CN113912735A - Polypeptide dimer and use thereof - Google Patents

Polypeptide dimer and use thereof Download PDF

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CN113912735A
CN113912735A CN202010651373.0A CN202010651373A CN113912735A CN 113912735 A CN113912735 A CN 113912735A CN 202010651373 A CN202010651373 A CN 202010651373A CN 113912735 A CN113912735 A CN 113912735A
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沈健
周家宏
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Nanjing Normal University
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Abstract

The present invention provides a polypeptide dimer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first dimerization domain and a polypeptide of interest, wherein the polypeptide of interest is located at a first end of the first dimerization domain, wherein the second polypeptide comprises a second dimerization domain and a binding domain, wherein the binding domain is located at a first end of the second dimerization domain, wherein the first polypeptide and the second polypeptide form a dimer through the first dimerization domain and the second dimerization domain, and the first end of the first dimerization domain is adjacent to the first end of the second dimerization domain, the binding domain being capable of binding to a site of interest on the polypeptide of interest. The invention also provides nucleic acids and vectors encoding the polypeptide dimers of the invention, host cells comprising the nucleic acids and vectors, and the use of the polypeptide dimers for treating diseases, such as cancer.

Description

Polypeptide dimer and use thereof
Technical Field
The present invention relates to polypeptide dimers. 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, but it has also been found that IL-15 can also bind to and function with moderate affinity to CD122/CD132 without binding to IL-15 Ra.
In addition, cytokines, such as IL-2 and IL-15, have short half-lives in vivo and require continuous injection.
Thus, there remains a need to develop engineered recombinant cytokines such as IL-2 and IL-15 that are capable of specifically activating immunity, have an extended half-life, have longer lasting immune stimulation 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 polypeptide dimer comprising a first polypeptide and a second polypeptide,
wherein the first polypeptide comprises a first dimerization domain and a polypeptide of interest, wherein the polypeptide of interest is located at a first end of the first dimerization domain,
wherein the second polypeptide comprises a second dimerization domain and a binding domain, wherein the binding domain is located at a first end of the second dimerization domain,
wherein the first polypeptide and the second polypeptide form a dimer through the first dimerization domain and the second dimerization domain, and the first end of the first dimerization domain is adjacent to the first end of the second dimerization domain, the binding domain being capable of binding to a site of interest on the polypeptide of interest.
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 native cytokine in the cyclically rearranged cytokine is linked to the amino acid corresponding to the C-terminus of the native cytokine 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 a cyclically rearranged IL-2 or 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. In some embodiments, the site of interest is a CD25 binding site and the binding domain is the extracellular domain of CD 25.
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 4. In some embodiments, the site of interest is a CD215 binding site and the binding domain is the extracellular domain of CD 215.
In some embodiments, the first and second dimerization domains comprise the heavy chain constant regions CH2 and CH3 of an immunoglobulin (Ig). In some embodiments, the Ig is a human Ig, e.g., human IgG 1. In some embodiments, the first dimerization domain and the second dimerization domain form an Fc region of human IgG 1. In some embodiments, the Fc region of the human IgG1 is modified. In some embodiments, the first dimerization domain comprises the amino acid sequence of SEQ ID No. 5 and the second dimerization domain comprises the amino acid sequence of SEQ ID No. 6; or the first dimerization domain comprises the amino acid sequence of SEQ ID NO 6 and the second dimerization domain comprises the amino acid sequence of SEQ ID NO 5.
In some embodiments, the first end of the first dimerization domain is C-terminal and the first end of the second dimerization domain is C-terminal.
In some embodiments, the polypeptide dimers of the invention comprise a first polypeptide and a second polypeptide,
wherein the first polypeptide comprises a first chain of the Fc region of human IgG1 and a cyclically rearranged IL-2, said cyclically rearranged IL-2 being linked C-terminal to the first chain of the Fc region of human IgG1,
wherein the second polypeptide comprises a second chain of an Fc region of human IgG1 and a CD25 extracellular domain, the CD25 extracellular domain is linked to the C-terminus of the second chain of an Fc region of human IgG1, and
wherein the cyclically rearranged IL-2 comprises, 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 cyclically rearranged IL-2 comprises the amino acid sequence of SEQ ID NO 2.
In some embodiments, the polypeptide dimers of the invention comprise a first polypeptide and a second polypeptide,
wherein the first polypeptide comprises a first chain of the Fc region of human IgG1 and a cyclically rearranged IL-15, said cyclically rearranged IL-15 being linked C-terminal to the first chain of the Fc region of human IgG1,
wherein the second polypeptide comprises a second chain of the Fc region of human IgG1 and a CD215 extracellular domain, the CD215 extracellular domain being linked to the C-terminus of the second chain of the Fc region of human IgG1, and
wherein 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 4.
