WO2025252012A1 - Variants d'il-7 modifiés et leurs procédés d'utilisation - Google Patents

Variants d'il-7 modifiés et leurs procédés d'utilisation

Info

Publication number
WO2025252012A1
WO2025252012A1 PCT/CN2025/098273 CN2025098273W WO2025252012A1 WO 2025252012 A1 WO2025252012 A1 WO 2025252012A1 CN 2025098273 W CN2025098273 W CN 2025098273W WO 2025252012 A1 WO2025252012 A1 WO 2025252012A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
engineered
corresponds
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/098273
Other languages
English (en)
Inventor
Huan-Ching LIN
Shih-Cheng Yang
Hsiang-Chun CHUANG
Chia-Yeh CHUANG
Yi-Syuan LIANG
Pei-Lun Tsai
Tzu-Lin KUO
Yen-Cheng Li
Yung-Chu Lin
Rui-ling LIANG
Yu-Chien Chen
Chieh-Hsin HO
Tsai-Kuei Shen
I-Yin CHEN
Zong Sean Juo
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
FBD Biologics Ltd
Original Assignee
FBD Biologics Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by FBD Biologics Ltd filed Critical FBD Biologics Ltd
Publication of WO2025252012A1 publication Critical patent/WO2025252012A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5418—IL-7
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • This disclosure relates to engineered IL-7 variants, and methods of use thereof.
  • T-cell development occurs through several stages in the thymus. Immature T-cells need to reach sufficient levels because their development has several stages known as positive and negative selection, contributing to the loss of 98%of T cells. Where regulation of human T cell maturation was achieved by decreasing IL-7 and increasing anti-CD3 levels. These effects are supported by studies demonstrating how intrathymic T cell development is dependent in some part on the presence of IL-7. Specifically, IL-7 along with thymic stromal lymphopoietin seem to be necessary for Treg maturation.
  • engineered IL-7 variants e.g., fusion proteins or protein complexes
  • the engineered IL-7 variants and protein constructs include a non-native disulfide bond formed by mutating two or more residues (e.g., a pair of residues) of wildtype IL-7 to cysteines, without interfering the overall structure or residues critical for IL-7 receptor interaction.
  • the variants and protein constructs include a disrupted MMP9 cleavage site. As a result, the engineered IL-7 variants or protein constructs thereof can be more stable than a wildtype IL-7 or protein construct thereof.
  • the protein constructs showed enhanced thermostability (e.g., increased T m ) , a medium to high potency on T cell response, and/or a similar or low CD127-binding affinity than a wildtype IL-7 or protein construct thereof.
  • the protein constructs showed significant anti-tumor effects with minimal toxicity using a tumor-bearing mouse model. Therefore, the engineered IL-7 variants and protein constructs thereof described herein can be used for cancer therapies.
  • the engineered IL-7 variants described herein include one or more non-native disulfide bonds, which can stabilize the internal core of IL-7, with purposes of increasing the thermostability of the engineered IL-7 variants.
  • the one or more non-native disulfide bonds can also exhibit beneficial effects on pharmacokinetics (PK) .
  • PK pharmacokinetics
  • by gently modifying the internal core of IL-7 subtle structural changes and/or unique functional properties (e.g., different potencies, different binding affinities, etc. ) can be achieved. Without wishing to be bound by theory, it is contemplated that such effects are not achievable by making surface mutations of IL-7.
  • IL-7 e.g., human IL-7
  • increased stability and/or anti-tumor efficacy are also provided herein.
  • the disclosure is related to an engineered IL-7 polypeptide, in some embodiments, the engineered IL-7 polypeptide comprises a non-native disulfide bond. In some embodiments, the engineered IL-7 polypeptide described herein comprises an amino acid sequence that is at least 80%identical to SEQ ID NO: 2.
  • the engineered IL-7 polypeptide comprises one or more of the following: (a) the amino acid that corresponds to position 9 of SEQ ID NO: 2 is C (cysteine) , and the amino acid that corresponds to position 146 of SEQ ID NO: 2 is C; (b) the amino acid that corresponds to position 16 of SEQ ID NO: 2 is C, and the amino acid that corresponds to position 82 of SEQ ID NO: 2 is C; (c) the amino acid that corresponds to position 19 of SEQ ID NO: 2 is C, and the amino acid that corresponds to position 78 of SEQ ID NO: 2 is C; (d)
  • the amino acid that corresponds to position 24 of SEQ ID NO: 2 is C, and the amino acid that corresponds to position 132 of SEQ ID NO: 2 is C;
  • the amino acid that corresponds to position 56 of SEQ ID NO: 2 is C, and the amino acid that corresponds to position 138 of SEQ ID NO: 2 is C;
  • the amino acid that corresponds to position 57 of SEQ ID NO: 2 is C, and the amino acid that corresponds to position 87 of SEQ ID NO: 2 is C;
  • the amino acid that corresponds to position 65 of SEQ ID NO: 2 is C, and the amino acid that corresponds to position 124 of SEQ ID NO: 2 is C;
  • the amino acid that corresponds to position 65 of SEQ ID NO: 2 is C, and the amino acid that corresponds to position 125 of SEQ ID NO: 2 is C;
  • the amino acid that corresponds to position 66 of SEQ ID NO: 2 is C, and the amino acid that corresponds to position 124 of SEQ ID NO: 2 is C is
  • the non-native disulfide bond can stabilize the internal core of IL-7.
  • the engineered IL-7 polypeptide has an increased thermostability and/or an improved pharmacokinetic profile as compared to a wildtype IL-7 or a functional fragment thereof.
  • the engineered IL-7 polypeptide comprises a disrupted matrix metalloproteinase-9 cleavage site. In some embodiments, the engineered IL-7 polypeptide comprises either one or both of the following: (a) the amino acid that corresponds to position 101 of SEQ ID NO: 2 is not proline, and (b) the amino acid that corresponds to position 104 of SEQ ID NO: 2 is not leucine. In some embodiments, the engineered IL-7 polypeptide comprises either one or both of the following: (a) the amino acid that corresponds to position 101 of SEQ ID NO: 2 is S, T, N, or Q, and (b) the amino acid that corresponds to position 104 of SEQ ID NO: 2 is S, T, N, or Q.
  • the engineered IL-7 polypeptide comprises either one or both of the following: (a) the amino acid that corresponds to position 101 of SEQ ID NO: 2 is S, and (b) the amino acid that corresponds to position 104 of SEQ ID NO: 2 is S.
  • the engineered IL-7 polypeptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26.
  • the engineered IL-7 polypeptide comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 9.
  • the engineered IL-7 polypeptide comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 15.
  • the engineered IL-7 polypeptide comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 20.
  • the engineered IL-7 polypeptide comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 21.
  • the engineered IL-7 polypeptide can induce IL-7/IL-7R signaling pathways (e.g., activation of the JAK/STAT5 pathway) and/or induce proliferation of immune cells (e.g., primary T cells) .
  • the engineered IL-7 polypeptide can bind to CD127 (e.g., human CD127) and/or CD132 (e.g., human CD132) .
  • the disclosure is related to an engineered IL-7 polypeptide
  • the engineered IL-7 polypeptide comprises a disrupted matrix metalloproteinase-9 cleavage site.
  • the engineered IL-7 polypeptide comprises either one or both of the following: (a) the amino acid that corresponds to position 101 of SEQ ID NO: 2 is not proline, and (b) the amino acid that corresponds to position 104 of SEQ ID NO: 2 is not leucine.
  • the engineered IL-7 polypeptide comprises either one or both of the following: (a) the amino acid that corresponds to position 101 of SEQ ID NO: 2 is S, T, N, or Q, and (b) the amino acid that corresponds to position 104 of SEQ ID NO: 2 is S, T, N, or Q. In some embodiments, the engineered IL-7 polypeptide comprises either one or both of the following: (a) the amino acid that corresponds to position 101 of SEQ ID NO: 2 is S, and (b) the amino acid that corresponds to position 104 of SEQ ID NO: 2 is S.
  • the disclosure is related to a fusion protein comprising the engineered IL-7 polypeptide described herein.
  • the fusion protein further comprises a His-tag, optionally at the N-terminus.
  • the fusion protein comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46.
  • the fusion protein can induce IL-7/IL-7R signaling pathways (e.g., activation of the JAK/STAT5 pathway) and/or induce proliferation of immune cells (e.g., primary T cells) .
  • the fusion protein can bind to CD127 (e.g., human CD127) and/or CD132 (e.g., human CD132) .
  • the fusion protein described herein further comprises an Fc region.
  • the fusion protein comprises or consists of an immunocytokine.
  • the disclosure is related to a protein complex comprising the engineered IL-7 polypeptide described herein, in some embodiments, the protein complex comprises an immunocytokine.
  • the disclosure is related to a protein complex comprising: (a) a first polypeptide comprising from N-terminus to C-terminus: an optional first hinge region, a first Fc region, an optional linker peptide, and the engineered IL-7 polypeptide described herein; and (b) a second polypeptide comprising from N-terminus to C-terminus: an optional second hinge region, and a second Fc region.
  • the disclosure is related to a protein complex comprising: (a) a first polypeptide comprising from N-terminus to C-terminus: the engineered IL-7 polypeptide of any one of claims 1-18, an optional first hinge region, and a first Fc region; and (b) a second polypeptide comprising from N-terminus to C-terminus: an optional second hinge region, and a second Fc region.
  • the first hinge region, the first Fc region, the second hinge region, and/or the second Fc region are derived from IgG4 (e.g., human IgG4) .
  • the first Fc region and/or the second Fc region comprise one or more knob-into-hole (KIH) mutations.
  • the first Fc region comprises a sequence that is at least 80%, 90%, 95%, or 100%to SEQ ID NO: 49
  • the second Fc region comprises a sequence that is at least 80%, 90%, 95%, or 100%to SEQ ID NO: 50
  • the first hinge region and/or the second hinge region comprise a sequence that is at least 80%, 90%, 95%, or 100%to SEQ ID NO: 48
  • the linker peptide comprises a sequence that is at least 80%, 90%, 95%, or 100%to SEQ ID NO: 4.
  • the first polypeptide comprises a sequence that is at least 80%, 90%, 95%, or 100%to SEQ ID NO: 55, and the first polypeptide comprises a sequence that is at least 80%, 90%, 95%, or 100%to SEQ ID NO: 53;
  • the first polypeptide comprises a sequence that is at least 80%, 90%, 95%, or 100%to SEQ ID NO: 56, and the first polypeptide comprises a sequence that is at least 80%, 90%, 95%, or 100%to SEQ ID NO: 53;
  • the first polypeptide comprises a sequence that is at least 80%, 90%, 95%, or 100%to SEQ ID NO: 57, and the first polypeptide comprises a sequence that is at least 80%, 90%, 95%, or 100%to SEQ ID NO: 53;
  • the first polypeptide comprises a sequence that is at least 80%, 90%, 95%, or 100%to SEQ ID NO: 58, and the first polypeptide comprises a sequence that is at least 80%, 90%, 95%, or 100%to S
  • the protein complex can induce IL-7/IL-7R signaling pathways (e.g., activation of the JAK/STAT5 pathway) and/or induce proliferation of immune cells (e.g., primary T cells) .
  • the protein complex can bind to CD127 (e.g., human CD127) and/or CD132 (e.g., human CD132) .
  • the disclosure is related to a pharmaceutical composition
  • a pharmaceutical composition comprising the engineered IL-7 polypeptide, the fusion protein, or the protein complex described herein; and a pharmaceutically acceptable carrier.
  • the disclosure is related to a nucleic acid encoding the engineered IL-7 polypeptide, the fusion protein, or the protein complex described herein.
  • the disclosure is related to a vector comprising the nucleic acid described herein.
  • the disclosure is related to a cell comprising the nucleic acid or the vector described herein.
  • the cell is a Expi293 cell or a CHO cell (e.g., a CHO-Scell) .
  • the disclosure is related to a method of producing an engineered IL-7 polypeptide or a fusion protein comprising the engineered IL-7 polypeptide, the method comprising (a) culturing the cell described herein under conditions sufficient for the cell to produce the engineered IL-7 polypeptide, the fusion protein, or the protein complex; and (b) collecting the engineered IL-7 polypeptide, the fusion protein, or the protein complex produced by the cell.
  • the disclosure is related to a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the engineered IL-7 polypeptide, the fusion protein, or the protein complex described herein to the subject.
  • the subject has a solid tumor or a hematologic cancer.
  • the cancer is lung cancer, melanoma, colorectal cancer, glioma, pancreatic cancer, lymphoma, leukemia, prostate cancer, renal cell carcinoma (RCC) , hepatocellular carcinoma, cholangiocarcinoma, gallbladder cancer, gastric cancer, endometrial carcinoma, ovarian cancer, bladder cancer, or glioblastoma.
  • the disclosure is related to a method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising the engineered IL-7 polypeptide, the fusion protein, or the protein complex described herein.
  • the disclosure is related to a method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the engineered IL-7 polypeptide, the fusion protein, or the protein complex described herein.
  • the disclosure is related to a method of improving the stability of IL-7, comprising (a) providing a 3D structure of IL-7, (b) measuring distance of the C alpha atoms of one or more of amino acid residues in the 3D structure; and (c) selecting two amino acid residues from the one or more amino acid residues, in some embodiments, the C alpha atoms of the two selected amino acid residues are within 3-7 angstroms.
  • the method described herein further comprises expressing a IL-7 variant, in some embodiments, the IL-7 variant comprises a non-native disulfide bond formed by mutating the two selected amino acid residues to cysteines. In some embodiments, the mutating the two selected amino acid residues to cysteines does not substantially change the 3D structure of IL-7.
  • the disclosure is related to a method of screening a IL-7 variant with an improved anti-tumor efficacy, comprising (a) providing a 3D structure of IL-7, (b) measuring distance of the C alpha atoms of one or more of amino acid residues in the 3D structure; and (c) selecting two amino acid residues from the one or more amino acid residues, in some embodiments, the C alpha atoms of the two selected amino acid residues are within 3-7 angstroms.
  • the method described herein further comprises (d) expressing a IL-7 variant, in some embodiments, the IL-7 variant comprises a non-native disulfide bond formed by mutating the two selected amino acid residues to cysteines; (e) administering the IL-7 variant to a tumor-bearing animal; and (f) determining tumor growth (e.g., by measuring tumor volume) in the tumor-bearing animal.
  • the IL-7 is human IL-7.
  • the IL-7 comprises an amino acid sequence that is at least 80%, 90%, 95%, or 100%to SEQ ID NO: 2.
  • non-native disulfide bond refers to a disulfide bond that does not naturally exist in a wild-type protein.
  • the non-native disulfide bond is formed by two cysteine residues, wherein at least one of them is a mutation. In some embodiments, two of them are mutations. In some embodiments, at least one or two cysteine residues are introduced by insertion. In some embodiments, a deletion changes the distance between two existing cysteine residues, which then forms a disulfide bond that does not exist in a wild-type protein.
  • the term “engineered IL-7 polypeptide” refers to a polypeptide derived from a wildtype IL-7 polypeptide or a portion thereof, optionally with one or more mutations (e.g., insertions, deletions, or substitutions) .
  • the engineered IL-7 polypeptide comprises or consists of an amino acid sequence corresponding to amino acids 26-177 of human IL-7 (SEQ ID NO: 1) .
