptable carrier. The fusion polypeptide of the present invention may be combined with a pharmaceutically acceptable carrier according to a known method to prepare the pharmaceutical composition.
Pharmaceutically acceptable carriers include, but are not limited to, solvents, emulsifiers, buffers, stabilizers and the like. The solvent includes water, aqueous solutions, non-aqueous solvents (e.g., vegetable oils).
The pharmaceutical compositions of the present invention may be administered by any suitable route, including subcutaneous, intramuscular, intraarticular, intravenous, intradermal, intraperitoneal, intranasal, intracranial, parenteral administration. Preferably, the pharmaceutical composition of the invention is administered intravenously. It is understood that the route of administration may vary with the therapeutic agent, the condition and age of the recipient, and the disease being treated.
The pharmaceutical composition of the present invention may be in the form of a solution or a lyophilized formulation.
In some embodiments, the pharmaceutical compositions of the present invention are provided in the form of a lyophilized powder for reconstitution prior to administration. The pharmaceutical compositions of the present invention may also be provided in liquid form, which may be administered directly to a patient. In some embodiments, the composition is provided in liquid form in a pre-filled syringe.
In some embodiments, the compositions of the present invention are encapsulated in liposomes. In some embodiments, liposomes can be coated with flexible, water-soluble polymers that avoid uptake by organs of the mononuclear phagocyte system, mainly the liver and spleen. Hydrophilic polymers suitable for coating liposomes include, but are not limited to, PEG, polyvinylpyrrolidone, polyvinyl methyl ether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose hydroxyethylamide, hydrophilic polyvinyl alcohol, and the like.
The pharmaceutical compositions of the present invention may be administered in one or more doses and at a frequency that is desired and tolerated by the patient. In any event, the pharmaceutical composition administered should provide a sufficient amount of the protein of the invention to effectively treat the patient.
Sixth, therapeutic application
The invention also provides methods of using the fusion polypeptides of the invention to treat diseases, such as diseases involving immunosuppression.
In some embodiments, there is provided a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a fusion polypeptide or pharmaceutical composition of the invention. Such cancers include, but are not limited to, lung cancer, liver cancer, kidney cancer, head and neck cancer, colorectal cancer, gastric cancer, nasopharyngeal cancer, glioma, melanoma, and osteosarcoma.
In some embodiments, there is provided a method of activating or increasing proliferation of an immune cell, comprising administering to a subject in need thereof an effective amount of a fusion polypeptide of the invention or a pharmaceutical composition of the invention. In some embodiments, the immune cell is a T cell or an NK cell.
In some embodiments, there is provided a use of a fusion polypeptide or a pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of cancer. Such cancers include, but are not limited to, lung cancer, liver cancer, kidney cancer, head and neck cancer, colorectal cancer, gastric cancer, nasopharyngeal cancer, glioma, melanoma, and osteosarcoma.
In some embodiments, there is provided a use of a fusion polypeptide or a pharmaceutical composition of the invention in the preparation of a medicament for activating an immune cell or increasing the proliferation of an immune cell. In some embodiments, the immune cell is a T cell or an NK cell.
Examples
The present invention will be more clearly understood by those skilled in the art from the following examples. It is to be understood that the examples are for illustration only and do not limit the scope of the invention. Unless otherwise indicated, the experimental procedures used in the present invention are conventional, and specific examples of genetic Cloning procedures are described in Sambrook, et al (Molecular Cloning: A Laboratory Manual,2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
Example 1 construction of expression vector
The construction of the expression vector was carried out by Nanjing Kingsrei Biotech Co., Ltd, and included: synthesizing a nucleic acid encoding the amino acid sequence of SEQ ID NO 12-15 additionally comprising a nucleotide sequence encoding the signal peptide of SEQ ID NO 18; the nucleic acid is constructed into a mammalian cell expression vector pcDNA3.4 by a molecular cloning method, and the amplification and purification of the plasmid and the like.
Example 2 expression of fusion Polypeptides
In this example, 293-6E cells were transfected with nucleic acid encoding a fusion polypeptide for eukaryotic expression. The expression and purification of the fusion polypeptide are completed by Nanjing Kingsrei Biotech Co., Ltd, the process is briefly described as follows:
-293-6E cells in serum-free FreeStyleTM293Expression medium (Thermo Fisher Scientific, Carlsbad, Calif., USA), placed in Erlenmeyer flasks (Corning Inc., Acton, MA), and cultured on a shaking incubator (VWR Scientific, Chester, Pa.) at 37 ℃ and 5% CO2。
The day before transfection, cells are diluted to the appropriate density.