In some embodiments, the first strand comprises the amino acid sequence of SEQ ID No. 5 and the second strand comprises the amino acid sequence of SEQ ID No. 6; or the first strand comprises the amino acid sequence of SEQ ID NO 6 and the second strand comprises the amino acid sequence of SEQ ID NO 5.
The invention also provides pharmaceutical compositions comprising the polypeptide dimers of the invention.
In a second aspect, the present invention also provides a method for treating cancer or activating immune cells or increasing the proliferation of immune cells (e.g., T cells or NK cells), comprising administering to a subject in need thereof an effective amount of a polypeptide dimer of the present invention or a pharmaceutical composition of the present invention.
The invention also provides the use of a polypeptide dimer or 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 polypeptide dimer 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 polypeptide dimers 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. 3(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. 3.
FIGS. 3A-3C and 4A-4C show the structural model of the polypeptide dimer of the present invention and its expression, and the amino acid sequence of the circularly rearranged IL-2 is shown in SEQ ID NO. 2.
FIGS. 5 and 6 show the results of in vitro activity assays of the polypeptide dimers of the present invention, and the amino acid sequence of the circularly rearranged IL-2 is shown in SEQ ID NO 2.
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.
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-GDA0002730778420000041
Figure RE-GDA0002730778420000051
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. For example, the cyclic rearrangement involves designing the polypeptide 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. 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 (in structure) is equivalent to the residue at position 97 of native IL-2, and the new C-terminus (in structure) is equivalent to the residue at position 96 of native IL-2; in IL-15(105/102), the new N-terminus (structurally) is identical to the residue at position 105 of native IL-15, and the new C-terminus (structurally) is identical 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 protein present on the surface of a cell that binds to a ligand, and also encompasses soluble receptors that are not present on the surface of a cell but 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 "immunoglobulin (Ig)" refers to a globulin having antibody activity or chemical structure, similar to an antibody molecule. Natural immunoglobulins are tetrapeptide chain structures consisting of two identical light chains and two identical heavy chains linked by interchain disulfide bonds.
As used herein, the term "Fc fragment" or "Fc region" is a portion of an immunoglobulin that is cleaved by papain to two identical Fab fragments and one Fc fragment. The Fc fragment of a natural antibody comprises two identical polypeptides linked by disulfide bonds. Each polypeptide comprises two heavy chain constant regions (CH2 and CH3, e.g., the Fc region of IgG), or three heavy chain constant regions (CH2, CH3, and CH4, e.g., the Fc regions of IgM and IgE).
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 Pharmaceuticals 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-GDA0002730778420000091
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 "Loop 1" and "Loop 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 S95-L100 or N50-P54 to form a linear molecule with new N and C termini. In some embodiments, a new N-terminus is formed at N97 and a new C-terminus is formed at K96, i.e., IL-2 (N97/K96).
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: 12).
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 is substituted with A. In some embodiments, the residue C145 of native IL-2 is substituted with S.
In some embodiments, the cyclically rearranged IL-2 comprises the amino acid sequence set forth in SEQ ID NO 2.
In some embodiments, the cyclically rearranged IL-15 comprises the amino acid sequence set forth in SEQ ID NO 4.
Polypeptide dimer
The polypeptide dimers of the present invention comprise a first polypeptide and a second polypeptide,
wherein the first polypeptide comprises a first dimerization domain and a polypeptide of interest, wherein the polypeptide of interest is located at a first end of the first dimerization domain,
wherein the second polypeptide comprises a second dimerization domain and a binding domain, wherein the binding domain is located at a first end of the second dimerization domain,
wherein the first polypeptide and the second polypeptide form a dimer through the first dimerization domain and the second dimerization domain, and the first end of the first dimerization domain is adjacent to the first end of the second dimerization domain, the binding domain being capable of binding to a site of interest on the polypeptide of interest.
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, the cyclic rearrangement being as described hereinbefore.
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, the binding domain is the extracellular domain of CD25, and the polypeptide dimer has comparable or improved activity as native IL-2, e.g., 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, the binding domain is an extracellular domain of CD215, and the polypeptide dimer has comparable or improved activity as native IL-15, such as activity to activate JAK1/JAK3 and STAT3/STAT5 signaling pathways.
In the polypeptide dimers of the invention, the site of interest and the binding domain are located in different (first and second) polypeptides. The first and second dimerization domains form a dimer such that the site of interest and the binding domain are in proximity. The first and second dimerization domains are preferably covalently linked by, for example, but not limited to, covalent linkage, hydrogen bonding, electrostatic interaction, and/or van der waals forces to form a dimer.