  • the engineered IL-7 polypeptide has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mutations (e.g., amino acids are substituted by cysteine) .
  • the term “protein construct” refers to a complex having one or more polypeptides.
  • the protein construct is a fusion protein, e.g., a fusion protein including a His tag and an engineered IL-7 polypeptide (e.g., any of the engineered IL-7 polypeptides described herein) .
  • the protein construct is a protein complex.
  • the protein construct has two or more polypeptides, wherein the polypeptides can associate with each other, forming a dimer or a multimer (e.g., a trimer) .
  • the protein construct is a heterodimeric Fc-fused IL-7 (e.g., any of the heterodimeric Fc-fused IL-7 or its variants described herein) .
  • cancer refers to cells having the capacity for uncontrolled autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • malignancies of the various organ systems such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, and endocrine systems; as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, and cancer of the small intestine.
  • Cancer that is “naturally arising” includes any cancer that is not experimentally induced by implantation of cancer cells into a subject, and includes, for example, spontaneously arising cancer, cancer caused by exposure of a patient to a carcinogen (s) , cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infections, e.g., viral infections.
  • a carcinogen s
  • cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene and cancer caused by infections, e.g., viral infections.
  • the term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • an “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • the term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.
  • hematopoietic neoplastic disorders includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin.
  • a hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
  • a hematologic cancer is a cancer that begins in blood-forming tissue, such as the bone marrow, or in the cells of the immune system. Examples of hematologic cancer include e.g., leukemia, lymphoma, and multiple myeloma etc.
  • the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided.
  • Veterinary and non-veterinary applications are contemplated in the present disclosure.
  • Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old) .
  • patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates.
  • non-human primates e.g., monkey, chimpanzee, gorilla, and the like
  • rodents e.g., rats, mice, gerbils, hamsters, ferrets, rabbits
  • lagomorphs e.g., swine (e.g., pig, miniature pig)
  • equine canine, feline, bovine, and other domestic, farm, and zoo animals.
  • polypeptide, ” “peptide, ” and “protein” are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.
  • nucleic acid molecule As used herein, the terms “polynucleotide, ” “nucleic acid molecule, ” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, and include, without limitation, DNA, RNA, DNA/RNA hybrids, and modifications thereof.
  • FIGS. 1A-1D show the OD value at 630 nm of His-tagged IL-7 variants measured using the HEK-Blue TM IL-7 reporter assay.
  • FIGS. 1A-1B show the results of high-potency variants.
  • FIG. 1C shows the results of medium-potency variants.
  • FIG. 1D shows the results of low-potency variants. His-tagged wildtype IL-7 without signal peptide (His-IL7_wt; SEQ ID NO: 47) was used as a control. An anti-mesothelin antibody was used as a negative control.
  • FIG. 2 shows the luminescence signal of His-tagged IL-7 variants measured using the primary T cell proliferation assay. His-tagged wildtype IL-7 without signal peptide (His-IL7_wt; SEQ ID NO: 47) was used as a control. An anti-mesothelin antibody was used as a negative control.
  • FIGS. 3A-3B show the OD value at 630 nm of His-tagged IL-7 variants and heterodimeric Fc-fused IL-7 variants measured using the HEK-Blue TM IL-7 reporter assay, respectively.
  • His-tagged wildtype IL-7 without signal peptide His-IL7_wt; SEQ ID NO: 47
  • His-tagged wildtype IL-7 without signal peptide His-IL7_wt; SEQ ID NO: 47
  • Heterodimer G4ssFc-IL7_wt hole chain: SEQ ID NO: 59; knob chain: SEQ ID NO: 53
  • An anti-mesothelin antibody was used as a negative control.
  • FIG. 4 shows the luminescence signal of heterodimeric Fc-fused IL-7 variants measured using the primary T cell proliferation assay.
  • Heterodimer G4ssFc-IL7_wt hole chain: SEQ ID NO: 59; knob chain: SEQ ID NO: 53
  • An anti-mesothelin antibody was used as a negative control.
  • FIG. 5 shows the absorbance value at 450 nm of the heterodimeric Fc-fused IL-7 variants binding to CD127, as measured by ELISA.
  • Heterodimer G4ssFc-IL7_wt hole chain: SEQ ID NO: 59; knob chain: SEQ ID NO: 53
  • An anti-mesothelin antibody was used as a negative control.
  • FIG. 6 shows the body weight of CT26-bearing BALB/c mice that were treated with heterodimer G4ssFc-IL7_C03_MMP9 (G1) , heterodimer G4ssFc-IL7_C09_MMP9 (G2) , heterodimer G4ssFc-IL7_C14_MMP9 (G3) , or heterodimer G4ssFc-IL7_wt (G4) .
  • the control group mice (G5) were injected with 10 mL/kg vehicle (isotonic sodium chloride solution) . After grouping (Day 0) , the mice were injected with heterodimeric Fc-fused IL-7 and its variants on Day 4, Day11, and Day 18 (indicated by arrows) .
  • FIG. 7A-7E show individual tumor growth curves of CT26-bearing BALB/c mice that were treated with heterodimer G4ssFc-IL7_C03_MMP9 (FIG. 7A) , heterodimer G4ssFc-IL7_C09_MMP9 (FIG. 7B) , heterodimer G4ssFc-IL7_C14_MMP9 (FIG. 7C) , heterodimer G4ssFc-IL7_wt (FIG. 7D) , or vehicle (FIG. 7E) .
  • FIG. 8 is a table showing amino acid sequences of His-tagged IL-7 variants.
  • FIG. 9 lists amino acid sequences of heterodimeric G4ssFc-fused IL-7 variants.
  • FIG. 10 lists sequences discussed in the disclosure.
  • Interleukin-7 is a four- ⁇ -helical cytokine that binds to a receptor comprising IL-7R ⁇ (CD127) and the common cytokine receptor ⁇ chain (CD132) .
  • IL-7 is critical for development and maintenance of the entire lymphoid compartment, including T cells, B cells and ILCs. IL-7 promotes the survival and expansion of naive T cells or memory T cells, but not Treg cells. IL-7 can decrease PD-1 expression and restore functional capacity of exhausted T cells.
  • IL-7 is a cytokine essential for the adaptive immune system. T lymphopoiesis in the thymus has been shown to be highly IL-7 dependent in mice. In addition, IL-7 is important for T cell homeostasis and lymphopenia-driven proliferation. It regulates lymph nodes (LN) organogenesis by controlling the pool of lymphoid tissue inducer (LTi) cells. By activating several intracellular signal pathways, IL-7 promotes the cell survival and proliferation of both and memory T cells.
  • LN lymph nodes
  • LTi lymphoid tissue inducer
  • IL-7 is mainly produced by non-hematopoietic cells including keratinocytes in the skin, fibroblastic stromal cells in the bone marrow and lymphoid organs, epithelial cells in the thymus, prostatic epithelium and the intestine.
  • Immune cells such as dendritic cells (DCs) can also produce IL-7.
  • DCs dendritic cells
  • IL-7 transcripts and proteins have also been found in normal adult human hepatic tissue produced by cells of lymphoid morphology.
  • the receptor of IL-7 is a heterodimer that consists of two chains: IL-7R ⁇ (CD127) , which is shared with thymic stromal lymphopoietin (TSLP) , and the common ⁇ chain (CD132) for IL-2, IL-4, IL-9, IL-15 and IL-21.
  • IL-7R ⁇ is expressed on all hematopoietic cell types, while IL-7R ⁇ is mainly expressed by lymphocytes, including common T/B lymphoid precursors, developing T and B cells, T cells and memory T cells.
  • Innate lymphoid cells are critical in lymphoid organ development and innate immune responses to pathogens.
  • IL-7R ⁇ is also found in ILCs, such as NK cells and gut-associated lymphoid tissue (GALT) -derived LTi cells. IL-7 can also regulate lymphoid organogenesis by controlling the pool of LTi cells. IL-7R ⁇ is regulated by stimulative transcription factors, GABP ⁇ and Foxo1 as well as inhibitory Gfi-1. TGF- ⁇ promotes IL-7R ⁇ expression via the inhibition of Gfi-1 expression. There is another type of IL-7 receptor: soluble IL-7R, which competes with cell-associated IL-7R to reduce excessive IL-7 consumption by IL-7R expressing target cells and enhances the bioactivity of IL-7 when the cytokine is limited.
  • IL-7R ⁇ is associated with the protein tyrosine kinase Janus kinase 1 (Jak1) , and the cytosolic tail of the ⁇ chain is associated with Jak3. Binding of IL-7 to its receptor causes activation of Jaks in the cytosol, phosphorylating signal transducer and activator of transcription (STAT) proteins. The dimeric phosphorylated STAT (pSTAT) proteins subsequently translocate into the nucleus to activate gene expression.
  • STAT protein tyrosine kinase Janus kinase 1
  • IL-7 activates the anti-apoptotic genes, Bcl-2 and Mcl-1, and suppresses pro-apoptotic proteins, such as Bax and Bak. Consequently, and memory T cells survive. This function is dose-dependent, such that a higher concentration of IL-7 induces thymic emigrant T cell proliferation, while lower concentrations sustain cell survival.
  • IL-7 downregulates the cell cycle inhibitor p27 kip1 to induce the expression of cyclin D1 for cell cycle progression. Moreover, it promotes glucose transporter 1 expression, glucose uptake and mitochondrial integrity to positively regulate cell metabolism and size.
  • IL-7 IL-7 and its function can be found, e.g., in Gao, J., et al. "Mechanism of action of IL-7 and its potential applications and limitations in cancer immunotherapy. " International Journal of Molecular Sciences 16.5 (2015) : 10267-10280; Lin, J., et al. "The role of IL-7 in Immunity and Cancer. " Anticancer Research 37.3 (2017) : 963-967; and McElroy, C. A., et al. "Structural and biophysical studies of the human IL-7/IL-7R ⁇ complex. " Structure 17.1 (2009) : 54-65; each of which is incorporated by reference herein in the entirety.
  • the present disclosure provides engineered IL-7 variants having at least one non-native disulfide bond.
  • two amino acid residues in a wildtype human IL-7 sequence e.g., SEQ ID NO: 2 are selectively mutated to cysteines, which can form the non-native disulfide bond.
  • the mutations do not substantially change the overall structure of IL-7, e.g., the relative position of the four alpha helices of IL-7.
  • the non-native disulfide bond formed by the cysteine mutations can stabilize IL-7.
  • the engineered IL-7 variants described herein can bind to the IL-7R ⁇ / ⁇ c complex, and induce downstream signaling pathways (e.g., the JAK/STAT, PI3K, and/or MAPK/ERK pathways) and/or immune cell (e.g., primary T cell) proliferation.
  • the engineered IL-7 variants comprises or consists of any of the engineered IL-7 polypeptides described herein.
  • protein constructs e.g., fusion proteins
  • protein constructs that further include a His tag that is fused (e.g., at the N-terminal end) to the engineered IL-7 variants described herein.
  • the protein constructs can induce downstream signaling pathways (e.g., the JAK/STAT, PI3K, and/or MAPK/ERK pathways) and/or immune cell (e.g., primary T cell) proliferation.
  • protein constructs e.g., protein complexes that an engineered IL-7 variant (e.g., any of the engineered IL-7 variants described herein) is linked (e.g., at the C-terminal end) to a human IgG4 Fc, forming a heterodimer (e.g., any of the heterodimeric Fc-fused IL-7 variants described herein) .
  • the protein complexes have a medium to high potency to induce downstream signaling pathways (e.g., the JAK/STAT, PI3K, and/or MAPK/ERK pathways) and/or immune cell (e.g., primary T cell) proliferation.
  • the protein complexes can bind to CD127 (IL-7R ⁇ ) with an affinity that is comparable or lower than that of a control protein complex (e.g., heterodimer G4ssFc-IL7_wt) .
  • a control protein complex e.g., heterodimer G4ssFc-IL7_wt
  • the protein complexes can have lower off-target effects and/or better pharmacokinetics (PK) than a control protein complex (e.g., heterodimer G4ssFc-IL7_wt) .
  • the protein complexes have a higher stability (e.g., a higher T m ) and/or improved anti-tumor efficacy than a control protein complex (e.g., heterodimer G4ssFc-IL7_wt) .
  • a control protein complex e.g., heterodimer G4ssFc-IL7_wt
  • the disclosure also provides methods of screening cytokine (e.g., IL-7) variants having a higher stability and/or improved anti-tumor efficacy.
  • cytokine e.g., IL-7
  • the human IL-7 gene locus is 72 kb in length, resides on chromosome 8q12-13 and encodes for a protein of 177 amino acids (SEQ ID NO: 1) with a molecular weight of 20 kDa.
  • Human IL-7 includes, from N-terminus to C-terminus, a signal peptide, and a soluble chain.
  • the signal peptide of human IL-7 corresponds to amino acids 1-25 of SEQ ID NO: 1
  • the soluble chain of human IL-7 corresponds to amino acids 26-177 of SEQ ID NO: 1.
  • the soluble chain of human IL-7 is also shown as SEQ ID NO: 2.
  • the soluble chain contains four highly conserved helical domains (helices) , i.e., from N-terminus to C-terminus, helix A, helix B, helix C, and helix D.
  • helices helical domains
  • helix A corresponds to amino acids 31-52 of SEQ ID NO: 1
  • helix B corresponds to amino acids 77-92 of SEQ ID NO: 1
  • helix C corresponds to amino acids 98-117 of SEQ ID NO: 1
  • helix D corresponds to amino acids 152-172 of SEQ ID NO: 1.
  • helix A corresponds to G6 to M27 of SEQ ID NO: 2
  • helix B corresponds to E52 to L67 of SEQ ID NO: 2
  • helix C corresponds to G73 to C92 of SEQ ID NO: 2
  • helix D corresponds to D127 to M147 of SEQ ID NO: 2.
  • the AB loop is between helix A and helix B.
  • the BC loop is between helix B and helix C.
  • the CD loop is between helix C and helix D.
  • residues of IL-7 were identified as positions potentially critical for IL-7R or ⁇ c binding: amino acids corresponding to positions 9, 16, 19, 20, 24, 45, 55, 56, 57, 60, 63, 65, 66, 78, 82, 83, 85, 87, 124, 125, 126, 132, 135, 138, and 146 of SEQ ID NO: 2.
  • these residues are G9, L16, S19, I20, L24, H45, F55, L56, F57, A60, L63, Q65, F66, H78, V82, S83, G85, T87, K124, L125, N126, K132, L135, I138, and L146 in SEQ ID NO: 2.
  • distance of the C alpha atoms of one or more amino acid residues (e.g., those in close proximity) in the 3D structure can be determined.
  • Two residues whose C alpha atoms are within 3-7 angstroms (e.g., 4.5-6.5 angstroms) can be selectively mutated to cysteines, without interfering the overall structure (e.g., the core structure formed by the four-helical bundle) of IL-7. It is contemplated that the newly introduced cysteines can form a disulfide bond non-native to the wild-type IL-7, which can stabilize IL-7 and/or improve the functional potencies of IL-7.
  • the engineered IL-7 variants includes a first cysteine mutation and a second cysteine mutation, such that the two cysteines can form a non-native disulfide bond.
  • the first cysteine mutation occurs at an amino acid residue in helix A that corresponds to position 9, 16, 19, 20, or 24 of SEQ ID NO: 2; and the second cysteine mutation occurs at an amino acid residue in helix D that corresponds to position 132, 135, 138, or 146 of SEQ ID NO: 2.
  • the first cysteine mutation occurs at an amino acid residue in helix A that corresponds to position 9, 16, 19, 20, or 24 of SEQ ID NO: 2; and the second cysteine mutation occurs at an amino acid residue in helix C that corresponds to position 78, 82, 83, 85, or 87 of SEQ ID NO: 2.
  • the first cysteine mutation occurs at an amino acid residue in helix B that corresponds to position 55, 56, 57, 60, 63, 65, or 66 of SEQ ID NO: 2; and the second cysteine mutation occurs at an amino acid residue in helix D that corresponds to position 132, 135, 138, or 146 of SEQ ID NO: 2.
  • the first cysteine mutation occurs at an amino acid residue in helix B that corresponds to position 55, 56, 57, 60, 63, 65, or 66 of SEQ ID NO: 2; and the second cysteine mutation occurs at an amino acid residue in helix C that corresponds to position 78, 82, 83, 85, or 87 of SEQ ID NO: 2.
  • the first cysteine mutation occurs at an amino acid residue in helix B that corresponds to position 55, 56, 57, 60, 63, 65, or 66 of SEQ ID NO: 2; and the second cysteine mutation occurs at an amino acid residue in CD loop that corresponds to position 124, 125, or 126 of SEQ ID NO: 2.