On the day of transfection, the plasmid mixture (plasmid mass ratio 1: 1 encoding the first and second polypeptides) is mixed with a transfection reagent (e.g. Polyetherimide, polyethylimide, PEI) in a suitable ratio (e.g. 1: 3 mass ratio) in the culture medium, and then added to the cell culture broth for transfection for secretory expression of the target protein.
After six days, the survival rate of the cells is about 50% to 80%, and the supernatant is obtained by centrifugation, which contains the protein of interest (the expressed polypeptide does not contain the signal peptide).
The supernatant was filtered through a 0.22 μm filter and loaded onto HisTrap at a rate of 3 ml/minTMAfter washing and elution, the purified proteins were pooled together and stored in phosphate buffered saline (PBS pH 7.2) in FF Crude 5ml (GE, cat # 17-5286-01) chromatography column.
The molecular weight, purity and sequence coverage of the purified proteins were assessed by SDS-PAGE, immunoblotting (results not shown), high performance liquid chromatography in combination with molecular sieves (SEC-HPLC) and liquid chromatography mass spectrometry (LC-MS). The primary antibody used in the immunoblot was either a murine anti-His tag antibody (GenScript, cat # a00186) or a murine anti-FLAG antibody (GenScript, cat # a00187), and the secondary antibody was a horseradish peroxidase-modified goat anti-mouse IgG antibody (GenScript, cat # a 00160).
The results are shown in FIGS. 4A-4C, 5A-5C, 6A-6C and 7A-7C, FIGS. 4A-4C: opening in Loop 1 of IL-2 to form a circularly rearranged IL-2 at the insertion site C361 of HSA (SEQ ID NO: 12); FIGS. 5A-5C: opening in Loop 2 of IL-2 to form a circularly rearranged IL-2 at the insertion site D56 of HSA (SEQ ID NO: 13); FIGS. 6A-6C: opening in Loop 2 of IL-2 to form a circularly rearranged IL-2, at the insertion site A300 of HSA (SEQ ID NO: 14); and FIGS. 7A-7C: opening in Loop 1 of IL-2 forms a circularly rearranged IL-2 with the insertion site at A362 of HSA (SEQ ID NO: 15). FIGS. 4A, 5A, 6A and 7A show structural models of HSA masking the CD25 binding site on IL-2, FIGS. 4B, 5B, 6B and 7B show SDS-PAGE results for reduced and non-reduced fusion polypeptides, and FIGS. 4C, 5C, 6C and 7C show results of molecular sieve analysis.
As shown in FIGS. 4A-4C, 5A-5C, 6A-6C and 7A-7C, high expression levels were achieved for all four fusion polypeptides, and SDS-PAGE and SEC-HPLC showed high purity and high homogeneity; high sequence coverage was detected by LC-MS confirming the integrity and correctness of the purified fusion polypeptide.
Example 3 detection of fusion polypeptide Activity
This example utilizes HEK-BlueTMIL-2 cell line (InvivoGen), the activity of these fusion proteins to activate downstream signaling pathways was examined at the cellular level. HEK-BlueTMThe IL-2 cell line is constructed by stably transfecting genes encoding molecules required by an IL-2 signal pathway in human embryonic kidney 293 cells (HEK-293), wherein the molecules comprise CD25, CD122 and CD132 receptor molecules, human JAK3 kinase and a transcription factor STAT5, and Secreted Embryonic Alkaline Phosphatase (SEAP) under the regulation of STAT5 is transferred into the IL-2 cell lineA reporter gene. When stimulated by IL-2, this cell line activates the downstream signaling pathway to activate STAT5, which up-regulates SEAP secretory expression.
The detection is completed by Nanjing topologic information Biotechnology Limited, and the process is briefly described as follows:
1. preparation of the experiment
1.1. Preparing QUANTI-Blue solution: thawing QB reagent and QB buffer at room temperature, adding into 98mL sterile water, mixing, packaging into 10mL tubes, and storing at-20 deg.C in dark place.
1.2. Inactivation of serum: taking 45mL serum (FBS) to a 50mL centrifuge tube, thermally inactivating in 56 deg.C water bath for 30min (mixing every 10 min), and temporarily storing at 4 deg.C.
1.3. Culture medium
Growth medium: DMEM + 10% FBS + 1% PS + 100. mu.g/mL Normolin + 1. mu.g/mL puromycin +1 XHEK-BlueTMCLR Selection; test medium: DMEM + 10% FBS (inactivated) + 1% PS +100 μ g/mL Normocin; freezing the culture medium: DMEM + 20% FBS + 10% DMSO.
2. Culture, passage and preservation of cells
2.1. The cells were placed in growth medium at 37 ℃ with 5% CO2Culturing and subculturing in an incubator;
2.2. when the confluence degree of the cells reaches 70-80% and the state is good, discarding the supernatant, suspending the cells by using a growth culture medium preheated in a water bath at 37 ℃, and centrifuging for 5min at 200 Xg after counting the cells; discarding the supernatant, resuspending the cells in a 4 ℃ pre-cooled cryoculture medium, and adjusting the cell density to 5-7X 106cells/mL, 1mL per tube, were placed in a programmed cooling box and left overnight at-80 ℃ for long term storage in liquid nitrogen.
HEK-Blue IL-2 System validation
3.1. Preparation of HEK-Blue IL-2 cell suspension: gently rinsing the cells twice with preheated DPBS, resuspending the cells with preheated DPBS to form a single cell suspension, counting the cells, and centrifuging the cells at 200 Xg for 5 min; resuspension with pre-warmed test medium at a cell density of 2.8X 105cell/mL;
3.2. human IL-2 protein (positive control, Acrobiosystems, Inc. of Baiposi biosciences, Beijing, cat # IL2-H4113) and HAS (negative control) were serially diluted as follows: starting concentration 650pM, 5-fold dilution, 8 gradients.