In some embodiments, the first and second dimerization domains form a dimer through disulfide bonds. In some embodiments, the first and second dimerization domains comprise heavy chain constant regions CH2 and CH3 of an immunoglobulin (Ig), such as a human Ig (e.g., human IgG 1). In some embodiments, the first dimerization domain and the second dimerization domain form an Fc region of human IgG 1.
Different mutations can be introduced in the Fc region to achieve different functions, e.g., increasing the affinity of Fc for FcRn under acidic conditions; decreasing or increasing the affinity of Fc for different Fc γ receptors as well as C1q to decrease or enhance Antibody Dependent Cellular Cytotoxicity (ADCC), Antibody Dependent Cellular Phagocytosis (ADCP), Complement Dependent Cytotoxicity (CDC), and the like. In some embodiments, the Fc region in the polypeptide dimers of the invention comprises Fc silent mutations, e.g., L234A + L235A + P329G, to reduce ADCC, ADCP and CDC, wherein the Numbering of the amino acid positions of the Fc region follows the IMGT EU Numbering convention (http:// www.imgt.org/imgtscientific chart/number/Hu _ IGH gnber. html).
In some embodiments, the Fc region is a homodimer, i.e., the first dimerization domain and the second dimerization domain have the same sequence. In some embodiments, the Fc region is a heterodimer, i.e., the first dimerization domain and the second dimerization domain differ in sequence. In some embodiments, the Fc region is modified with a knob and hole structure. For example, the mutation T366Y (first generation knob structure) or S354C + T366W (second generation knob structure) is introduced into one polypeptide chain to form a knob (knob) chain; a hole (hole) chain is formed in the other polypeptide chain Y407T (first generation knob structure) or Y349C + T366S + L368A + Y407V (second generation knob structure), and the numbering rules are the same as above. In some embodiments, the first polypeptide comprises a knob chain and the second polypeptide comprises a hole chain. In some embodiments, the first polypeptide comprises a hole chain and the second polypeptide comprises a knob chain.
In some embodiments, the pestle chain comprises SEQ ID No. 5 or an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 5. In some embodiments, the mortar chain comprises SEQ ID No. 6 or an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 6.
In some embodiments, the first dimerization domain comprises SEQ ID No. 5 or an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 5, and the second dimerization domain comprises SEQ ID No. 6 or an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 6; or said first dimerization domain comprises SEQ ID No. 6 or an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 6 and said second dimerization domain comprises SEQ ID No. 5 or an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 5.
In some embodiments involving an immunoglobulin Fc region, the polypeptide of interest is C-terminal to a first dimerization domain (i.e., a first chain of the Fc region), and the binding domain is C-terminal to the second dimerization domain (i.e., a second chain of the Fc region). In some embodiments involving an immunoglobulin Fc region, the polypeptide of interest is N-terminal to a first dimerization domain (i.e., a first chain of the Fc region), and the binding domain is N-terminal to the second dimerization domain (i.e., a second chain of the Fc region).
In some embodiments, the polypeptide of interest is linked to the first dimerization domain by a linker and the binding domain is linked to the second dimerization domain by a linker. In some embodiments, the linker is a flexible linker. 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 has 1-20, 2-18, 3-16, 5-15, 6-12, or 8-10 amino acids. In some embodiments, the linker has the amino acid sequence GGGGSGGGGS (SEQ ID NO: 13).
In some embodiments, the polypeptide dimers of the invention comprise a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first chain of the Fc region of human IgG1 and a cyclically rearranged IL-2, the cyclically rearranged IL-2 being linked to the C-terminus of the first chain of the Fc region of human IgG1, wherein the second polypeptide comprises a second chain of the Fc region of human IgG1 and a CD25 extracellular domain, the CD25 extracellular domain being linked to the C-terminus of the second chain of the Fc region of human IgG1, and wherein the cyclically rearranged IL-2 is as described hereinbefore, e.g., the cyclically rearranged IL-2 consists of the amino acid sequence SEQ ID NO: 2. In some embodiments, the first strand consists of the amino acid sequence of SEQ ID No. 5 and the second strand consists of the amino acid sequence of SEQ ID No. 6; or the first strand consists of the amino acid sequence of SEQ ID NO. 6 and the second strand consists of the amino acid sequence of SEQ ID NO. 5.
In some embodiments, the polypeptide dimers of the invention comprise a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first chain of an Fc region of human IgG1 and a cyclically rearranged IL-15, the cyclically rearranged IL-15 being linked to the C-terminus of the first chain of the Fc region of human IgG1, wherein the second polypeptide comprises a second chain of an Fc region of human IgG1 and a CD215 extracellular domain, the CD215 extracellular domain being linked to the C-terminus of the second chain of the Fc region of human IgG1, and wherein the cyclically rearranged IL-15 is as previously described, e.g., the cyclically rearranged IL-15 comprises the amino acid sequence SEQ ID No. 4. In some embodiments, the first strand comprises the amino acid sequence of SEQ ID No. 5 and the second strand comprises the amino acid sequence of SEQ ID No. 6; or the first strand comprises the amino acid sequence of SEQ ID NO 6 and the second strand comprises the amino acid sequence of SEQ ID NO 5.