  • the first cysteine mutation occurs at an amino acid residue in AB loop that corresponds to position 45 of SEQ ID NO: 2; and the second cysteine mutation occurs at an amino acid residue in helix B that corresponds to position 55, 56, 57, 60, 63, 65, or 66 of SEQ ID NO: 2.
  • the C alpha atoms of the two selected amino acid residues are within
  • any of the first cysteine mutations can be paired with any of the second cysteine mutations described herein, when distance of the C alpha atoms of the two selected amino acid residues is within the range described above.
  • the non-native disulfide bond can further stabilize the overall structure of IL-7, e.g., the relative position and angles of the four-helical bundle in IL-7 are maintained.
  • the matrix metalloproteinase-9 (MMP9) cleavage site of a wildtype IL-7 sequence is disrupted. Details of the MMP9 cleavage site in IL-7 can be found, e.g., in Vandooren, J., et al. "Internal Disulfide bonding and glycosylation of Interleukin-7 protect against proteolytic inactivation by neutrophil Metalloproteinases and serine proteases. " Frontiers in Immunology 12 (2021) : 701739, which is incorporated herein by reference in its entirety.
  • the engineered IL-7 variant (e.g., any of the engineered IL-7 variants described herein) includes a disrupted MMP9 cleavage site.
  • MMP9 a disrupted MMP9 cleavage site.
  • disrupting the MMP9 cleavage site in the engineered IL-7 variant can protect it from being cleaved by MMP9 within tumor microenvironment, thereby increasing its half-life and/or stability.
  • the MMP9 cleavage site corresponds to positions 101-104 of SEQ ID NO: 2.
  • at least 1, 2, 3, or 4 amino acids in the MMP9 cleavage site are disrupted (e.g., mutated) .
  • the disruption involves mutating two amino acids corresponding to P101 and L104 of SEQ ID NO: 2.
  • the amino acid residue corresponding to P101 of SEQ ID NO: 2 is mutated to S, T, N, or Q; and/or the amino acid residue corresponding to L104 of SEQ ID NO: 2 is mutated to S, T, N, or Q.
  • either one or both amino acid residues corresponding to P101 and L104 of SEQ ID NO: 2 are mutated to S.
  • the disrupted MMP9 cleavage site includes an amino acid sequence of “SAAS” (SEQ ID NO: 6) .
  • the disrupted MMP9 cleavage site does not include an amino acid sequence of “PAAL” (SEQ ID NO: 5) .
  • the disrupted MMP9 cleavage site has an increased hydrophilicity as compared the corresponding wildtype sequence, e.g., to prevent aggregation.
  • disruption of the MMP9 cleavage site can prevent the cleavage of the engineered IL-7 variants (e.g., any of the engineered IL-7 variants described herein) or protein constructs thereof (e.g., any of the His-tagged IL-7 variants described herein or heterodimeric Fc-fused IL-7 variants described herein) by MMP9 within tumor microenvironment, thereby increasing the half-life and/or stability of the engineered IL-7 variants or protein constructs thereof.
  • the engineered IL-7 variants e.g., any of the engineered IL-7 variants described herein
  • protein constructs thereof e.g., any of the His-tagged IL-7 variants described herein or heterodimeric Fc-fused IL-7 variants described herein
  • disruption of the MMP9 cleavage site can decrease the cleavage level of the engineered IL-7 variants (e.g., any of the engineered IL-7 variants described herein) or protein constructs thereof (e.g., any of the His-tagged IL-7 variants described herein or heterodimeric Fc-fused IL-7 variants described herein) within tumor microenvironment to less than about 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1%, as compared to that of a wildtype IL-7 or protein construct thereof.
  • the engineered IL-7 variants e.g., any of the engineered IL-7 variants described herein
  • protein constructs thereof e.g., any of the His-tagged IL-7 variants described herein or heterodimeric Fc-fused IL-7 variants described herein
  • disruption of the MMP9 cleavage site can increase the half-life of the engineered IL-7 variants (e.g., any of the engineered IL-7 variants described herein) or protein constructs thereof (e.g., any of the His-tagged IL-7 variants described herein or heterodimeric Fc-fused IL-7 variants described herein) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold, as compared to that of a wildtype IL-7 or protein construct thereof.
  • the engineered IL-7 variants e.g., any of the engineered IL-7 variants described herein
  • protein constructs thereof e.g., any of the His-tagged IL-7 variants described herein or heterodimeric Fc-fused
  • disruption of the MMP9 cleavage site can enhance the pharmacokinetics (PK) of the engineered IL-7 variants (e.g., any of the engineered IL-7 variants described herein) or protein constructs thereof (e.g., any of the His-tagged IL-7 variants described herein or heterodimeric Fc-fused IL-7 variants described herein) .
  • PK pharmacokinetics
  • an engineered IL-7 variant IL7_C01_MMP9 which includes a first cysteine residue corresponding to position 9 (e.g., G9) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 146 (L146) of SEQ ID NO: 2.
  • IL7_C01_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C01_MMP9 is set forth in SEQ ID NO: 7.
  • an engineered IL-7 variant IL7_C02_MMP9 which includes a first cysteine residue corresponding to position 16 (e.g., L16) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 82 (V82) of SEQ ID NO: 2.
  • IL7_C02_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C02_MMP9 is set forth in SEQ ID NO: 8.
  • an engineered IL-7 variant IL7_C03_MMP9 which includes a first cysteine residue corresponding to position 19 (e.g., S19) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 78 (H78) of SEQ ID NO: 2.
  • IL7_C03_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C03_MMP9 is set forth in SEQ ID NO: 9.
  • an engineered IL-7 variant IL7_C04_MMP9 which includes a first cysteine residue corresponding to position 19 (e.g., S19) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 82 (V82) of SEQ ID NO: 2.
  • IL7_C04_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C04_MMP9 is set forth in SEQ ID NO: 10.
  • an engineered IL-7 variant IL7_C05_MMP9 which includes a first cysteine residue corresponding to position 20 (e.g., I20) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 82 (V82) of SEQ ID NO: 2.
  • IL7_C05_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C05_MMP9 is set forth in SEQ ID NO: 11.
  • an engineered IL-7 variant IL7_C06_MMP9 which includes a first cysteine residue corresponding to position 20 (e.g., I20) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 135 (L135) of SEQ ID NO: 2.
  • IL7_C06_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C06_MMP9 is set forth in SEQ ID NO: 12.
  • an engineered IL-7 variant IL7_C07_MMP9 which includes a first cysteine residue corresponding to position 24 (e.g., L24) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 132 (K132) of SEQ ID NO: 2.
  • IL7_C07_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C07_MMP9 is set forth in SEQ ID NO: 13.
  • an engineered IL-7 variant IL7_C08_MMP9 which includes a first cysteine residue corresponding to position 56 (e.g., L56) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 138 (I138) of SEQ ID NO: 2.
  • IL7_C08_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C08_MMP9 is set forth in SEQ ID NO: 14.
  • an engineered IL-7 variant IL7_C09_MMP9 which includes a first cysteine residue corresponding to position 57 (e.g., F57) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 87 (T87) of SEQ ID NO: 2.
  • IL7_C09_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C09_MMP9 is set forth in SEQ ID NO: 15.
  • an engineered IL-7 variant IL7_C10_MMP9 which includes a first cysteine residue corresponding to position 65 (e.g., Q65) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 124 (K124) of SEQ ID NO: 2.
  • IL7_C10_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C10_MMP9 is set forth in SEQ ID NO: 16.
  • an engineered IL-7 variant IL7_C11_MMP9 which includes a first cysteine residue corresponding to position 65 (e.g., Q65) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 125 (L125) of SEQ ID NO: 2.
  • IL7_C11_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C11_MMP9 is set forth in SEQ ID NO: 17.
  • an engineered IL-7 variant IL7_C12_MMP9 which includes a first cysteine residue corresponding to position 66 (e.g., F66) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 124 (K124) of SEQ ID NO: 2.
  • IL7_C12_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C12_MMP9 is set forth in SEQ ID NO: 18.
  • an engineered IL-7 variant IL7_C13_MMP9 which includes a first cysteine residue corresponding to position 66 (e.g., F66) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 125 (L125) of SEQ ID NO: 2.
  • IL7_C13_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C13_MMP9 is set forth in SEQ ID NO: 19.
  • an engineered IL-7 variant IL7_C14_MMP9 which includes a first cysteine residue corresponding to position 66 (e.g., F66) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 126 (N126) of SEQ ID NO: 2.
  • IL7_C14_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C14_MMP9 is set forth in SEQ ID NO: 20.
  • an engineered IL-7 variant IL7_C15_MMP9 which includes a first cysteine residue corresponding to position 16 (e.g., L16) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 85 (G85) of SEQ ID NO: 2.
  • IL7_C15_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C15_MMP9 is set forth in SEQ ID NO: 21.
  • an engineered IL-7 variant IL7_C16_MMP9 which includes a first cysteine residue corresponding to position 45 (e.g., H45) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 55 (F55) of SEQ ID NO: 2.
  • IL7_C16_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C16_MMP9 is set forth in SEQ ID NO: 22.
  • an engineered IL-7 variant IL7_C17_MMP9 which includes a first cysteine residue corresponding to position 60 (e.g., A60) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 83 (S83) of SEQ ID NO: 2.
  • IL7_C17_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C17_MMP9 is set forth in SEQ ID NO: 23.
  • an engineered IL-7 variant IL7_C18_MMP9 which includes a first cysteine residue corresponding to position 60 (e.g., A60) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 87 (T87) of SEQ ID NO: 2.
  • IL7_C18_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C18_MMP9 is set forth in SEQ ID NO: 24.
  • an engineered IL-7 variant IL7_C19_MMP9 which includes a first cysteine residue corresponding to position 63 (e.g., L63) of SEQ ID NO: 2, and a second cysteine residue corresponding to position 83 (S83) of SEQ ID NO: 2.
  • IL7_C19_MMP9 also includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_C19_MMP9 is set forth in SEQ ID NO: 25.
  • an engineered IL-7 variant IL7_wt_MMP9 which includes serine residues corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2.
  • the sequence of IL7_wt_MMP9 is set forth in SEQ ID NO: 26.
  • an engineered IL-7 variant comprising one or more of the following: (a) the amino acid that corresponds to G9 of SEQ ID NO: 2 is C, and the amino acid that corresponds to L146 of SEQ ID NO: 2 is C; (b) the amino acid that corresponds to L16 of SEQ ID NO: 2 is C, and the amino acid that corresponds to V82 of SEQ ID NO: 2 is C; (c) the amino acid that corresponds to S19 of SEQ ID NO: 2 is C, and the amino acid that corresponds to H78 of SEQ ID NO: 2 is C; (d) the amino acid that corresponds to S19 of SEQ ID NO: 2 is C, and the amino acid that corresponds to V82 of SEQ ID NO: 2 is C; (e) the amino acid that corresponds to I20 of SEQ ID NO: 2 is C, and the amino acid that corresponds to V82 of SEQ ID NO: 2 is C; (f) the amino acid that corresponds to I20 of SEQ ID NO: 2 is
  • the engineered IL-7 variant comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to any one of SEQ ID NOs: 7-26, as shown in Table 1.
  • the engineered IL-7 variant described herein comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 1 or SEQ ID NO: 2, wherein the amino acid sequence comprises one or more of the mutations described herein, .
  • the engineered IL-7 variant described herein includes at least 1, at least 2, at least 3, at least 4, or at least 5 pairs of the cysteine mutations and/or at least 1 or at least 2 of the two serine mutations (corresponding to position 101 (e.g., P101) and position 104 (e.g., L104) of SEQ ID NO: 2) described in Table 2.
  • the disclosure also provides a nucleic acid comprising a polynucleotide encoding an engineered IL-7 variant comprising a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to any sequence of SEQ ID NOs: 7-26.
  • the engineered IL-7 variant can have at least or about 1 (e.g., at least or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40) amino acid insertions, deletions, or substitutions as compared to any one of SEQ ID NOs: 7-26.
  • the engineered IL-7 variant can have additional modifications.
  • the engineered IL-7 variant can have a CH2 domain and/or a CH3 domain of Fc.
  • the engineered IL-7 variant can be linked to the N-terminus of the CH2 domain (e.g., optionally through a hinge region, a GS linker, or any of the linker peptides described herein) .
  • the engineered IL-7 variant can be linked to the C-terminus of the CH3 domain (e.g., optionally through a GS linker or any of the linker peptides described herein) .
  • the hinge region is an IgG hinge region (e.g., IgG4 hinge region) .
  • the CH2 domain is an IgG CH2 domain (e.g., IgG4 CH2 domain) .
  • the CH3 domain is an IgG CH3 domain (e.g., IgG4 CH3 domain) .
  • the engineered IL-7 variant described herein can be expressed in Expi293 or CHO (e.g., CHO-S) cells.
  • the cells are Expi293F TM cells or ExpiCHO-S TM cells.
  • the disclosure provides protein constructs (e.g., fusion proteins or protein complexes) comprising the engineered IL-7 variants described herein (e.g., any of the engineered IL-7 variants described herein) .
  • the protein constructs described herein comprise or consist of an immunocytokine.
  • the protein construct further includes a tag (e.g., a His tag) that is fused to the engineered IL-7 variant, e.g., to facilitate screening and/or detection.
  • the His tag comprises or consists of at least 6, at least 7, or at least 8 contiguous histidine residues.
  • the His tag has a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 3.
  • the tag e.g., the His tag
  • the tag is connected to the N-terminus or C-terminus of any engineered IL-7 variants described herein.
  • the fusion proteins described herein comprises or consists of, optionally from N-terminus to C-terminus, a His tag (e.g., any of the His tags described herein) and an engineered IL-7 variant (e.g., any of the engineered IL-7 variants described herein) .
  • the fusion proteins described herein comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%to any one of SEQ ID NOs: 27-46, as shown in FIG. 8.
  • the disclosure also provides a nucleic acid comprising a polynucleotide encoding a fusion protein comprising a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to any sequence of SEQ ID NOs: 27-46.
  • the protein constructs can be expressed in Expi293 or CHO (e.g., CHO-S) cells.
  • the cells are Expi293F TM cells or ExpiCHO-S TM cells.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) .
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the engineered IL-7 variants can further comprises an Fc region of an antibody.
  • These antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) , class or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE1, IgE2) .
  • the Fc region is derived from human IgG (e.g., IgG1, IgG2, IgG3, or IgG4) .
  • the Fc region is an IgG4 Fc region (e.g., human IgG4 Fc region) .
  • the engineered IL-7 variant is linked to the Fc region through an antibody hinge region (e.g., IgG, IgE hinge region) .
  • the Fc region can be modified to provide desired effector functions or serum half-life.
  • the protein constructs as described herein include a functional Fc region.
  • the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.
  • effector function of a functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC) .