3.3. 20 μ L of serial concentration gradient samples and 180 μ L of cell suspension (triplicate for each sample) were added to each well of the plate, placed at 37 ℃ and 5% CO2Culturing in an incubator for 20-24 hours.
3.4. The supernatant of each culture was added to a 96-well plate at 20. mu.L/well, and 180. mu.L/well of QUANTI-Blue solution thawed and returned to room temperature was added thereto, incubated at 37 ℃ for 1 to 3 hours, and then the absorbance OD 620-655nm was read with a microplate reader to analyze the data.
4. And (4) detecting the activity of the fusion polypeptide.
The activity of the fusion polypeptides prepared in step 2 was tested using the method in step 3, wherein each fusion polypeptide was also prepared as a serial gradient dilution sample according to the method in step 3.2.
The results are shown in FIGS. 8-11, FIG. 8: opening in Loop 1 of IL-2 to form a circularly rearranged IL-2 at the insertion site C361 of HSA (SEQ ID NO: 12); FIG. 9: opening in Loop 2 of IL-2 to form a circularly rearranged IL-2 at the insertion site D56 of HSA (SEQ ID NO: 13); FIG. 10: opening in Loop 2 of IL-2 to form a circularly rearranged IL-2, at the insertion site A300 of HSA (SEQ ID NO: 14); and FIG. 11: opening in Loop 1 of IL-2 forms a circularly rearranged IL-2 with the insertion site at A362 of HSA (SEQ ID NO: 15).
A 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.
TABLE 1 comparison of the fusion polypeptide of SEQ ID NO 12 with human IL-2
Human IL-2 SEQ ID NO12 fusion polypeptides
EC50(pM) 5.123 1.750
TABLE 2 comparison of the fusion polypeptide of SEQ ID NO 13 with human IL-2
Human IL-2 Fusion polypeptide of SEQ ID NO 13
EC50(pM) 7.934 6.356
TABLE 3 comparison of the fusion polypeptide of SEQ ID NO. 14 with human IL-2
Human IL-2 Fusion polypeptide of SEQ ID NO. 14
EC50(pM) 2.583 5.859
TABLE 4 comparison of the fusion polypeptide of SEQ ID NO. 15 with human IL-2
Human IL-2 Fusion polypeptide of SEQ ID NO. 15
EC50(pM) 2.755 5.253
The results show that the fusion polypeptides of the invention show comparable or higher activity due to wild-type IL-2, whereas no corresponding response was shown with HSA (results not shown).
Sequence listing
<110> university of Nanjing university
<120> fusion polypeptide and use thereof
<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 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 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
Ser Ala Lys Gln Arg Leu Lys Cys Ala 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 Lys 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 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> 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
Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala 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 Ala
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 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr
485 490 495
Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr 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
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 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
Ser Ala Lys Gln Arg Leu Lys Cys Ala 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 Lys 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 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 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 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
Ser Ala Lys Gln Arg Leu Lys Cys Ala 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 Lys 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 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
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 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 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
Ser Ala Lys Gln Arg Leu Lys Cys Ala 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 Lys 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 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
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 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
Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala 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 Ala
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 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr
485 490 495
Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr 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 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 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
Ser Ala Lys Gln Arg Leu Lys Cys Ala 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 Lys 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 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 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 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
Ser Ala Lys Gln Arg Leu Lys Cys Ala 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 Lys 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 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
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
His His His His His His
725
<210> 16
<211> 609
<212> PRT
<213> Homo sapiens
<400> 16
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe 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 Ala 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 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 Lys 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 (31)