One skilled in the art will recognize that modifications can be made to the amino acid sequence of a cyclically rearranged polypeptide without reducing its biological activity. Such modifications are well known to those skilled in the art and include the addition of residues, such as methionine at the amino terminus to provide an initiation site, or the addition of additional amino acids at either terminus to protect the protein from exopeptidases.
Those skilled in the art will recognize that other modifications may be made. For example, amino acid substitutions, such as conservative substitutions, may be made without affecting the activity of the protein. Alternatively, non-essential regions of the molecule may be shortened or eliminated altogether. Thus, in regions of the molecule not themselves involved in the activity of the molecule, they may be eliminated or replaced by shorter fragments which serve only to maintain the correct spatial relationship between the active components of the molecule.
In some embodiments, the cyclically rearranged IL-2 comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 2, wherein the first polypeptide comprises a native CD25 binding site, and the polypeptide dimer has comparable or improved activity to native IL-2.
In some embodiments, the cyclically rearranged IL-15 comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 4, wherein the first polypeptide comprises a native CD215 binding site, and the polypeptide dimer has comparable or improved activity to a native IL-15.
In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO. 7 and the second polypeptide comprises the amino acid sequence of SEQ ID NO. 8. In some embodiments, the first polypeptide comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 7, and the second polypeptide comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 8, wherein the first polypeptide comprises a native CD25 binding site, and the polypeptide dimer has comparable or improved activity to native IL-2.
In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO 9 and the second polypeptide comprises the amino acid sequence of SEQ ID NO 10. In some embodiments, the first polypeptide comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 9, and the second polypeptide comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 10, wherein the first polypeptide comprises a native CD25 binding site, and the polypeptide dimer has comparable or improved activity to native IL-2.
In some embodiments, the polypeptide dimers of the invention have a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or more increase in half-life compared to native IL-2.
In some embodiments, the polypeptide dimers of the invention have a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or more increase in half-life compared to native IL-15.
Expression of tetra, polypeptide dimer
To express the polypeptide dimers of the present invention, the present invention also provides polynucleotides encoding the polypeptide dimers of the present invention. In some embodiments, the first and second polypeptides are encoded by a single polynucleotide. In some embodiments, the first and second polypeptides are encoded by different polynucleotides.
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 polypeptide dimers of the invention are expressed using a single expression vector. In some embodiments, the first and second polypeptides of the polypeptide dimers of the invention are expressed using different expression vectors.
The choice of expression vector is relevant to the host cell used to express the polypeptide dimer. Host cells that can be used to express the polypeptide dimers 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: 14).
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 polypeptide dimers of the invention may further comprise a tag sequence, including but not limited to a His6 tag, FLAG tag, and the like.
In some embodiments, the polynucleotide or vector of the invention, such as an expression vector, encodes the amino acid sequence of SEQ ID NO. 15, and the amino acid sequence of SEQ ID NO. 16. In some embodiments, a polynucleotide or vector of the invention, such as an expression vector, encodes an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 15 and an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 16, wherein the first polypeptide expressed comprises a native CD25 binding site and the polypeptide dimer has comparable or improved activity as native IL-2, e.g., activating the activity of JAK1/JAK3 and STAT3/STAT5 signaling pathways.
In some embodiments, a polynucleotide or vector of the invention, such as an expression vector, encodes the amino acid sequence of SEQ ID NO. 17 and the amino acid sequence of SEQ ID NO. 18. In some embodiments, a polynucleotide or vector of the invention, such as an expression vector, encodes an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 17 and an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 18, wherein the first polypeptide expressed comprises a native CD25 binding site and the polypeptide dimer has comparable or improved activity as native IL-2, e.g., activating the activity of JAK1/JAK3 and STAT3/STAT5 signaling pathways.
Medicine composition
The present invention provides pharmaceutical compositions comprising the polypeptide dimers of the invention. In one embodiment, the pharmaceutical composition comprises the polypeptide dimer of the present invention and at least one pharmaceutically acceptable carrier. The polypeptide dimer 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 polypeptide dimers of the invention to treat diseases, such as diseases involving immunosuppression.
In some embodiments, a method of treating cancer is provided, comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide dimer 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 an immune cell or increasing the proliferation of an immune cell, comprising administering to a subject in need thereof an effective amount of a polypeptide dimer 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 polypeptide dimer or a pharmaceutical composition of the present invention in the manufacture of a medicament for treating 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 polypeptide dimer or a pharmaceutical composition of the present invention in the preparation of a medicament for activating an immune cell or increasing 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: Arabidopsis 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. 15-18 additionally comprising a nucleotide sequence encoding the signal peptide of SEQ ID NO. 14; 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 polypeptide dimer
In this example, 293-6E cells were transfected with nucleic acids encoding polypeptide dimers for eukaryotic expression. The expression and purification of the polypeptide dimer are completed by Nanjing Kingsrei Biotech, Inc., and 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. 3A-3C and 4A-4C. FIGS. 3A-3C: the cyclically rearranged IL-2 was fused to the knob (knob) chain of the Fc region of human IgG1 modified by a knob-in-hole structure (SEQ ID NO:15), and the extracellular domain of CD25 was fused to the knob (hole) chain of the Fc region of human IgG1 modified by a knob-in-hole structure (SEQ ID NO: 16); FIGS. 4A-4C: the cyclically rearranged IL-2 was fused to the hole chain of the human IgG1 Fc region modified by the knob and hole structure (SEQ ID NO:17), and the extracellular domain of CD25 was fused to the knob chain of the human IgG1 Fc region modified by the knob and hole structure (SEQ ID NO: 18). FIGS. 3A and 4A show structural models of the polysaccharide dimers of the invention, FIGS. 3B and 4B show SDS-PAGE results for reduced and non-reduced polypeptide dimers, and FIGS. 3C and 4C show molecular sieve analysis results.
The results show that high expression levels are realized by both polypeptide dimers, and SDS-PAGE and SEC-HPLC show high purity and high homogeneity; high sequence coverage was detected by LC-MS confirming the integrity and correctness of the purified dimer.
Example 3 detection of dimer Activity of polypeptide
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 a Secretory Embryonic Alkaline Phosphatase (SEAP) reporter gene regulated and controlled by STAT5 is also transferred. 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-BlueTM CLR Selection；
-test medium: DMEM + 10% FBS (inactivated) + 1% PS +100 μ g/mL Normocin;
-freezing 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 finenessCell 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 Peking Baiprosperius Biotechnology Ltd., cat # IL2-H4113) and human IgG1 Fc fragment (negative control, Acrobiosystems, Inc. of Peking Baiprosperius Biotechnology Ltd., cat # FCC-H5214) were serially diluted in the following formulation series: 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 polypeptide dimer.
The activity of the polypeptide dimers prepared in step 2 was assayed using the method in step 3, wherein each polypeptide dimer was also prepared as a serial gradient dilution sample according to the method in step 3.2.
The results are shown in FIGS. 5 and 6, FIG. 5: the first polypeptide comprises a cyclically rearranged IL-2 fused to the knob chain of the human IgG1 Fc region modified by the knob structure (SEQ ID NO:15), and the second polypeptide comprises the extracellular domain of CD25 fused to the knob chain of the human IgG1 Fc region modified by the knob structure (SEQ ID NO: 16); FIG. 6: the first polypeptide comprises a cyclically rearranged IL-2 fused to the hole chain of the human IgG1 Fc region modified by a knob and hole structure (SEQ ID NO:17), and the second polypeptide comprises the extracellular domain of CD25 fused to the knob chain of the human IgG1 Fc region modified by a knob and hole structure (SEQ ID NO: 18).
A comparison of the 50% maximal effect concentration (EC50) of each polypeptide dimer detected with the EC50 of human IL-2 is shown in tables 1 and 2 below.
TABLE 1 comparison of polypeptide dimer 1(SEQ ID NO:15+ SEQ ID NO:16) with human IL-2
Human IL-2 Polypeptide dimer 1
EC50(pM) 6.117 10.83
TABLE 2 comparison of polypeptide dimer 2(SEQ ID NO:17+ SEQ ID NO:18) with human IL-2
Human IL-2 Polypeptide dimer 2
EC50(pM) 5.406 10.17
The results show that the dimers of the invention show comparable activity due to wild-type IL-2, whereas no corresponding response was shown using the Fc region fragment (results not shown).
Sequence listing
<110> university of Nanjing university
<120> polypeptide dimer and use thereof
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<170> PatentIn version 3.5
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Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
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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
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Cys Gln Ser Ile Ile Ser Thr Leu Thr
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Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile
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Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala
20 25 30
Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe
35 40 45
Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly Ser Gly Pro Ala Ser Ser
50 55 60
Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu
65 70 75 80
Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr
85 90 95
Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu
100 105 110
Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val
115 120 125
Leu Asn Leu Ala Gln Ser Lys
130 135
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Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr
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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
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Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn
145 150 155 160
Thr Ser
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Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser
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Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu
20 25 30
Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His
35 40 45
Ile Val Gln Met Phe Ile Asn Thr Ser Gly Gly Gly Gly Asn Trp Val
50 55 60
Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met
65 70 75 80
His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys
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Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser
100 105 110
Leu Glu Ser
115
<210> 5
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His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
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Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
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Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
35 40 45
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
50 55 60
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
65 70 75 80
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
85 90 95
Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr
100 105 110
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
115 120 125
Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys
130 135 140
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
145 150 155 160
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
165 170 175
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
180 185 190
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
195 200 205
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
210 215 220
<210> 6
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His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
1 5 10 15
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
20 25 30
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
35 40 45
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
50 55 60
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
65 70 75 80
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
85 90 95
Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr
100 105 110
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu
115 120 125
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys
130 135 140
Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
145 150 155 160
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
165 170 175
Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser
180 185 190
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
195 200 205
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
210 215 220
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<220>
<223> IL-2-knob
<400> 7
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
1 5 10 15
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
20 25 30
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
35 40 45
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
50 55 60
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
65 70 75 80
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
85 90 95
Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr
100 105 110
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
115 120 125
Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys
130 135 140
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
145 150 155 160
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
165 170 175
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
180 185 190
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
195 200 205
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Phe His Leu Arg Pro Arg
225 230 235 240
Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser
245 250 255
Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val
260 265 270
Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr
275 280 285
Leu Thr Gly Ser Gly Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu
290 295 300
Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile
305 310 315 320
Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe
325 330 335
Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu
340 345 350
Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys
355 360 365
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<220>
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His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
1 5 10 15
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
20 25 30
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
35 40 45
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
50 55 60
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
65 70 75 80
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
85 90 95
Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr
100 105 110
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu
115 120 125
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys
130 135 140
Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
145 150 155 160
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
165 170 175
Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser
180 185 190
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
195 200 205
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Cys Asp Asp Asp Pro
225 230 235 240
Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly
245 250 255
Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser
260 265 270
Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp
275 280 285
Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys
290 295 300
Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu
305 310 315 320
Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His
325 330 335
Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr
340 345 350
His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr
355 360 365
Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His
370 375 380
Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly
385 390 395
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<220>
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His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
1 5 10 15
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
20 25 30
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
35 40 45
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
50 55 60
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
65 70 75 80
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
85 90 95
Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr
100 105 110
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu
115 120 125
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys
130 135 140
Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
145 150 155 160
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
165 170 175
Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser
180 185 190
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
195 200 205
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Phe His Leu Arg Pro Arg
225 230 235 240
Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser
245 250 255
Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val
260 265 270
Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr
275 280 285
Leu Thr Gly Ser Gly Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu
290 295 300
Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile
305 310 315 320
Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe
325 330 335
Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu
340 345 350
Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys
355 360 365
<210> 10
<211> 398
<212> PRT
<213> Artificial Sequence
<220>
<223> CD25-knob
<400> 10
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
1 5 10 15
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
20 25 30
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
35 40 45
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
50 55 60
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
65 70 75 80
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
85 90 95
Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr
100 105 110
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
115 120 125
Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys
130 135 140
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
145 150 155 160
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
165 170 175
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
180 185 190
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
195 200 205
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Cys Asp Asp Asp Pro
225 230 235 240
Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly
245 250 255
Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser
260 265 270
Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp
275 280 285
Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys
290 295 300
Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu
305 310 315 320
Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His
325 330 335
Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr
340 345 350
His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr
355 360 365
Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His
370 375 380
Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly
385 390 395
<210> 11
<211> 165
<212> PRT
<213> Artificial Sequence
<220>
<223> CD25ecd
<400> 11
Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys
1 5 10 15
Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg
20 25 30
Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly
35 40 45
Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser
50 55 60
Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln
65 70 75 80
Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp
85 90 95
Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn
100 105 110
Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr
115 120 125
Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu
130 135 140
Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln
145 150 155 160
Leu Ile Cys Thr Gly
165
<210> 12
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> linker
<400> 12
Gly Gly Gly Gly
1
<210> 13
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> linker
<400> 13
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 14
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> signal peptide
<400> 14
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> 15
<211> 373
<212> PRT
<213> Artificial Sequence
<220>
<223> IL-2-knob-full
<400> 15
His His His His His His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
1 5 10 15
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala
100 105 110
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Phe
225 230 235 240
His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu
245 250 255
Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu
260 265 270
Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln
275 280 285
Ser Ile Ile Ser Thr Leu Thr Gly Ser Gly Pro Ala Ser Ser Ser Thr
290 295 300
Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met
305 310 315 320
Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met
325 330 335
Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His
340 345 350
Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn
355 360 365
Leu Ala Gln Ser Lys
370
<210> 16
<211> 406
<212> PRT
<213> Artificial Sequence
<220>
<223> CD25-hole-full
<400> 16
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
1 5 10 15
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
20 25 30
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
35 40 45
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
50 55 60
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
65 70 75 80
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
85 90 95
Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr
100 105 110
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu
115 120 125
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys
130 135 140
Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
145 150 155 160
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
165 170 175
Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser
180 185 190
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
195 200 205
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Cys Asp Asp Asp Pro
225 230 235 240
Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly
245 250 255
Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser
260 265 270
Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp
275 280 285
Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys
290 295 300
Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu
305 310 315 320
Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His
325 330 335
Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr
340 345 350
His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr
355 360 365
Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His
370 375 380
Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly Asp Tyr
385 390 395 400
Lys Asp Asp Asp Asp Lys
405
<210> 17
<211> 373
<212> PRT
<213> Artificial Sequence
<220>
<223> IL-2-hole-full
<400> 17
His His His His His His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
1 5 10 15
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala
100 105 110
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Phe
225 230 235 240
His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu
245 250 255
Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu
260 265 270
Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln
275 280 285
Ser Ile Ile Ser Thr Leu Thr Gly Ser Gly Pro Ala Ser Ser Ser Thr
290 295 300
Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met
305 310 315 320
Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met
325 330 335
Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His
340 345 350
Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn
355 360 365
Leu Ala Gln Ser Lys
370
<210> 18
<211> 406
<212> PRT
<213> Artificial Sequence
<220>
<223> CD25-knob-full
<400> 18
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
1 5 10 15
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
20 25 30
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
35 40 45
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
50 55 60
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
65 70 75 80
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
85 90 95
Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr
100 105 110
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
115 120 125
Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys
130 135 140
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
145 150 155 160
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
165 170 175
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
180 185 190
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
195 200 205
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Cys Asp Asp Asp Pro
225 230 235 240
Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly
245 250 255
Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser
260 265 270
Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp
275 280 285
Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys
290 295 300
Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu
305 310 315 320
Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His
325 330 335
Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr
340 345 350
His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr
355 360 365
Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His
370 375 380
Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly Asp Tyr
385 390 395 400
Lys Asp Asp Asp Asp Lys
405

Claims (30)

1. A polypeptide dimer comprising a first polypeptide and a second polypeptide,
wherein the first polypeptide comprises a first dimerization domain and a polypeptide of interest, wherein the polypeptide of interest is located at a first end of the first dimerization domain,
wherein the second polypeptide comprises a second dimerization domain and a binding domain, wherein the binding domain is located at a first end of the second dimerization domain,
wherein the first polypeptide and the second polypeptide form a dimer through the first dimerization domain and the second dimerization domain, and the first end of the first dimerization domain is adjacent to the first end of the second dimerization domain, the binding domain being capable of binding to a site of interest on the polypeptide of interest.
2. The polypeptide dimer 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 polypeptide dimer 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 polypeptide dimer of claim 3, wherein the amino acid corresponding to the N-terminus of the native cytokine in the cyclically rearranged cytokine is linked to the amino acid corresponding to the C-terminus of the native cytokine by a linker.
5. The polypeptide dimer of claim 4, wherein the linker is a GS linker or polyglycine linker of 1-10 amino acids in length.
6. The polypeptide dimer of any one of claims 1-5, wherein the polypeptide of interest is a cyclically rearranged IL-2 or IL-15.
7. The polypeptide dimer 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 polypeptide dimer of claim 7, wherein the cyclically rearranged IL-2 comprises the amino acid sequence of SEQ ID No. 2.
9. The polypeptide dimer of claim 7 or 8, wherein the site of interest is a CD25 binding site and the binding domain is the extracellular domain of CD 25.
10. The polypeptide dimer 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 polypeptide dimer of claim 10, wherein the cyclically rearranged IL-15 comprises the amino acid sequence of SEQ ID No. 4.
12. The polypeptide dimer of claim 10 or 11, wherein the site of interest is a CD215 binding site and the binding domain is the extracellular domain of CD 215.
13. The polypeptide dimer of any one of claims 1-12, wherein the first and second dimerization domains comprise the heavy chain constant regions CH2 and CH3 of an immunoglobulin (Ig).
14. The polypeptide dimer of claim 13, wherein the Ig is a human Ig.
15. The polypeptide dimer of claim 14, wherein the Ig is human IgG 1.
16. The polypeptide dimer of any one of claims 1-15, wherein the first dimerization domain and the second dimerization domain form an Fc region of human IgG 1.
17. The polypeptide dimer of any one of claims 1-16, wherein the first dimerization domain comprises the amino acid sequence of SEQ ID No. 5 and the second dimerization domain comprises the amino acid sequence of SEQ ID No. 6; or the first dimerization domain comprises the amino acid sequence of SEQ ID NO 6 and the second dimerization domain comprises the amino acid sequence of SEQ ID NO 5.
18. The polypeptide dimer of any one of claims 13-17, wherein the first end of the first dimerization domain is C-terminal and the first end of the second dimerization domain is C-terminal.
19. A polypeptide dimer comprising a first polypeptide and a second polypeptide,
wherein the first polypeptide comprises a first chain of the Fc region of human IgG1 and a cyclically rearranged IL-2, said cyclically rearranged IL-2 being linked C-terminal to the first chain of the Fc region of human IgG1,
wherein the second polypeptide comprises a second chain of an Fc region of human IgG1 and a CD25 extracellular domain, the CD25 extracellular domain is linked to the C-terminus of the second chain of an Fc region of human IgG1, and
wherein the cyclically rearranged IL-2 comprises, in order from N-terminus to C-terminus, H3, H4, H1, and H2; or four alpha-helical bundles of H4, H1, H2 and H3.
20. The polypeptide dimer of claim 19, wherein the cyclically rearranged IL-2 comprises the amino acid sequence of SEQ ID No. 2.
21. A polypeptide dimer comprising a first polypeptide and a second polypeptide,
wherein the first polypeptide comprises a first chain of the Fc region of human IgG1 and a cyclically rearranged IL-15, said cyclically rearranged IL-15 being linked C-terminal to the first chain of the Fc region of human IgG1,
wherein the second polypeptide comprises a second chain of the Fc region of human IgG1 and a CD215 extracellular domain, the CD215 extracellular domain being linked to the C-terminus of the second chain of the Fc region of human IgG1, and
wherein the cyclically rearranged IL-15 comprises four α -helical bundles of H3, H4, H1 and H2 in order from N-terminus to C-terminus.
22. The polypeptide dimer of claim 21, wherein the cyclically rearranged IL-15 comprises the amino acid sequence of SEQ ID No. 4.
23. The polypeptide dimer of any one of claims 19-22, wherein the first strand comprises the amino acid sequence of SEQ ID No. 5 and the second strand comprises the amino acid sequence of SEQ ID No. 6; or the first strand comprises the amino acid sequence of SEQ ID NO 6 and the second strand comprises the amino acid sequence of SEQ ID NO 5.
24. A pharmaceutical composition comprising the polypeptide dimer of any one of claims 1-23.
25. Use of the polypeptide dimer of any one of claims 1-23 in the manufacture of a medicament for the treatment of cancer.
26. Use of the polypeptide dimer of any one of claims 1-23 in the manufacture of a medicament for activating or increasing proliferation of immune cells.
27. The use of claim 26, wherein the immune cell is a T cell or NK cell.
28. One or more isolated polynucleotides encoding the polypeptide dimer of any one of claims 1-23.
29. One or more expression vectors comprising the polynucleotide of claim 28.
30. A host cell comprising the polynucleotide of claim 28 or the vector of claim 29.
CN202010651373.0A 2020-07-08 2020-07-08 Polypeptide dimer and use thereof Pending CN113912735A (en)

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EP21838465.9A EP4186928A1 (en) 2020-07-08 2021-07-08 Fusion polypeptide and polypeptide dimer, and use 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

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Citations (4)

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Publication number Priority date Publication date Assignee Title
US20150218260A1 (en) * 2014-02-06 2015-08-06 Hoffman-La Roche Inc. Interleukin-2 fusion proteins and uses thereof
CN110325205A (en) * 2016-12-22 2019-10-11 库尔生物制药有限公司 T cell modulability multimeric polypeptide and its application method
WO2019222283A1 (en) * 2018-05-14 2019-11-21 Harpoon Therapeutics, Inc. Binding moiety for conditional activation of immunoglobulin molecules
CN110724198A (en) * 2018-07-17 2020-01-24 上海一宸医药科技有限公司 Long-acting fibronectin type III domain fusion proteins

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150218260A1 (en) * 2014-02-06 2015-08-06 Hoffman-La Roche Inc. Interleukin-2 fusion proteins and uses thereof
CN110325205A (en) * 2016-12-22 2019-10-11 库尔生物制药有限公司 T cell modulability multimeric polypeptide and its application method
WO2019222283A1 (en) * 2018-05-14 2019-11-21 Harpoon Therapeutics, Inc. Binding moiety for conditional activation of immunoglobulin molecules
CN110724198A (en) * 2018-07-17 2020-01-24 上海一宸医药科技有限公司 Long-acting fibronectin type III domain fusion proteins

Non-Patent Citations (2)

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Title
NATASHA C. WARD等: "IL-2/CD25: A long-acting fusion protein that promotes immune tolerance by selectively targeting the IL-2R on regulatory T cells", 《J IMMUNOL》, vol. 9, 1 November 2019 (2019-11-01), pages 2579 - 2592 *
RAJAT VARMA等: "Regulatory T Cell Selective IL-2-Fc Fusion Proteins for the Treatment of Autoimmune Diseases", 《BLOOD》, vol. 132, 29 November 2018 (2018-11-29), pages 1 - 2 *

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