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • effector function of a functional Fc region is phagocytosis.
  • effector function of a functional Fc region is ADCC and phagocytosis.
  • the protein constructs as described herein have an Fc region without effector function.
  • the Fc is a human IgG4 Fc.
  • the Fc does not have a functional Fc region.
  • the Fc region has LALA mutations (L234A and L235A mutations in EU numbering) , or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering) .
  • the engineered IL-7 variant (e.g., any of the engineered IL-7 variants described herein) is linked to the N-terminus or C-terminus of the Fc region. In some embodiments, the engineered IL-7 variant is linked to the Fc region via a linker peptide (e.g., any of the linker peptides described herein) .
  • protein constructs that include, from N-terminus to C-terminus, a human IgG4 hinge region (e.g., with a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 48) , a human IgG4 Fc region (with a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 49, 50, or 51) , a linker peptide (e.g., any of the linker peptides described herein) , and an engineered IL-7 variant (e.g., any of the engineered IL-7 variants described herein) .
  • a human IgG4 hinge region e.g., with a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO:
  • the human IgG4 hinge region and human IgG4 Fc region together comprise or consist of a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 52, 53, or 54.
  • the Fc region has S228P mutation (EU numbering) .
  • the S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange.
  • Fc regions are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such Fc region composition may be from 1%to 80%, from 1%to 65%, from 5%to 65%or from 20%to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues; or position 314 in Kabat numbering) ; however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in Fc region sequences. Such fucosylation variants may have improved ADCC function.
  • the Fc region can be further engineered to replace the Asparagine at position 297 with Alanine (N297A) .
  • the disclosure is related to a protein construct comprising the engineered IL-7 variant described herein.
  • the protein construct comprises two or more engineered IL-7 variants. In some embodiments, at least two of the engineered IL-7 variants are identical. In some embodiments, at least two of the engineered IL-7 variants are different.
  • the protein construct further comprises an Fc region. In some embodiments, the Fc region is an IgG4 Fc region. In some embodiments, the Fc region is an IgG1 Fc region (e.g., with LALA mutations or LALA-PG mutations) .
  • the engineered IL-7 variant is linked to the C-terminus of the Fc region. In some embodiments, the engineered IL-7 variant is linked to the C-terminus of the Fc region via a linker peptide (e.g., any of the linker peptides described herein) .
  • the disclosure provides protein complexes comprising the engineered IL-7 variants described herein (e.g., any of the engineered IL-7 variants described herein) .
  • the disclosure is related to a protein complex comprising a first polypeptide and a second polypeptide.
  • the first polypeptide comprises or consists of, optionally from N-terminus to C-terminus: an optional first hinge region (e.g., a human IgG4 hinge region) , a first Fc region (e.g., a human IgG4 Fc region) , an optional linker peptide (e.g., any of the linker peptides described herein) , and an engineered IL-7 variant (e.g., any of the engineered IL-7 variants described herein) .
  • an optional first hinge region e.g., a human IgG4 hinge region
  • a first Fc region e.g., a human IgG4 Fc region
  • an optional linker peptide e.g., any of the linker peptides described herein
  • an engineered IL-7 variant e.g., any of the engineered IL-7 variants described herein
  • the second polypeptide comprises or consists of, optionally from N-terminus to C-terminus: an optional second hinge region (e.g., a human IgG4 hinge region) , and a second Fc region (e.g., a human IgG4 Fc region) .
  • an optional second hinge region e.g., a human IgG4 hinge region
  • a second Fc region e.g., a human IgG4 Fc region
  • the first Fc region and/or the second Fc region described herein do not include a hinge region.
  • the first Fc region and/or the second Fc region described herein consists of a CH2 domain (e.g., a human IgG4 CH2 domain) and a CH3 domain (e.g., a human IgG4 CH3 domain) .
  • the first and/or the second hinge regions include all or a portion of the hinge region of an immunoglobulin, e.g., human IgG4 hinge region (SEQ ID NO: 48) .
  • the first and/or the second hinge regions include an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 48.
  • the first and the second hinge regions are identical. In some embodiments, the first and the second hinge regions are different.
  • the first and/or the second Fc region are different.
  • the first and/or the second Fc regions can form a Fc heterodimer by introducing one or more mutations.
  • the first and/or the second Fc region can include one or more knob-into-hole (KIH) mutations.
  • the first Fc region can include a cysteine at position 349, a serine at position 366, an alanine at position 368, and/or a valine at position 407 according to EU numbering
  • the second Fc region can include a cysteine at position 354 and/or a tryptophan at position 366 according to EU numbering.
  • the first Fc region includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 49
  • the second Fc region includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 50.
  • the first and/or the second Fc region can form a Fc heterodimer using other technologies. Details of the KIH mutations and other heterodimeric Fc technologies can be found, e.g., in Ha, et al.
  • the first and/or the second Fc regions described herein are derived from human IgG (e.g., IgG1, IgG2, IgG3, or IgG4) .
  • the first and/or second Fc regions are IgG4 Fc regions (e.g., human IgG4 Fc regions) .
  • the first and/or second Fc regions are IgG Fc regions (e.g., human IgG1 Fc regions) whose effector function is silenced.
  • the hole chain described herein includes a cysteine at position 349, a serine at position 366, an alanine at position 368, and/or a valine at position 407 according to EU numbering.
  • the knob chain described herein includes a cysteine at position 354 and/or a tryptophan at position 366 according to EU numbering.
  • the KIH mutations described herein include any of the mutations at the positions described above.
  • the linker peptide described herein includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 4. In some embodiments, the linker peptide described herein includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) repeats of GGGGS (SEQ ID NO: 60) . In some embodiments, the linker peptide is a GS linker. As used herein, the term “GS linker” refers to a linker having sequences comprising primarily of glycine and serine residues.
  • the GS linker consists of glycine and serine residues.
  • the linker peptide is a flexible linker. Details of flexible linkers can be found, e.g., Chen, X., et al. "Fusion protein linkers: property, design and functionality. " Advanced Drug Delivery Reviews 65.10 (2013) : 1357-1369, which is incorporated herein by reference in its entirety.
  • the first polypeptide described herein includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 55, 56, 57, 58, 59, 61, or 62; and the second polypeptide described herein includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 53.
  • a protein complex with a heterodimeric structure e.g., any of the heterodimeric Fc-fused IL-7 variants described herein. Sequences of some exemplary protein complexes are shown in FIG. 9.
  • the heterodimeric Fc-fused IL-7 variant is heterodimer G4ssFc-IL7_C03_MMP9, which includes (a) a first polypeptide comprising from N-terminus to C-terminus: a first hinge region, a first Fc region, a linker peptide, and the engineered IL-7 variant IL7_C03_MMP9; and (b) a second polypeptide comprising from N-terminus to C-terminus: a second hinge region and a second Fc region.
  • the first polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 55
  • the second polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 53
  • the first and/or second hinge regions include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 48.
  • the linker peptide includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 4.
  • the first and/or second Fc regions include one or more KIH mutations (e.g., any of the KIH mutations described herein) .
  • the heterodimeric Fc-fused IL-7 variant is heterodimer G4ssFc-IL7_C09_MMP9, which includes (a) a first polypeptide comprising from N-terminus to C-terminus: a first hinge region, a first Fc region, a linker peptide, and the engineered IL-7 variant IL7_C09_MMP9; and (b) a second polypeptide comprising from N-terminus to C-terminus: a second hinge region and a second Fc region.
  • the first polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 56
  • the second polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 53
  • the first and/or second hinge regions include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 48.
  • the linker peptide includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 4.
  • the first and/or second Fc regions include one or more KIH mutations (e.g., any of the KIH mutations described herein) .
  • the heterodimeric Fc-fused IL-7 variant is heterodimer G4ssFc-IL7_C14_MMP9, which includes (a) a first polypeptide comprising from N-terminus to C-terminus: a first hinge region, a first Fc region, a linker peptide, and the engineered IL-7 variant IL7_C14_MMP9; and (b) a second polypeptide comprising from N-terminus to C-terminus: a second hinge region and a second Fc region.
  • the first polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 57
  • the second polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 53
  • the first and/or second hinge regions include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 48.
  • the linker peptide includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 4.
  • the first and/or second Fc regions include one or more KIH mutations (e.g., any of the KIH mutations described herein) .
  • the heterodimeric Fc-fused IL-7 variant is heterodimer G4ssFc-IL7_C15_MMP9, which includes (a) a first polypeptide comprising from N-terminus to C-terminus: a first hinge region, a first Fc region, a linker peptide, and the engineered IL-7 variant IL7_C15_MMP9; and (b) a second polypeptide comprising from N-terminus to C-terminus: a second hinge region and a second Fc region.
  • the first polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 58
  • the second polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 53
  • the first and/or second hinge regions include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 48.
  • the linker peptide includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 4.
  • the first and/or second Fc regions include one or more KIH mutations (e.g., any of the KIH mutations described herein) .
  • the heterodimeric Fc-fused IL-7 variant is heterodimer G4ssFc-IL7_wt, which includes (a) a first polypeptide comprising from N-terminus to C-terminus: a first hinge region, a first Fc region, a linker peptide, and IL7_wt (SEQ ID NO: 2) ; and (b) a second polypeptide comprising from N-terminus to C-terminus: a second hinge region and a second Fc region.
  • the first polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 59
  • the second polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 53
  • the first and/or second hinge regions include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 48.
  • the linker peptide includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 4.
  • the first and/or second Fc regions include one or more KIH mutations (e.g., any of the KIH mutations described herein) .
  • the heterodimeric Fc-fused IL-7 variant is heterodimer G4ssFc-IL7_C10_MMP9, which includes (a) a first polypeptide comprising from N-terminus to C-terminus: a first hinge region, a first Fc region, a linker peptide, and the engineered IL-7 variant IL7_C10_MMP9; and (b) a second polypeptide comprising from N-terminus to C-terminus: a second hinge region and a second Fc region.
  • the first polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 61
  • the second polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 53
  • the first and/or second hinge regions include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 48.
  • the linker peptide includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 4.
  • the first and/or second Fc regions include one or more KIH mutations (e.g., any of the KIH mutations described herein) .
  • the heterodimeric Fc-fused IL-7 variant is heterodimer G4ssFc-IL7_C11_MMP9, which includes (a) a first polypeptide comprising from N-terminus to C-terminus: a first hinge region, a first Fc region, a linker peptide, and the engineered IL-7 variant IL7_C11_MMP9; and (b) a second polypeptide comprising from N-terminus to C-terminus: a second hinge region and a second Fc region.
  • the first polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 62
  • the second polypeptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 53
  • the first and/or second hinge regions include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 48.
  • the linker peptide includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 4.
  • the first and/or second Fc regions include one or more KIH mutations (e.g., any of the KIH mutations described herein) .
  • the disclosure is related to a protein complex comprising a first polypeptide and a second polypeptide.
  • the first polypeptide comprises or consists of, optionally from N-terminus to C-terminus: an optional first hinge region (e.g., a human IgG4 hinge region) , and a first Fc region (e.g., a human IgG4 Fc region) .
  • the second polypeptide comprises or consists of, optionally from N-terminus to C-terminus: an optional second hinge region (e.g., a human IgG4 hinge region) , a second Fc region (e.g., a human IgG4 Fc region) , an optional linker peptide (e.g., any of the linker peptides described herein) , and an engineered IL-7 variant (e.g., any of the engineered IL-7 variants described herein) .
  • an optional second hinge region e.g., a human IgG4 hinge region
  • a second Fc region e.g., a human IgG4 Fc region
  • an optional linker peptide e.g., any of the linker peptides described herein
  • an engineered IL-7 variant e.g., any of the engineered IL-7 variants described herein
  • the first Fc region includes a cysteine at position 349, a serine at position 366, an alanine at position 368, and/or a valine at position 407 according to EU numbering; and the second Fc region includes a cysteine at position 354 and/or a tryptophan at position 366 according to EU numbering.
  • the first and/or second hinge regions include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 48.
  • the linker peptide includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 4.
  • the first Fc region includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 49
  • the second Fc region includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 50.
  • the disclosure is related to a protein complex comprising a first polypeptide and a second polypeptide.
  • the first polypeptide comprises or consists of, optionally from N-terminus to C-terminus: an optional first hinge region (e.g., a human IgG4 hinge region) , a first Fc region (e.g., a human IgG4 Fc region) , an optional first linker peptide (e.g., any of the linker peptides described herein) , and a first engineered IL-7 variant (e.g., any of the engineered IL-7 variants described herein) .
  • an optional first hinge region e.g., a human IgG4 hinge region
  • a first Fc region e.g., a human IgG4 Fc region
  • an optional first linker peptide e.g., any of the linker peptides described herein
  • a first engineered IL-7 variant e.g., any of the
  • the second polypeptide comprises or consists of, optionally from N-terminus to C-terminus: an optional second hinge region (e.g., a human IgG4 hinge region) , a second Fc region (e.g., a human IgG4 Fc region) , an optional second linker peptide (e.g., any of the linker peptides described herein) , and a second engineered IL-7 variant (e.g., any of the engineered IL-7 variants described herein) .
  • an optional second hinge region e.g., a human IgG4 hinge region
  • a second Fc region e.g., a human IgG4 Fc region
  • an optional second linker peptide e.g., any of the linker peptides described herein
  • a second engineered IL-7 variant e.g., any of the engineered IL-7 variants described herein
  • the first Fc region includes a cysteine at position 349, a serine at position 366, an alanine at position 368, and/or a valine at position 407 according to EU numbering; and the second Fc region includes a cysteine at position 354 and/or a tryptophan at position 366 according to EU numbering.
  • the first and/or second hinge regions include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 48.
  • the first and/or second linker peptides include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 4.
  • the first Fc region includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 49
  • the second Fc region includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 50.
  • the first and/or second engineered IL-7 variants are identical. In some embodiments, the first and/or second engineered IL-7 variants are identical are different.
  • the disclosure provides protein complexes having a Fc homodimer structure.
  • the disclosure is related to a protein complex comprising two identical polypeptides.
  • each polypeptide comprises or consists of, optionally from N-terminus to C-terminus: an optional hinge region (e.g., a human IgG4 hinge region) , a Fc region (e.g., a human IgG4 Fc region) , an optional linker peptide (e.g., any of the linker peptides described herein) , and an engineered IL-7 variant (e.g., any of the engineered IL-7 variants described herein) .
  • an optional hinge region e.g., a human IgG4 hinge region
  • Fc region e.g., a human IgG4 Fc region
  • an optional linker peptide e.g., any of the linker peptides described herein
  • an engineered IL-7 variant e.g., any of the
  • the hinge region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 48.
  • the Fc region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 51.
  • the linker peptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 4.
  • the engineered IL-7 variant comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to any one of SEQ ID NOs: 7-26.
  • the protein complexes with a Fc heterodimer structure may exhibit a higher in vitro potency (e.g., induction of IL-7/IL-7R signaling pathways and/or induction of immune cell proliferation) and/or a higher in vivo efficacy (e.g., anti-tumor efficacy) than the corresponding protein complexes with a Fc homodimer structure described herein.
  • This can be attributed to the spatial hindrance when the two engineered IL-7 variants (e.g., connected to the C-terminus of the Fc region of the protein complexes) simultaneously interact with the IL-7R ⁇ / ⁇ c complex.
  • the engineered IL-7 variants e.g., any of the engineered IL-7 variants described herein
  • protein constructs e.g., fusion proteins or protein complexes thereof described herein
  • IL-7R ⁇ human IL-7 receptor
  • ⁇ c common cytokine ⁇ chain
  • the engineered IL-7 variants or protein constructs thereof can induce downstream signaling pathways, e.g., the JAK/STAT, PI3K, and/or MAPK/ERK pathways, by binding to the IL-7R/ ⁇ c complex expressed on immune cells (e.g., primary T cells) .
  • immune cells e.g., primary T cells
  • introduction of the non-native disulfide bond, or cysteine mutations can lead to protein conformational change with a RMSD (root-mean-square deviation of atomic positions) value of less than less than less than less than less than less than less than less than or less than
  • RMSD root-mean-square deviation of atomic positions
  • the RMSD value is calculated by structurally align the wild-type protein and the protein variants. In some embodiments, only C alpha atoms are used for determining the conformational change.
  • the engineered IL-7 variants e.g., any of the engineered IL-7 variants described herein
  • protein constructs thereof described herein e.g., any of the His-tagged IL-7 variants described herein or heterodimeric Fc-fused IL-7 variants described herein
  • the engineered IL-7 variants or protein constructs thereof can induce IL-7/IL-7R signaling pathways (e.g., activation of the JAK/STAT5 pathway) with a high potency that is at least about 80%, about 90%, about 100%, about 110%, or about 120%as compared to that of a wildtype IL-7 or protein construct thereof.
  • the engineered IL-7 variants or protein constructs thereof can induce IL-7/IL-7R signaling pathways (e.g., activation of the JAK/STAT5 pathway) with a medium potency that is at least about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%as compared to that of a wildtype IL-7 or protein construct thereof.
  • the engineered IL-7 variants or protein constructs thereof can induce IL-7/IL-7R signaling pathways (e.g., activation of the JAK/STAT5 pathway) with a low potency that is less than about 50%, about 40%, about 40%, about 20%, or about 10%as compared to that of a wildtype IL-7 or protein construct thereof.
  • IL-7/IL-7R signaling pathways e.g., activation of the JAK/STAT5 pathway
  • the engineered IL-7 variants or protein constructs thereof described herein have a high potency, a medium potency, or a low potency to induce proliferation of immune cells (e.g., primary T cells) .
  • the engineered IL-7 variants or protein constructs thereof can induce proliferation of immune cells (e.g., primary T cells) with a high potency that is at least about 80%, about 90%, about 100%, about 110%, or about 120%as compared to that of a wildtype IL-7 or protein construct thereof.
  • the engineered IL-7 variants or protein constructs thereof can induce proliferation of immune cells (e.g., primary T cells) with a medium potency that is at least about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%as compared to that of a wildtype IL-7 or protein construct thereof.
  • the engineered IL-7 variants or protein constructs thereof can induce proliferation of immune cells (e.g., primary T cells) with a low potency that is less than about 50%, about 40%, about 40%, about 20%, or about 10%as compared to that of a wildtype IL-7 or protein construct thereof.
  • the engineered IL-7 variants described herein includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 9, 15, 16, 17, 20, or 21.
  • the protein constructs (e.g., His-tagged IL-7 variants) described herein includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 29, 35, 36, 37, 40, or 41.
  • the protein constructs (e.g., heterodimeric Fc-fused IL-7 variants) includes a first polypeptide having an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 55, 56, 57, 58, 61, or 62; and second polypeptide having an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 53.
  • the engineered IL-7 variants or protein constructs thereof as described herein can increase immune response, activity or number of immune cells (e.g., primary T cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds.
  • immune cells e.g., primary T cells
  • the engineered IL-7 variants or protein constructs thereof can bind to IL-7R ⁇ , ⁇ c, or the complex thereof (e.g., human IL-7R/ ⁇ c complex) with a dissociation rate (k off ) of less than 0.1 s -1 , less than 0.01 s -1 , less than 0.001 s -1 , less than 0.0001 s -1 , or less than 0.00001 s -1 .
  • the dissociation rate (k off ) is greater than 0.01 s -1 , greater than 0.001 s -1 , greater than 0.0001 s -1 , greater than 0.00001 s -1 , or greater than 0.000001 s -1 .
  • kinetic association rates (k on ) is greater than 1 ⁇ 10 2 /Ms, greater than 1 ⁇ 10 3 /Ms, greater than 1 ⁇ 10 4 /Ms, greater than 1 ⁇ 10 5 /Ms, or greater than 1 ⁇ 10 6 /Ms. In some embodiments, kinetic association rates (k on ) is less than 1 ⁇ 10 5 /Ms, less than 1 ⁇ 10 6 /Ms, or less than 1 ⁇ 10 7 /Ms.
  • KD is less than 1 ⁇ 10 -6 M, less than 1 ⁇ 10 -7 M, less than 1 ⁇ 10 -8 M, less than 1 ⁇ 10 -9 M, or less than 1 ⁇ 10 -10 M.
  • the KD is less than 300 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM.
  • KD is greater than 1 ⁇ 10 -7 M, greater than 1 ⁇ 10 -8 M, greater than 1 ⁇ 10 -9 M, greater than 1 ⁇ 10 -10 M, greater than 1 ⁇ 10 -11 M, or greater than 1 ⁇ 10 -12 M.
  • the engineered IL-7 variants or protein constructs thereof described herein can bind to IL-7R ⁇ (CD127) alone (e.g., in the absence of ⁇ c) with a similar affinity that is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, or about 120%as compared to that of a wildtype IL-7 or protein construct thereof.
  • the engineered IL-7 variants or protein constructs thereof described herein can bind to IL-7R ⁇ (CD127) alone (e.g., in the absence of ⁇ c) with a low affinity that is less than about 100%, about 90%, about 80%, about 70%, about 60%, or about 50%as compared to that of a wildtype IL-7 or protein construct thereof.
  • the affinity is determined by cell-based assays.
  • the engineered IL-7 protein constructs thereof described herein can be expressed and purified by methods commonly used in the art, e.g., affinity chromatography.
  • the protein constructs can be purified by size-exclusive chromatography (SEC) coupled with HPLC.
  • SEC size-exclusive chromatography
  • the percentage of the main peak in the SEC-HPLC analysis result is at least 80%, at least 90%, at least 95%, at least 96%, at least 96%, at least 97%, at least 98%, or at least 99%.
  • the percentage of high molecular weight peak (HMW%) and/or low molecular weight peak (LMW%) is less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.
  • thermal stabilities of the engineered IL-7 variants or protein constructs thereof described herein are determined.
  • the engineered IL-7 variants and protein constructs thereof described herein can have a Tm greater than 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.
  • Tm is less than 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.
  • the melting temperature (T m ) of the engineered IL-7 variants and protein constructs thereof described herein can be measured by application on a heat ramp (e.g., from 20-95°C) based on differential scanning fluorimetry (DSF) .
  • DSF is a commonly used method for measuring protein thermal shifts that utilizes specialized fluorogenic dyes. With the increase of temperature, the sample will gradually unfold. The unfolded portion of the sample can bind to a specialized fluorogenic dye and emit fluorescence signals for determination of T m .
  • the melting temperature (T m ) is considered a key predictor of stability.
  • the T m of the engineered IL-7 variants or protein constructs thereof described herein is at least 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 °C.
  • the cysteine mutations e.g., any of the cysteine mutations or a combination thereof described herein
  • the disruption of MMP9 cleavage site e.g., by any of the disruption methods described herein
  • T m melting temperature
  • the cysteine mutations and/or the disruption of MMP9 cleavage site can increase the melting temperature (T m ) of the engineered IL-7 variants or protein constructs thereof described herein by at least 0.5°C, at least 1°C, at least 1.5°C, at least 2°C, at least 2.5°C, at least 3°C, at least 3.5°C, at least 4°C, at least 4.5°C, at least 5°C, at least 5.5°C, at least 6°C, at least 6.5°C, at least 7°C, at least 7.5°C, at least 8°C, at least 8.5°C, at least 9°C, at least 9.5°C, at least 10°C, at least 10.5°C, at least 11°C
  • the engineered IL-7 variants or protein constructs thereof described herein can inhibit tumor growth, e.g., when administered in a tumor-bearing animal.
  • the engineered IL-7 variants or protein constructs thereof has a tumor growth inhibition percentage (TGI%) that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%.
  • TGI% tumor growth inhibition percentage
  • the engineered IL-7 variants or protein constructs thereof described herein has a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%.
  • the TGI% can be determined, e.g., at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the treatment starts.
  • T final is the average tumor volume in the treatment group on the final day.
  • T initial is the average tumor volume in the treatment group on Day 0.
  • C final is the average tumor volume in the control group on the final day.
  • C initial is the average tumor volume in the control group on Day 0.
  • the TGI%of the engineered IL-7 variants or protein constructs thereof described herein is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold greater that of a wildtype IL-7 or protein construct thereof.
  • the percentage of tumor-bearing mice treated with the engineered IL-7 variants or protein constructs thereof described herein e.g., any of the His-tagged IL-7 variants described herein or heterodimeric Fc-fused IL-7 variants described herein
  • having a tumor volume that is less than half the value of the mean tumor volume of the control tumor-bearing mice is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%after 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days of tumor inoculation, relative to the total number of surviving mice on the same day.
  • the survival rate of tumor-bearing mice after treatment of the engineered IL-7 variants or protein constructs thereof described herein is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%after 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days of tumor inoculation.
  • the body weight of tumor-bearing mice after treatment of the engineered IL-7 variants or protein constructs thereof described herein is at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%as compared to that of tumor-bearing mice after treatment of a wildtype IL-7 or protein construct thereof.
  • the half-life of the engineered IL-7 variants or protein constructs thereof described herein can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 100-fold, as compared to that of a wildtype IL-7 or protein construct thereof.
  • the half-life is determined by measuring the in vivo concentration of the molecule over time, after administration into a subject.
  • the engineered IL-7 variants or protein constructs thereof described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a IL-7 peptide or a part thereof or by peptide synthesis.
  • Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acids sequences.
  • selective cysteine mutations can be introduced to one or more pairs of residues whose C alpha atoms are within angstroms of a 3D structure of human IL-7.
  • the 3D structure of human IL-7 has a PDB (protein data bank) ID of 3DI2.
  • the engineered IL-7 variants were fused with an N-terminal His tag generate His-tagged IL-7 variants (e.g., any of the His-tagged IL-7 variants described herein) .
  • the potency of the His-tagged IL-7 variants on T cell response can be determined by detecting activation of the JAK/STAT5 pathway and/or induction of primary T cell proliferation. Based on the experimental results above, some of the His-tagged IL-7 variants with a good expression/purification profile and a medium to high T cell response potency can be selected to generate heterodimeric Fc-fused IL-7 variants (e.g., any of the heterodimeric Fc-fused IL-7 variants described herein) .
  • the potency of the heterodimeric Fc-fused IL-7 variants on T cell response can be determined by detecting activation of the JAK/STAT5 pathway and/or induction of primary T cell proliferation.
  • the binding ability to human CD127 (IL-7R ⁇ ) was also determined.
  • heterodimeric Fc-fused IL-7 variants with a medium to high potency on T cell response e.g., a potency that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, or about 120%as compared to a wildtype IL-7 or protein construct thereof
  • a similar or low binding affinity to CD127 e.g., a binding affinity that is less than about 120%, about 110%, about 100%, about 90%, or about 80%as compared to a wildtype IL-7 or protein construct thereof
  • these candidates may have lower off-target effects and/or better pharmacokinetics (PK) as compared to that of a wildtype IL-7 or protein construct thereof.
  • heterodimeric Fc-fused IL-7 variants can also be performed by measuring their stability.
  • some heterodimeric Fc-fused IL-7 variants may have an increased melting temperature (T m ) determined by DSF as compared to that of a wildtype IL-7 or protein construct thereof.
  • heterodimeric Fc-fused IL-7 variants Based on the experimental results on heterodimeric Fc-fused IL-7 variants above, some heterodimeric Fc-fused IL-7 variants with a good expression/purification profile, a high potency on T cell response, and/or a similar or low binding affinity to CD127 can be selected to evaluate their anti-tumor efficacy and in vivo toxicity, e.g., in a tumor-bearing animal model.
  • the engineered IL-7 variants or protein constructs thereof may have comparable or increased affinity for the IL-7R/ ⁇ c complex. Any combination of deletions, insertions, and/or combinations can be made to arrive at a variant that has increased binding affinity for the binding partner (e.g., CD127) .
  • the amino acid changes introduced into the variant can also alter or introduce new post-translational modifications into the polypeptide, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence such that a different sugar is attached by enzymes present in a cell) , or introducing new glycosylation sites.
  • Engineered IL-7 variants can be derived from any species of animal, including mammals.
  • Non-limiting examples of IL-7 variants include IL-7 variants derived from humans, primates, e.g., monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas) , chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits) .
  • the present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) , host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide) , and the production of recombinant polypeptides or fragments thereof by recombinant techniques.
  • recombinant vectors e.g., an expression vectors
  • an isolated polynucleotide disclosed herein e.g., a polynucleotide that encodes a polypeptide disclosed herein
  • host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleot
  • a “vector” is any construct capable of delivering one or more polynucleotide (s) of interest to a host cell when the vector is introduced to the host cell.
  • An “expression vector” is capable of delivering and expressing the one or more polynucleotide (s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced.
  • the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-Atail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.
  • regulatory elements such as a promoter, enhancer, and/or a poly-Atail
  • a vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran) , transformation, transfection, and infection and/or transduction (e.g., with recombinant virus) .
  • vectors include viral vectors (which can be used to generate recombinant virus) , naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.
  • a polynucleotide disclosed herein e.g., a polynucleotide that encodes a polypeptide disclosed herein
  • a viral expression system e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • vaccinia or other pox virus, retrovirus, or adenovirus e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • vaccinia or other pox virus, retrovirus, or adenovirus e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art.
  • the DNA may also be “naked. ”
  • the uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells.
  • the DNA insert comprising a polypeptide-encoding polynucleotide disclosed herein can be operatively linked to an appropriate promoter (e.g., a heterologous promoter) , such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • an appropriate promoter e.g., a heterologous promoter
  • a heterologous promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • a promoter e.g., a heterologous promoter
  • the promoter is a cytomegalovirus (CMV) promoter.
  • the promoter is a human promoter, e.g.,
  • the human promoters can improve expression of proteins derived from human. Details of such human promoters can be found, e.g., in Antoniou, M., et al. "Transgenes encompassing dual-promoter CpG islands from the human TBP and HNRPA2B1 loci are resistant to heterochromatin-mediated silencing. " Genomics 82.3 (2003) : 269-279; and Zhang, F., et al. "Aubiquitous chromatin opening element (UCOE) confers resistance to DNA methylation–mediated silencing of lentiviral vectors. " Molecular Therapy 18.9 (2010) : 1640-1649; each of which is incorporated herein by reference in its entirety.
  • UCOE Aubiquitous chromatin opening element
  • the expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors can include at least one selectable marker.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HEK 293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.
  • Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
  • Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter.
  • Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV) , and metallothionein promoters, such as the mouse metallothionein-I promoter.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used.
  • Introduction of the construct into the host cell can be affected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods.
  • Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986) , which is incorporated herein by reference in its entirety.
  • the host cell is a human cell suitable for protein expression, e.g., HEK293 cells or CHO cells (e.g., CHO-Scells) .
  • the host cells are Expi293 cells.
  • the Expi293 Expression System is designed to deliver up to 6 ⁇ more protein in just one week, compared with other transient 293 expression systems that can take two weeks or more. This is in part due to the fact that Expi293F cells are adapted to achieve higher pg/cell/day productivity than standard HEK 293 cells, and the Expifectamine 293 Transfection Reagent and enhancers enable high-efficiency transfection and expression levels of high-density HEK 293 cultures. Additionally, the Expi293 Expression System requires less plasticware, which means less waste and more incubator space.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • secretion signals may be incorporated into the expressed polypeptide.
  • the signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide (e.g., engineered IL-7 variants) can be expressed in a modified form, such as a fusion protein (e.g., a HSA-fusion or GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
  • the engineered IL-7 variants and protein constructs thereof of the present disclosure can be used for various therapeutic purposes.
  • the disclosure provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject.
  • the treatment can halt, slow, retard, or inhibit progression of a cancer.
  • the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the cancer in a subject.
  • the disclosure features methods that include administering a therapeutically effective amount of engineered IL-7 variants and protein constructs disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, a cancer) , e.g., breast cancer (e.g., triple-negative breast cancer) , carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, stomach cancer, urothelial cancer, skin cancer, or hematologic malignancy.
  • a subject in need thereof e.g., a subject having, or identified or diagnosed as having, a cancer
  • breast cancer e.g., triple-negative breast cancer
  • carcinoid cancer e.g., cervical cancer
  • the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung carcinoma (NSCLC) , small cell lung cancer (SCLC) , bladder cancer, or metastatic hormone-refractory prostate cancer.
  • the subject has a solid tumor.
  • the cancer is squamous cell carcinoma of the head and neck (SCCHN) , renal cell carcinoma (RCC) , triple-negative breast cancer (TNBC) , or colorectal carcinoma.
  • the cancer is melanoma, pancreatic carcinoma, mesothelioma, hematological malignancies, especially Non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumors.
  • the cancer is lung cancer, melanoma, colorectal cancer, glioma, or pancreatic cancer.
  • compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer.
  • Patients with cancer can be identified with various methods known in the art.
  • compositions and methods described herein can be used for treatment of lymphopenia and/or infectious diseases.
  • an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a cancer.
  • An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the engineered IL-7 variants and protein constructs, vector comprising the polynucleotide encoding the engineered IL-7 variants and protein constructs, and/or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.
  • an effective amount can be administered in one or more administrations.
  • an effective amount of the engineered IL-7 variants and/or protein constructs is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and/or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line) ) in vitro.
  • a cell e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line)
  • an effective amount may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of the engineered IL-7 variants and protein constructs used.
  • Effective amounts and schedules for administering the engineered IL-7 variants or protein constructs thereof, the polynucleotides encoding the engineered IL-7 variants or protein constructs, and/or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the engineered IL-7 variants or protein constructs thereof, the polynucleotides, and/or compositions disclosed herein, the route of administration, the particular type of polynucleotides, and/or compositions disclosed herein used and other drugs being administered to the mammal.
  • a typical daily dosage of an effective amount of the engineered IL-7 variants and/or protein constructs is 0.1 mg/kg to 100 mg/kg (mg per kg of patient weight) .
  • the dosage can be less than 100 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.1 mg/kg.
  • the dosage can be greater than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.1 mg/kg. In some embodiments, the dosage is about 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, or 1 mg/kg. In some embodiments, the dosage is about 1 to 10 mg/kg, about 1 to 5 mg/kg, or about 2 to 5 mg/kg.
  • the engineered IL-7 variants or protein constructs thereof can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day) .
  • the one or more additional therapeutic agents can be administered to the subject prior to, or after administering the engineered IL-7 variants or protein constructs thereof. In some embodiments, the one or more additional therapeutic agents are administered to the subject such that there is an overlap in the bioactive period of the one or more additional therapeutic agents and the engineered IL-7 variants or protein constructs thereof in the subject.
  • compositions and routes of administration are provided.
  • compositions that contain the engineered IL-7 variants or protein constructs thereof described herein.
  • the pharmaceutical compositions can be formulated in any manner known in the art.
  • compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) .
  • the compositions can include a sterile diluent (e.g., sterile water or saline) , a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose) , polyalcohols (e.g., mannitol or
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations) , proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Absorption of the agents can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin) .
  • an agent that delays absorption e.g., aluminum monostearate and gelatin
  • controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid) .
  • biodegradable, biocompatible polymers e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid
  • compositions containing the engineered IL-7 variants or protein constructs thereof described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage) .
  • parenteral e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal
  • dosage unit form i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage
  • compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under Good Manufacturing Practice (GMP) conditions.
  • Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration) .
  • Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen.
  • the engineered IL-7 variants or protein constructs thereof can be formulated in aqueous solutions, preferably in physiologically-compatible buffers to reduce discomfort at the site of injection.
  • the solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the engineered IL-7 variants or protein constructs thereof can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys) .
  • Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects) .
  • Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.
  • Exemplary doses include milligram or microgram amounts of any of the engineered IL-7 variants or protein constructs thereof described herein per kilogram of the subject’s weight (e.g., about 1 ⁇ g/kg to about 500 mg/kg; about 100 ⁇ g/kg to about 500 mg/kg; about 100 ⁇ g/kg to about 50 mg/kg; about 10 ⁇ g/kg to about 5 mg/kg; about 10 ⁇ g/kg to about 0.5 mg/kg; about 1 ⁇ g/kg to about 50 ⁇ g/kg; about 1 mg/kg to about 10 mg/kg; or about 1 mg/kg to about 5 mg/kg) .
  • weight e.g., about 1 ⁇ g/kg to about 500 mg/kg; about 100 ⁇ g/kg to about 500 mg/kg; about 100 ⁇ g/kg to about 50 mg/kg; about 10 ⁇ g/kg to about 5 mg/kg; about 10 ⁇ g/kg to about 0.5 mg/kg; about 1 ⁇ g/kg to about 50 ⁇ g/kg
  • therapeutic agents can vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained.
  • the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the engineered IL-7 variants or protein constructs thereof in vivo.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • disclosure also provides methods of manufacturing the engineered IL-7 variants or protein constructs thereof for various uses as described herein.
  • a protein e.g., a cytokine
  • methods of improving the properties (e.g., stability, activity or affinity) of a protein comprising one or more of the following steps: (a) providing a 3D structure of the protein (e.g., the cytokine) , (b) measuring distance of the C alpha atoms of one or more of amino acid residues in the 3D structure; and (c) selecting two amino acid residues from the one or more amino acid residues, wherein the C alpha atoms of the two selected amino acid residues are within In some embodiments, the C alpha atoms of the two selected amino acid residues are within e.g., about or In some embodiments, the directionality from C alpha to C beta atoms of the two selected amino acid residues are in favor of forming a non-native disulfide bond.
  • the C beta atoms point to a direction (from C alpha to C beta ) that is possible to form a disulfide bond.
  • the angel between the direction of the C beta atom of the first selected amino acid residue (from C alpha to C beta of the first selected amino acid residue) and the direction of the C beta atom of the second selected amino acid residue (from C alpha to C beta of the second selected amino acid residue) is less than 120 degrees.
  • the C beta atoms dot not point away from each other.
  • the 3D structure of the protein is derived from a PDB structure of the protein (e.g., the cytokine) , a fragment thereof, or a complex of the protein (e.g., the cytokine) bound with its binding partner (s) .
  • the spatial coordinates of all atoms of the 3D structure can be loaded to a software (e.g., a software for modeling and simulation) , and the distance of any two atoms in the 3D structure can be determined.
  • the angel between the direction of the C beta atom of the first selected amino acid residue and the direction of the C beta atom of the second selected amino acid residue is less than 120 degrees, e.g., less than 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44,
  • the distance of the C alpha atoms of the two selected amino acid residues described herein is about 3 to about about 3 to about about 3 to about about 3 to about about 3 to about about 3 to about about 3 to about about 3 to about about 3.5 to about about 3.5 to about about 3.5 to about about 3.5 to about about 3.5 to about about 3.5 to about about 3.5 to about about 3.5 to about about 3.5 to about about about 3.5 to about about about 3.5 to about about about about about 3.5 to about about about about about 4 to about about 4 to about about about 4 to about about about 4.5 to about about 4.5 to about about about 4.5 to about about about 4.5 to about about about 4.5 to about about about 4.5 to about about about 4.5 to about about about 5 to about about 5 to about about 5 to about about 5.5 to about about about 5.5 to about about 6 to about about 6 to about or about 6.5 to about
  • the distance of the C alpha atoms of the two selected amino acid residues described herein is about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about about 4.5 to about about about 4.5 to about about about 4.5 to about about about 4.5 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about
  • the distance of the C beta atoms of the two selected amino acid residues described herein is about 3 to about about 3 to about about 3 to about about 3 to about about 3 to about about 3 to about about 3 to about about 3 to about about 3 to about about 3 to about about 3.5 to about about 3.5 to about about 3.5 to about about 3.5 to about about 3.5 to about about 3.5 to about about 3.5 to about about 3.5 to about about 3.5 to about about 3.5 to about about about 3.5 to about about about 3.5 to about about about 3.5 to about about about about 3.5 to about about about about 3.5 to about about about about about 3.5 to about about about about about 3.5 to about about about about about about 3.5 to about about about about about about about 3.5 to about about about about about about about 3.5 to about about about about about about 3.5 to about about about about about about about 3.5 to about about about about about about about about 3.5 to about about about about about about about about about 3.5 to about about about about about about about about 3.5 to about about about about about about about about about 4 to about about 4
  • the distance of the C beta atoms of the two selected amino acid residues described herein is about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about about 4.5 to about about about 4.5 to about about about 4.5 to about about about 4.5 to about about about 4.5 to about about about 4.5 to about about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about 4.5 to about about about 4.5 to about about about 4.5 to about about about 4.5 to about about about 4.5 to about about about 4.6 to about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to about about about 4.6 to
  • the distance of the C alpha atoms of the two selected amino acid residues are close enough to form a non-native disulfide bond.
  • the two selected amino acid residues are not involved in the protein (e.g., the cytokine) interaction with its binding partners (e.g., a receptor binding to the protein (e.g., the cytokine) ) .
  • the methods further comprise expressing a protein variant (e.g., a cytokine variant) , wherein the variant comprises a non-native disulfide bond formed by mutating the two selected amino acid residues to cysteines.
  • mutating the two selected amino acid residues to cysteines does not substantially change the 3D structure of the protein (e.g., the cytokine) .
  • the cysteine mutations do not substantially interfere the overall structure of the protein (e.g., cytokine) .
  • the mutated residues do not exhibit any spatial clashes with the un-mutated residues.
  • a skilled person in the art may perform a simulation of the mutated protein (e.g., the mutated cytokine) structure in silico, and determine the associated conformational change caused by the cysteine mutations.
  • the associated conformational change is minimal, e.g., with a RMSD value that is considered insignificant by a skilled person in the art.
  • Also provided herein are methods of screening a cytokine variant with an improved anti-tumor efficacy comprising (a) providing a 3D structure of the cytokine, (b) measuring distance of the C alpha atoms of one or more of amino acid residues in the 3D structure; and (c) selecting two amino acid residues from the one or more amino acid residues, wherein the C alpha atoms of the two selected amino acid residues are within or in any of the ranges described herein.
  • the protein or cytokine has no more than 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues.
  • the methods further comprise (d) expressing a cytokine variant, wherein the variant comprises a non-native disulfide bond formed by mutating the two selected amino acid residues to cysteines; (e) administering the cytokine variant to a tumor-bearing animal; and (f) determining tumor growth (e.g., by measuring tumor volume) in the tumor-bearing animal.
  • administering the cytokine variant does not cause substantial toxicity to the animal. For example, the body weight of the animal administered with the cytokine variant does not drop significantly as compared to that of a reference animal (e.g., an animal administered with vehicle) .
  • the cytokine described herein is IL-7 (e.g., human IL-7) .
  • functional assays are performed to compare the one or more expressed protein variants (e.g., IL-7 variants) .
  • Example 1 Design of IL-7 variants by introducing an artificial disulfide bond
  • Human IL-7 (SEQ ID NO: 1) is a 177 amino acid cytokine that includes four ⁇ -helical domains (helices) , from N-terminus to C-terminus: helix A, helix B, helix C, and helix D.
  • the protein includes a signal peptide that corresponds to amino acids 1-25 of SEQ ID NO: 1.
  • the mature IL-7 protein has an amino acid sequence set forth in SEQ ID NO: 2.
  • variants of wildtype IL-7 SEQ ID NO: 2; without signal peptide
  • SEQ ID NO: 2 variants of wildtype IL-7
  • cysteines were designed by selectively mutating two spatially proximate residues to cysteines, according to the distance of the C alpha atoms and directionalities of the C beta atoms of the two residues within the 3D structure of IL-7 (e.g., PDB ID: 3DI2) . It was contemplated that the two newly introduced cysteines can form an artificial disulfide bond, thereby stabilizing IL-7 and/or changing its functional potency.
  • MMP9 matrix metalloproteinase-9
  • PAAL SEQ ID NO: 5
  • SAAS SEQ ID NO: 6
  • the disrupted MMP9 cleavage site can further increase the stability of the IL-7 variants in tumor-microenvironment.
  • IL-7 variants are also named IL-7_MMP9 variants.
  • amino acid sequences of the IL-7 variants are shown in the table below.
  • All the IL-7 variants above contain a disrupted MMP9 cleavage site (with a proline at a position corresponding to position 101 of SEQ ID NO: 2 mutated to serine (P101S) ; and with a leucine at a position corresponding to position 104 of SEQ ID NO: 2 mutated to serine (L104S) ) .
  • IL7_C01_MMP9 contains a cysteine corresponding to position 9 of SEQ ID NO: 2, a serine corresponding to position 101 of SEQ ID NO: 2, a leucine corresponding to position 104 of SEQ ID NO: 2; and a cysteine corresponding to position 146 of SEQ ID NO: 2.
  • a His tag (H8; SEQ ID NO: 3) can be fused to the N-terminus to the IL-7 variants above. Sequences of the obtained His-tagged IL-7 variants are shown in FIG. 8.
  • HEK-Blue TM IL-7 reporter cell (InvivoGen, Cat#: hkb-IL-7) suspension was prepared at 2.77 ⁇ 10 5 cell/mL. 20 ⁇ L/well of the purified IL-7 variants were added to a flat-bottom 96-well plate. 180 ⁇ L/well of the reporter cell suspension (about 5 ⁇ 10 4 cells) was then added. The plate was incubated at 37°C in 5%CO 2 for 20 hours. After the incubation, the induced reporter cell supernatant was collected.
  • 1 ⁇ QUANTI-Blue TM Solution (InvivoGen, Cat#: rep-qbs) was prepared by diluting the 100 ⁇ stock reagents with water. 20 ⁇ L/well of the induced reporter cell supernatant was added to a flat-bottom 96-well plate, and 180 ⁇ L/well of the prepared 1 ⁇ QUANTI-Blue Solution TM was added. The plate was incubated at 37°C for 1 hour. After the incubation, OD 630 was measured using a spectrophotometer.
  • H8-IL7_C04_MMP9, H8-IL7_C05_MMP9, H8-IL7_C07_MMP9, H8-IL7_C09_MMP9, H8-IL7_C12_MMP9, H8-IL7_C15_MMP9, and H8-IL7_C16_MMP9 exhibited a medium potency (FIG. 1C) .
  • H8-IL7_C02_MMP9, H8-IL7_C06_MMP9, and H8-IL7_C08_MMP9 exhibited a low potency (FIG. 1D) .
  • the in vitro potency of the His-tagged IL-7 variants were also determined using the primary T cell proliferation assay. Induction of primary T cell proliferation was performed as follows. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy human blood, and the total T cells were isolated by MagniSort TM Human T cell Enrichment Kit (Invitrogen, Cat#: 8804-6810-74) and cultured in a complete RPMI medium containing 10%fetal bovine serum (FBS) and 1 ⁇ streptomycin/penicillin. The purified IL-7 variants were then serially diluted (10-fold) to final concentrations of 10 -5 to 10 2 nM.
  • PBMCs Peripheral blood mononuclear cells
  • IL-7 variants 5 ⁇ 10 4 cells/well of T cells were incubated with the diluted His-tagged IL-7 variants in a low-binding 96-well U-shaped plate at 37°C for 7 days.
  • the ability of the IL-7 variants to induce T cell proliferation was evaluated by the Luminescent Cell Viability Assay (Promega, Cat#: G7573) .
  • the tested His-tagged IL-7 variants exhibited similar potency ranking using the T cell proliferation assay, as compared to the results obtained using the HEK-Blue TM IL-7 reporter assay. Only those His-tagged IL-7 variants having a medium to high potency using the HEK-Blue TM IL-7 reporter assay were selected to test their abilities to induce primary T cell proliferation.
  • each heterodimeric Fc-fused IL-7 variant contains two polypeptide chains, i.e., a first polypeptide chain (or a “hole chain” ) comprising from N-terminus to C-terminus: a human IgG4 hinge region (SEQ ID NO: 48) , a human IgG4 Fc region with hole mutations (SEQ ID NO: 49) , a linker peptide (SEQ ID NO: 4) , and an IL-7 variant (e.g., any of the IL-7 variants described in Table 2) ; and a second polypeptide chain (or a “knob chain” ) comprising from N-terminus to C-terminus: a human IgG4 hinge region (SEQ ID NO: 48) , and a human IgG4 Fc region with knob mutations (SEQ ID NO: 50) .
  • a first polypeptide chain or a “hole chain”
  • SEQ ID NO: 48 a human IgG4 hinge region with hole
  • IL7_C03_MMP9 SEQ ID NO: 9
  • IL7_C14_MMP9 SEQ ID NO: 20
  • medium-potency IL-7 variants IL7_C09_MMP9 (SEQ ID NO: 15) and IL7_C15_MMP9 (SEQ ID NO: 21)
  • Plasmids expressing heterodimeric Fc-fused IL-7 and its variants were constructed.
  • Expi293F TM cells were transfected to express the following molecules: heterodimer G4ssFc-IL7_C03_MMP9 (hole chain: SEQ ID NO: 55; knob chain: SEQ ID NO: 53) , heterodimer G4ssFc-IL7_C09_MMP9 (hole chain: SEQ ID NO: 56; knob chain: SEQ ID NO: 53) , heterodimer G4ssFc-IL7_C14_MMP9 (hole chain: SEQ ID NO: 57; knob chain: SEQ ID NO: 53) , heterodimer G4ssFc-IL7_C15_MMP9 (hole chain: SEQ ID NO: 58; knob chain: SEQ ID NO: 53) , heterodimer G4ssFc-IL7_wt (hole chain: SEQ ID NO: 59; knob chain: SEQ ID NO: 53) , heterodimer G4ssFc-IL7_C10
  • the in vitro potency of the heterodimeric Fc-fused IL-7 variants were determined using the HEK-Blue TM IL-7 reporter assay as described in Example 2. As shown in FIGS. 3A-3B, the tested heterodimeric Fc-fused IL-7 variants exhibited similar potency ranking as compared to the corresponding His-tagged IL-7 variants.
  • the in vitro potency of the heterodimeric Fc-fused IL-7 variants were also determined using the primary T cell proliferation assay as described in Example 2. As shown in FIG. 4, the tested heterodimeric Fc-fused IL-7 variants exhibited similar potency ranking using the T cell proliferation assay, as compared to the results obtained using the HEK-Blue TM IL-7 reporter assay (shown in FIG. 3B) .
  • ELISA plates coated with 1 ⁇ g/ml anti-His tag antibody (R&D Systems, Cat#: MAB050-500) were prepared. 30 ⁇ l of His-0.5 ⁇ g/ml CD127 (Sino Biological, Cat#: 10975-H08H) was added to the ELISA plates and incubated at 25°C for 1 hour. After the incubation, the serially diluted Fc-fused IL-7 variants was added to the ELISA plates and incubated at 25°C for 1 hour.
  • heterodimer G4ssFc-IL7_C14_MMP9 exhibited a comparably high binding affinity as compared to heterodimer G4ssFc-IL7_wt, whereas heterodimer G4ssFc-IL7_C03_MMP9, heterodimer G4ssFc-IL7_C09_MMP9, and heterodimer G4ssFc-IL7_C15_MMP9 exhibited a relatively low binding affinity.
  • heterodimeric Fc-fused IL-7 variants with a medium to high potency on T cell response and a similar or low binding affinity to CD127 may be good candidates that have lower off-target effects and/or better pharmacokinetics (PK) .
  • the protein stability of heterodimeric Fc-fused IL-7 variants were determined by differential scanning fluorimetry (DSF) . Assays were performed with a Bio-Rad CFX Connect TM Real-Time PCR System (Hercules, CA, USA) into Multiplate TM 96-Well PCR Plates, low profile, unskirted, clear (Bio-Rad; Hercules, CA, USA) . Wells were filled to 25 ⁇ L, each composed of 2.5 ⁇ L of Orange (Sigma-Aldrich; Cat#: S5692) 5,000 ⁇ , for a final concentration of 5 ⁇ ; 10 ⁇ L of each tested sample at a final protein concentration of 0.4 mg/mL; and the remaining 12.5 ⁇ L with the appropriate buffer.
  • DFS differential scanning fluorimetry
  • the protocol was set with a temperature analysis range from 20°C to 95°C, with steps of 1°C increasing every 10 seconds and fluorescence recorded at the end of each step.
  • Raw fluorescence data were analyzed to determine the melting temperature (T m ) , from non-linear fitting of thermal denaturation data. The results are shown in the table below.
  • the melting temperature T m of the tested heterodimeric Fc-fused IL-7 variants including heterodimer G4ssFc-IL7_C03_MMP9, heterodimer G4ssFc-IL7_C09_MMP9, heterodimer G4ssFc-IL7_C10_MMP9, heterodimer G4ssFc-IL7_C11_MMP9, and heterodimer G4ssFc-IL7_C14_MMP9, was similar or higher than that of heterodimer G4ssFc-IL7_wt.
  • the results indicate that these heterodimeric Fc-fused IL-7 variants have similar or better protein stability as compared to heterodimer G4ssFc-IL7_wt.
  • the anti-tumor efficacy of heterodimeric Fc-fused IL-7 and its variants were evaluated in a CT26 tumor-bearing BALB/c mouse model.
  • the treatment plan and dosing schedule are shown in the table below.
  • mice 5 weeks old BALB/c mice were selected and subcutaneously (s. c. ) inoculated with 2 ⁇ 10 5 mouse colon carcinoma cell CT26. Mice were grouped, and treatment of the tested molecules started on Day 4 (D4) after the tumor inoculation, when tumor volume reached approximately 100 mm 3 . The treatment group mice were intraperitoneally (i.p.
  • heterodimer G4ssFc-IL7_C03_MMP9 G1
  • heterodimer G4ssFc-IL7_C09_MMP9 G2
  • heterodimer G4ssFc-IL7_C14_MMP9 G3
  • heterodimer G4ssFc-IL7_wt G4 mice
  • mice treated with heterodimer G4ssFc-IL7_C14_MMP9 (G3) and heterodimer G4ssFc-IL7_wt (G4) showed slight body weight loss on Day 11.
  • the results indicate that all tested heterodimeric Fc-fused IL-7 and its variants were well tolerated at 10 mg/kg.
  • heterodimer G4ssFc-IL7_C03_MMP9 (G1) and heterodimer G4ssFc-IL7_C09_MMP9 (G2) were better tolerated as compared to heterodimer G4ssFc-IL7_wt (G4) .
  • FIGS. 7A-7E Individual tumor growth curves of each mouse in the G1-G5 groups are shown in FIGS. 7A-7E, respectively.
  • the dotted line indicates half the value of the mean tumor volume of the vehicle group (G5) on Day 21.
  • the ratio next to the dashed line in each figure (e.g., “3/5” in FIG. 7A) shows a ratio of the number of mice with a tumor volume smaller than the dotted line over the total number of surviving mice on Day 21.
  • heterodimer G4ssFc-IL7_C03_MMP9, heterodimer G4ssFc-IL7_C09_MMP9, and heterodimer G4ssFc-IL7_C14_MMP9 exhibited a better anti-tumor efficacy than heterodimer G4ssFc-IL7_wt.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Toxicology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des variants d'IL-7 modifiés, des constructions protéiques de ceux-ci, et des procédés d'utilisation de ceux-ci. Dans certains modes de réalisation, les variants d'IL-7 modifiés comprennent une liaison disulfure non native.
PCT/CN2025/098273 2024-06-03 2025-05-30 Variants d'il-7 modifiés et leurs procédés d'utilisation Pending WO2025252012A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463655444P 2024-06-03 2024-06-03
US63/655444 2024-06-03

Publications (1)

Publication Number Publication Date
WO2025252012A1 true WO2025252012A1 (fr) 2025-12-11

Family

ID=97960064

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2025/098273 Pending WO2025252012A1 (fr) 2024-06-03 2025-05-30 Variants d'il-7 modifiés et leurs procédés d'utilisation

Country Status (1)

Country Link
WO (1) WO2025252012A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0155832A2 (fr) * 1984-03-16 1985-09-25 Genentech, Inc. Protéines stabiles, méthode pour leur préparation, plasmides et autres ADN codant pour celles-ci, cultures de cellules contenant de tels plasmides
CN108064236A (zh) * 2015-03-31 2018-05-22 免疫医疗有限公司 新颖的il33形式、il33的突变形式、抗体、测定及其使用方法
US20180271998A1 (en) * 2015-12-04 2018-09-27 Merrimack Pharmaceuticals, Inc. Disulfide-stabilized fabs
WO2023114829A1 (fr) * 2021-12-15 2023-06-22 Genentech, Inc. Polypeptides il-18 stabilisés et leurs utilisations

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0155832A2 (fr) * 1984-03-16 1985-09-25 Genentech, Inc. Protéines stabiles, méthode pour leur préparation, plasmides et autres ADN codant pour celles-ci, cultures de cellules contenant de tels plasmides
CN108064236A (zh) * 2015-03-31 2018-05-22 免疫医疗有限公司 新颖的il33形式、il33的突变形式、抗体、测定及其使用方法
US20180271998A1 (en) * 2015-12-04 2018-09-27 Merrimack Pharmaceuticals, Inc. Disulfide-stabilized fabs
WO2023114829A1 (fr) * 2021-12-15 2023-06-22 Genentech, Inc. Polypeptides il-18 stabilisés et leurs utilisations

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
COSENZA L, SWEENEY E, MURPHY J R: "Disulfide bond assignment in human interleukin-7 by matrix-assisted laser desorption/ionization mass spectroscopy and site-directed cysteine to serine mutational analysis.", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 272, no. 52, 26 December 1997 (1997-12-26), US , pages 32995 - 33000, XP002226701, ISSN: 0021-9258, DOI: 10.1074/jbc.272.52.32995 *
GREENSTEIN ANDREW E., ZAVODOVSKAYA MARIANNA, VELASQUEZ MAILE, WEISSBURG PERRY, JURIC VLADI, MIKELS-VIGDAL AMANDA, BRACHMANN CARRIE: "Effect of andecaliximab (anti-MMP9) on proteolysis of IL-7 in vitro, TCR diversity in mice, and serum IL-7 in gastric cancer patients in combination with chemotherapy.", JOURNAL OF CLINICAL ONCOLOGY, AMERICAN SOCIETY OF CLINICAL ONCOLOGY, UNITED STATES, vol. 36, no. 5_suppl, 10 February 2018 (2018-02-10), United States, pages 101 - 101, XP093378375, ISSN: 0732-183X, DOI: 10.1200/jco.2018.36.5_suppl.101 *

Similar Documents

Publication Publication Date Title
US11717559B2 (en) Interleukin 15 protein complex and use thereof
CN113038964B (zh) 超长效胰岛素-fc融合蛋白及使用方法
EP2016098B1 (fr) Cytokines ciblees par des anticorps pour therapie
ES2636668T3 (es) Polipéptidos de eritropoyetina de acción prolongada y usos de los mismos
US20220204587A1 (en) Tgf-b-receptor ectodomain fusion molecules and uses thereof
WO2025252012A1 (fr) Variants d'il-7 modifiés et leurs procédés d'utilisation
CN105229035A (zh) 生长激素化合物
US20240228584A1 (en) Engineered tgfbrii variants and methods of use thereof
WO2025251929A1 (fr) Variants d'il-15 modifiés et leurs procédés d'utilisation
WO2024251200A1 (fr) Variants d'il-7 de synthèse et leurs procédés d'utilisation
TW202613154A (zh) 工程化ｉｌ－７變異體及其使用方法
WO2024193546A1 (fr) Variants d'il-21 modifiés et leurs procédés d'utilisation
TW202613153A (zh) 工程化ｉｌ－１５變異體及其使用方法
WO2024088404A1 (fr) Variants de 4-1bbl modifiés et leurs procédés d'utilisation
US20250115658A1 (en) Engineered pd-1 variants and methods of use thereof
US12545741B2 (en) Anti-PD-1 and IL-15/IL-15Ra multifunctional antibody conjugate, preparation for same, and uses thereof
KR20240066490A (ko) 항체-사이토카인 융합단백질 및 이의 용도
HK40112257A (en) Engineered tgfbrii variants and methods of use thereof
RU2798845C2 (ru) Полипептиды длительного действия и способы их получения и введения
JP2026506309A (ja) インスリン－Ｆｃ融合タンパク質および癌を治療するための使用方法
CN121949562A (zh) 一种新型蛋白复合物的二聚体化合物及其制备方法和制药用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25819129

Country of ref document: EP

Kind code of ref document: A1