1. 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 masks a site of interest of the polypeptide of interest, thereby blocking the site's accessibility.
2. The fusion polypeptide of claim 1, wherein the polypeptide of interest is derived from a cytokine of the family of four α -helix bundle cytokines comprising, in order from N-terminus to C-terminus, four α -helix bundles of helix bundle 1(H1), helix bundle 2(H2), helix bundle 3(H3) and helix bundle 4 (H4).
3. The fusion polypeptide of claim 2, wherein the polypeptide of interest is a cytokine of the circularly rearranged tetra- α -helical bundle cytokine family comprising, in order from N-terminus to C-terminus, H2, H3, H4 and H1; h3, H4, H1 and H2; or four alpha-helical bundles of H4, H1, H2 and H3.
4. The fusion polypeptide of claim 3, wherein the amino acid corresponding to the N-terminus of the cytokine without cyclic rearrangement in the cyclic rearranged cytokine is linked to the amino acid corresponding to the C-terminus of the cytokine without cyclic rearrangement by a linker.
5. The fusion polypeptide of claim 4, wherein the linker is a GS linker or a polyglycine linker of 1-10 amino acids in length.
6. The fusion polypeptide of any one of claims 1-5, wherein the polypeptide of interest is selected from the group consisting of a cyclically rearranged IL-2 and a cyclically rearranged IL-15.
7. The fusion polypeptide of claim 6, wherein the cyclically rearranged IL-2 comprises four α -helix bundles of H3, H4, H1 and H2, or H4, H1, H2 and H3, in order from N-terminus to C-terminus.
8. The fusion polypeptide of claim 7, wherein the cyclically rearranged IL-2 comprises the amino acid sequence of SEQ ID NO 2, 3, 4 or 5.
9. The fusion polypeptide of claim 7 or 8, wherein the site of interest is the CD25 binding site.
10. The fusion polypeptide of claim 6, wherein the cyclically rearranged IL-15 comprises four α -helix bundles of H3, H4, H1, and H2 in order from N-terminus to C-terminus.
11. The fusion polypeptide of claim 10, wherein the cyclically rearranged IL-15 comprises the amino acid sequence of SEQ ID No. 7.
12. The fusion polypeptide of claim 10 or 11, wherein the site of interest is the CD215 binding site.
13. The fusion polypeptide of any one of claims 1-12, wherein the carrier protein is albumin.
14. The fusion polypeptide of claim 13, wherein the carrier protein is Human Serum Albumin (HSA).
15. The fusion polypeptide of claim 14, wherein the loop is selected from the group consisting of 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, numbered with reference to SEQ ID NO 16.
16. The fusion polypeptide of claim 15, wherein the polypeptide of interest is a cyclically rearranged IL-2 and the loop is selected from the group consisting of loops D56-L66, V293-L305 and a362-D365 located at HSA.
17. The fusion polypeptide of claim 16, wherein the insertion site for the polypeptide of interest is selected from the group consisting of D56, a300, C361, and a362 of HSA.
18. A fusion polypeptide comprising a carrier protein HSA and a cyclically rearranged IL-2, wherein said cyclically rearranged IL-2 comprises four alpha-helical bundles of H3, H4, H1 and H2, or H4, H1, H2 and H3 in order from N-terminus to C-terminus, and wherein said cyclically rearranged IL-2 is inserted into a loop of HSA selected from the group consisting of loops at D56-L66, a92-P96, D129-E131, Q170-a172, K281-L, V293-L305, E311-S312, E321-a322, a362-D365, L398-E400, K439-R445, E465-D471, P537-E542 and a561-T566, said positions being numbered with reference to SEQ ID NO:16, said HSA masks the CD25 binding site of said cyclically rearranged IL-2, thereby blocking the accessibility to said cyclic rearrangement site.
19. The fusion polypeptide of claim 18, wherein the cyclically rearranged IL-2 comprises the amino acid sequence of SEQ ID NO 2, 3, 4 or 5.
20. The fusion polypeptide of claim 18 or 19, wherein the loop is selected from the group consisting of loops D56-L66, V293-L305 and a362-D365 located at HSA.
21. The fusion polypeptide of any one of claims 18-20, wherein the insertion site for the circularly rearranged IL-2 is selected from the group consisting of D56, a300, C361, and a362 of HSA.
22. A fusion polypeptide comprising a carrier protein HSA and a circularly rearranged IL-15, wherein said circularly rearranged IL-15 comprises four α -helix bundles of H3, H4, H1 and H2 in order from N-terminus to C-terminus, and wherein said circularly rearranged IL-15 is inserted into a loop of HSA selected from the group consisting of 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, said positions being numbered with reference to SEQ ID NO:16, said HSA CD215 binding site of said circularly rearranged IL-15 thereby blocking the accessibility of said site.
23. The fusion polypeptide of claim 22, wherein the cyclically rearranged IL-15 comprises the amino acid sequence of SEQ ID No. 7.
24. A fusion polypeptide comprising the amino acid sequence of one of SEQ ID NOs 8-11.
25. A pharmaceutical composition comprising the fusion polypeptide of any one of claims 1-24.
26. Use of the fusion polypeptide of any one of claims 1-24 in the manufacture of a medicament for the treatment of cancer.
27. Use of the fusion polypeptide of any one of claims 1-24 in the manufacture of a medicament for activating an immune cell or increasing the proliferation of an immune cell.
28. The use of claim 27, wherein the immune cell is a T cell or NK cell.
29. An isolated polynucleotide encoding the fusion polypeptide of any one of claims 1-24.
30. An expression vector comprising the polynucleotide of claim 29.
31. A host cell comprising the polynucleotide of claim 29 or the expression vector of claim 30.
CN202010650886.XA 2020-07-08 2020-07-08 Fusion polypeptides and uses thereof Pending CN113912734A (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CN202010650886.XA CN113912734A (en) 2020-07-08 2020-07-08 Fusion polypeptides and uses thereof
US18/004,634 US20230322882A1 (en) 2020-07-08 2021-07-08 Fusion Polypeptide and Polypeptide Dimer, and Use Thereof
PCT/CN2021/105187 WO2022007885A1 (en) 2020-07-08 2021-07-08 Fusion polypeptide and polypeptide dimer, and use thereof
EP21838465.9A EP4186928A1 (en) 2020-07-08 2021-07-08 Fusion polypeptide and polypeptide dimer, and use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010650886.XA CN113912734A (en) 2020-07-08 2020-07-08 Fusion polypeptides and uses thereof

Publications (1)

Publication Number Publication Date
CN113912734A true CN113912734A (en) 2022-01-11

Family

ID=79231683

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010650886.XA Pending CN113912734A (en) 2020-07-08 2020-07-08 Fusion polypeptides and uses thereof

Country Status (1)

Country Link
CN (1) CN113912734A (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104507504A (en) * 2012-08-10 2015-04-08 罗切格利卡特公司 Interleukin-2 fusion proteins and uses thereof
US20200040052A1 (en) * 2018-05-14 2020-02-06 Werewolf Therapeutics, Inc. Activatable interleukin-2 polypeptides and methods of use thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104507504A (en) * 2012-08-10 2015-04-08 罗切格利卡特公司 Interleukin-2 fusion proteins and uses thereof
US20200040052A1 (en) * 2018-05-14 2020-02-06 Werewolf Therapeutics, Inc. Activatable interleukin-2 polypeptides and methods of use thereof

Similar Documents

Publication Publication Date Title
US11717559B2 (en) Interleukin 15 protein complex and use thereof
JP6723982B2 (en) Interleukin-2/interleukin-2 receptor alpha fusion protein and methods of use
JP7423607B2 (en) Fusion protein composed of interleukin 2 mutant protein and type I interferon
WO2017190684A1 (en) Interleukin combination and use thereof
KR20220113492A (en) Designed ankyrin repeat domains with altered surface residues
CN108430492B (en) Single-chain CD27 receptor agonist proteins
CN112142859A (en) Human interleukin 10-Fc fusion protein and coding gene and application thereof
US11041157B2 (en) Slit2D2-HSA fusion protein and use thereof against tumours
JP4493854B2 (en) Method for enhancing biological activity of a ligand
TWI758884B (en) Fusion protein containing human interleukin 10 and fc fragment and medical use thereof
WO2015149708A1 (en) New recombinant bifunctional fusion protein, preparation method therefor and use thereof
WO2017071173A1 (en) Tumor therapeutic agent modified by il-12/cd62l fusion protein and preparation method and use thereof
CN111378046B (en) Immune effector cell conversion receptor
WO2023109835A1 (en) Vegf-crm197 recombinant fusion protein vaccine, and preparation method therefor and use thereof
CN113912734A (en) Fusion polypeptides and uses thereof
US20230322882A1 (en) Fusion Polypeptide and Polypeptide Dimer, and Use Thereof
CN113912735A (en) Polypeptide dimer and use thereof
JP3646927B2 (en) A novel human thrombopoietin derivative with improved platelet growth efficacy in vivo
CN115850387A (en) Ultrahigh-affinity PD-L1-targeting small protein and pharmaceutical composition
CN109384849B (en) Fusion protein of thrombopoietin and preparation method and application thereof
KR20220131529A (en) CD122 with altered ICD STAT signaling
CN111825770B (en) Long-acting interleukin 21-Fc fusion protein and application thereof
US20170252435A1 (en) Combination
CA2408034C (en) Recombinant sequence, its preparation and use
JP3689111B2 (en) Interleukin 15

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination