WO2021031998A1 - Polypeptides recombinés et leurs utilisations - Google Patents

Polypeptides recombinés et leurs utilisations Download PDF

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Publication number
WO2021031998A1
WO2021031998A1 PCT/CN2020/109183 CN2020109183W WO2021031998A1 WO 2021031998 A1 WO2021031998 A1 WO 2021031998A1 CN 2020109183 W CN2020109183 W CN 2020109183W WO 2021031998 A1 WO2021031998 A1 WO 2021031998A1
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Prior art keywords
cancer
polypeptide
recombinant polypeptide
subject
cells
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PCT/CN2020/109183
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English (en)
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Keng-Li Lan
Cheng-Liang Tsai
Fei-Ting HSU
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Lan Keng Li
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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]
    • C07K14/5443—IL-15
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • the present disclosure in general relates to the field of cancer treatment. More particularly, the present disclosure relates to recombinant polypeptides and their uses for treating cancers.
  • Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Over the years, cancer has become a severe problem to all human beings and is a heavy burden for health care system of a society.
  • IL-15 is a member of the common ⁇ chain receptor ( ⁇ c) cytokine family, regulating T cell activation and proliferation and promoting natural killer (NK) cell expansion and development.
  • IL-15 has been shown to enhance the anti-tumor immunity of CD8 + T cells in some pre-clinical models (Klebanoff CA et al., PNAS 101 (7) : 1969–74; Teague RM et al., Nature Medicine 12 (3) : 335–41) .
  • production of recombinant IL-15 with suitable pharmacokinetics profile remains an obstacle which limits its clinical use for treating cancers.
  • Several studies have shown that IL-15 per se is rapidly metabolized in circulation.
  • bioactivity of IL-15 fused to an extra functional domain that is isolated from mammalian cells is generally compromised compared to that of a natural IL-15 or a recombinant IL-15 produced by E. coli.
  • one aspect of the disclosure is directed to a recombinant polypeptide, which comprises,
  • a second polypeptide operably linked to the N-or C-terminus of the first polypeptide, wherein the second polypeptide has an amino acid sequence at least 85%identical to SEQ ID NO: 2 or SEQ ID NO: 3.
  • the first polypeptide has the amino acid sequence 100%identical to SEQ ID NO: 1;
  • the second polypeptide has the amino acid sequence 100%identical to SEQ ID NO: 2 or SEQ ID NO: 3.
  • the N-terminus of the recombinant polypeptide is acetylated, formylated, methylated, carbamylated, pegylated, phosphorylated, or glycosylated, and/or
  • the C-terminus of the recombinant polypeptide is amidated, glypiated, biotinylated, or glycosylated.
  • the present recombinant polypeptide further comprises a third polypeptide operably linked to the N- or C-terminus of the first or the second polypeptides, wherein the third polypeptide is ⁇ -galactosidase (lacZ) , chloramphenicol acetyltransferase (CAT) , luciferase, green fluorescent protein (GFP) , red fluorescent protein (RFP) , cyan fluorescent protein (CFP) , yellow fluorescent protein (YFP) , ⁇ -lactamase, aminoglycoside-3’-phosphotransferase (APH (3’) ) , orotidine-5’-phosphate decarboxylase (ODCase) , chitin binding protein (CBP) , maltose binding protein (MBP) , Strep-tag, glutathione-S-transferase (GST) , thioredoxin (TRX) , alpha-galactosidase
  • the recombinant polypeptide further comprises a linker configured to link any two of the first, the second, and the third polypeptides.
  • compositions for preventing and/or treating a cancer in a subject in need thereof pertains to a pharmaceutical composition for preventing and/or treating a cancer in a subject in need thereof.
  • the pharmaceutical composition comprises the present recombinant polypeptide and a pharmaceutically acceptable carrier.
  • the present pharmaceutical composition further comprises an anti-PD-L1 antibody.
  • the present disclosure is related to a method for preventing and/or treating a cancer in a subject in need thereof.
  • the method comprises administering to the subject a therapeutically effective amount of the present pharmaceutical composition as described above.
  • examples of the cancers treatable by the present method include, but are not limited to, bladder cancer, biliary cancer, bone cancer, brain tumor, breast cancer, cervical cancer, colorectal cancer, colon cancer, esophageal cancer, epidermal carcinoma, gastric cancer, gastrointestinal stromal tumor (GIST) , glioma, hematopoietic tumors of lymphoid lineage, hepatic cancer, non-Hodgkin’s lymphoma, Kaposi’s sarcoma, leukemia, lung cancer, lymphoma, intestinal cancer, melanoma, myeloid leukemia, pancreatic cancer, prostate cancer, retinoblastoma, ovary cancer, renal cell carcinoma, spleen cancer, squamous cell carcinoma, thyroid cancer, and thyroid follicular cancer.
  • the cancer is a colon cancer.
  • the cancer is a melanoma
  • the subject is preferably a human.
  • the present method further comprises the step of subjecting the subject to a surgery, a radiotherapy, a chemotherapy, an immunotherapy, a hormone therapy, a targeted therapy, a thermal therapy, or a combination therapy thereof, prior to, concurrently with or after administering the present pharmaceutical composition to the subject.
  • the immunotherapy of the present method comprises administering an anti-PD-L1 antibody to the subject.
  • FIGs. 1A-1C illustrate the recombinant polypeptides of the present disclosure and their pharmacokinetics in vivo according to one embodiment of the present disclosure.
  • FIG. 1A a schematic diagram showing the constructs of the present recombinant polypeptides
  • FIG. 1B the pharmacokinetics of IL-15-albumin binding domain (ABD)
  • FIG. 1C the pharmacokinetics of IL-15-albumin binding peptide (ABP)
  • IL-15 was included as a comparison reference.
  • FIGs. 2A-2E depict the results of biological activities of the recombinant polypeptides of the present disclosure.
  • FIGs. 2A-2C the binding activity of the present recombinant polypeptides toward the serum albumin of human (FIG. 2A) , dog (FIG. 2B) , and mouse (FIG. 2C) ;
  • FIG. 2D induction of CTLL-2 cell proliferation by the present recombinant polypeptides (5 ng/ml) ;
  • FIG. 2E induction of STAT-5 phosphorylation in CTLL-2 cells by the present recombinant polypeptides.
  • FIGs. 3A-3D depict the effect of the present recombinant polypeptides on melanoma.
  • FIG. 3A tumor volume of the mice treated with specified treatments
  • FIG. 3B tumor weight of the mice treated with specified treatments
  • FIG. 3C tumor metastases in the intestines of the mice monitored by an in vivo imaging system
  • FIG. 3D tumor weight of the metastatic lesions in the intestines of the mice treated with PBS or IL15-ABP on day 28.
  • FIGs. 4A-4D depict the effect of the present recombinant polypeptides on colon cancer.
  • FIG. 4A tumor volume of the mice treated with specified treatments
  • FIG. 4B tumor weight on day 18 of the mice treated with specified treatments
  • FIG. 4C number of the tumor lumps in the lung of the mice treated with specified treatments
  • FIG. 4D number of the tumor lumps in the lung, colon and liver of the mice treated with specified treatments.
  • a 1 p ⁇ 0.05 vs. control a 2 p ⁇ 0.01 vs. control
  • b 1 p ⁇ 0.05 vs. IL-15 b 2 p ⁇ 0.01 vs. IL-15.
  • FIGs. 5A-5H depict the impact of the present recombinant polypeptides on alteration of the immune cell populations within the mice bearing colon cancer treated with specified treatments.
  • FIGs. 5A-5B percentage (%) of CD11b + /GR-1 + myeloid-derived suppressor cell (MDSC) population from bone marrow (FIG. 5A) and spleen (FIG. 5B) ;
  • FIGs. 5C-5D percentage (%) of CD4 + /CD25 + /FOXP3 + regulatory T (T reg ) cell population from tumor-draining lymph node (TDLN) (FIG. 5C) and spleen (FIG. 5D) ;
  • 5E-5F percentage (%) of CD8 + T cell population from TDLN (FIG. 5E) and spleen (FIG. 5D) ;
  • FIG. 5G percentage (%) of CD3 - /CD49b + , CD3 - /CD335 + , and CD3 - /CD49b + /CD335 + NK cell population from spleen;
  • FIG. 5H mice body weight measured during the course of treatments. a 1 p ⁇ 0.05 vs. control; a 2 p ⁇ 0.01 vs. control; b 1 p ⁇ 0.05 vs. IL-15; b 2 p ⁇ 0.01 vs. IL-15.
  • FIGs. 6A-6J depict the effect of the combination therapy on tumor growth according to one embodiment of the present disclosure.
  • FIG. 6A tumor volume of colon cancer (CT26) -bearing mice treated with specified treatments
  • FIG. 6B tumor volume of melanoma (B16F10) -bearing mice treated with specified treatments
  • FIGs. 6C-6D tumor weight measured from colon cancer (FIG. 6C) and melanoma (FIG. 6D) on day 18
  • FIG. 6E quantification of the bioluminescence images for measuring B16F10/luc2 tumor volume within the mice treated with specified treatments
  • FIGs. 6F-6G body weight of the colon cancer (CT26) -bearing mice (FIG.
  • FIGs. 6H-6J protein expressions in colon cancer or melanoma measured by immunohistochemistry (IHC) staining: BAX and cleaved caspase 3 in colon cancer (FIG. 6H) and in melanoma (FIG. 6I) , and KI-67 in both colon cancer and melanoma (FIG. 6J) .
  • IHC immunohistochemistry
  • FIGs. 7A-7F depict the effect of the combination therapy on induction of CD8 + T cell activation according to one embodiment of the present disclosure.
  • FIGs. 7A-7B percentage (%) of CD8 + /IFN- ⁇ + T cell population in the TDLN (FIG. 7A) or spleen (FIG. 7B) of the tumor-bearing mice treated with specified treatments
  • FIGs. 7C-7D percentage (%) of CD8 + /IL-2 + T cell population in the TDLN (FIG. 7C) or spleen (FIG. 7D) of the tumor-bearing mice treated with specified treatments;
  • FIGs. 7A-7B percentage (%) of CD8 + /IFN- ⁇ + T cell population in the TDLN (FIG. 7A) or spleen (FIG. 7B) of the tumor-bearing mice treated with specified treatments
  • FIGs. 7C-7D percentage (%) of CD8 + /IL-2 + T cell population in the TDLN (FIG. 7C
  • FIG. 7E-7F protein expressions in colon cancer or melanoma measured by IHC staining: Granzyme B and CD8 in colon cancer (FIG. 7E) and in melanoma (FIG. 7F) .
  • a 1 p ⁇ 0.05 vs. control a 2 p ⁇ 0.01 vs. control
  • FIGs. 8A-8H depict the effect of the combination therapy on induction of NK cell accumulation and function according to one embodiment of the present disclosure.
  • FIG. 8A percentage (%) of CD3 - /CD49b + , CD3 - /CD335 + , and CD3 - /CD49b + /CD335 + NK cell population in the spleen of colon cancer (CT26) -bearing mice treated with specified treatments;
  • CT26 colon cancer
  • FIG. 8B percentage (%) of CD3 - /CD49b + , CD3 - /NK1.1 + , and CD3 - /CD49b + /NK1.1 + NK cell population in the spleen of melanoma (B16F10) -bearing mice treated with specified treatments
  • FIG. 8C percentage (%) of CD3 - /CD335 + /IFN- ⁇ + NK cell population in the spleen of colon cancer (CT26) -bearing mice treated with specified treatments
  • FIG. 8D percentage (%) of CD3 - /NK1.1 + /IFN- ⁇ + NK cell population in the spleen of melanoma (B16F10) -bearing mice treated with specified treatments
  • FIGs. 8E-8F protein expressions in colon cancer or melanoma measured by IHC staining: CD49b and IFN- ⁇ in colon cancer (FIG. 8E) and in melanoma (FIG. 8F) ;
  • FIGs. 8G-8H protein expressions in serum measured by enzyme-linked immunosorbent assay (ELISA) : secreted VEGF (FIG. 8G) and secreted IL-15 (FIG. 8H) .
  • a 1 p ⁇ 0.05 vs. control a 2 p ⁇ 0.01 vs. control
  • FIGs. 9A-9F depict the effect of the combination therapy on suppression of immunosuppressive cell accumulation according to one embodiment of the present disclosure.
  • FIGs. 9A-9B percentage (%) of CD4 + /CD25 + /FOXP3 + T reg cell population from TDLN (FIG. 9A) and spleen (FIG. 9B) in tumor-bearing mice treated with specified treatments;
  • FIGs. 9C-9D percentage (%) of CD11b + /GR-1 + MDSC population from bone marrow (FIG. 9C) and spleen (FIG. 9D) in tumor-bearing mice treated with specified treatments;
  • FIGs. 9A-9B percentage (%) of CD4 + /CD25 + /FOXP3 + T reg cell population from TDLN (FIG. 9A) and spleen (FIG. 9B) in tumor-bearing mice treated with specified treatments
  • FIGs. 9C-9D percentage (%) of CD11b + /GR-1 + MDSC population from bone
  • 9E-9F protein expressions in colon cancer or melanoma measured by IHC staining: FOXP3 and IDO in colon cancer (FIG. 9E) and in melanoma (FIG. 9F) .
  • a 1 p ⁇ 0.05 vs. control a 2 p ⁇ 0.01 vs. control
  • sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid sequence for optimal alignment with a second amino acid sequence) , and not considering any conservative substitutions as part of the sequence identity.
  • the amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position.
  • Alignment for purposes of determining percentage sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, sequence comparison between two amino acid sequences was carried out by computer program Blastp (protein-protein BLAST) provided online by National Center for Biotechnology Information (NCBI) .
  • Blastp protein-protein BLAST
  • NCBI National Center for Biotechnology Information
  • the two sequences are the same length.
  • linker refers to a peptide having natural or synthetic amino acid residues for connecting two polypeptides.
  • the linker may be used to connect human IL-15 to albumin binding domain (ABD) or albumin binding peptide (ABP) , or to connect ABD or ABP to 6His (i.e., poly-histidine tag) to form a recombinant polypeptide of the present disclosure.
  • the linker is a peptide having at least 5 amino acid residues in length, such as 5 to 100 amino acid residues in length, more preferably 10 to 30 amino acid residues in length.
  • the linker within the present recombinant polypeptide is a peptide of at least 5 amino acid residues in length, preferably 5 to 15 amino acid residues in length.
  • cancers and tumors are used alternatively in the present disclosure, and refer to the physiological condition in mammals and especially in humans that is typically characterized by un-regulated cell growth. Cancers in this respect include in situ cancers, metastatic cancers, and/or drug-resistant cancers. Accordingly, cancers or tumors treatable by the present disclosure are those occurred in/on breast, lung, colon, colorectal, spleen, kidney, liver, bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone, blood, thymus, uterus, testicles, cervix, and neuron.
  • subject refers to an animal including the human species that is treatable with the recombinant polypeptide, the pharmaceutical composition and/or methods of the present disclosure.
  • subject or “patient” intended to refer to both the male and female gender unless one gender is specifically indicated. Accordingly, the term “subject” or “patient” comprises any mammal which may benefit from treatment of cancer. Examples of a “subject” or “patient” include, but are not limited to, a human, a rat, a mouse, a guinea pig, a monkey, a pig, a goat, a cow, a horse, a dog, a cat, a bird and a fowl.
  • the patient is a human.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically acceptable material, composition or vehicle such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • the carrier can be in the form of a solid, semi-solid or liquid diluent, cream or a capsule.
  • a therapeutically effective amount refers to a predetermined amount calculated to achieve the therapeutically desired effect, i.e., to discourage, combat, ameliorate, improve, prevent, inhibit, block, or reverse an unwanted condition, disease or symptom of a patient, and for periods of time necessary, as may be indicated by the particular embodiment.
  • a “therapeutically effective amount” as recited in a “method of treating” embodiment is a predetermined amount calculated to achieve the desired treatment effect, i.e., to discourage, combat, ameliorate, or improve an unwanted condition, disease or symptom.
  • a “therapeutically effective amount” as recited in a “method of preventing” embodiment is a predetermined amount calculated to achieve the desired treatment effect, i.e., to prevent or inhibit or block an unwanted condition, disease or symptom prior to its occurrence.
  • the therapeutically effective amount may therefore be in an amount sufficient for a certain exposure of the present recombinant polypeptide in the patient.
  • the activity contemplated by the present methods includes both medical therapeutic and/or prophylactic treatment, as appropriate.
  • a therapeutically effective amount of the present recombinant polypeptide is typically an amount such that when it is administered in a physiologically tolerable excipient composition, it is sufficient to achieve an effective systemic concentration or local concentration in the tissue.
  • administered means either directly administering a recombinant polypeptide or a pharmaceutical composition of the present disclosure, or a combination therapy comprising the same (i.e., the pharmaceutical kit) .
  • treatment may refer to a curative or palliative measure.
  • treating refers to the administration of a recombinant polypeptide or a pharmaceutical composition of the present disclosure, or a combination therapy comprising the same (i.e., the pharmaceutical kit) to a subject, who has a cancer, a symptom associated with a cancer, a disease or disorder secondary to a cancer, with the purpose to partially or completely alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a cancer.
  • the term “synergistic effect” or grammatical variations thereof means and includes a cooperative action encountered in a combination of two or more active therapies in which the combination activity of the two or more active therapies exceeds the sum of the activity of each active therapy alone.
  • the present disclosure is based, at least in part, on the discovery that the polypeptide of ABD or ABP can greatly prolong the half-life of natural IL-15 (i.e., IL-15 wild-type) in circulation when operably linked to the N-or C-terminus of the IL-15.
  • IL-15 possesses an anti-cancer activity by enhancing the ability of anti-tumor CD8 + T cells in some pre-clinical models.
  • the obstacle for utilizing IL-15 to treat cancers lies in that IL-15 is rapidly degraded in circulation.
  • the present disclosure aims to prolong the half-life of IL-15, in which ABD or ABP is connected to IL-5 to protect IL-15 from degradation, thereby increasing its half-life in circulation and achieving desirable pharmacokinetics profiling.
  • the first aspect of the present disclosure is directed to a recombinant polypeptide, which comprises,
  • a second polypeptide operably linked to the N-or C-terminus of the first polypeptide, wherein the second polypeptide has an amino acid sequence at least 85%identical to SEQ ID NO: 2 or SEQ ID NO: 3.
  • the first polypeptide has an amino acid sequence at least 85%identical to SEQ ID NO: 1, such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 1.
  • the first polypeptide has the amino acid sequence 100%identical to SEQ ID NO. 1.
  • the second polypeptide has an amino acid sequence at least 85%identical to SEQ ID NO: 2, such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 2.
  • the second polypeptide has the amino acid sequence 100%identical to SEQ ID NO. 2.
  • the second polypeptide has an amino acid sequence at least 85%identical to SEQ ID NO: 3, such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 3.
  • the second polypeptide has the amino acid sequence 100%identical to SEQ ID NO. 3.
  • the present recombinant polypeptide may have some modifications either on the N-terminus or the C-terminus, or both.
  • the N-terminus of the present recombinant polypeptide may be acetylated, formylated, methylated, carbamylated, pegylated, phosphorylated, or glycosylated; additionally or alternatively, the C-terminus of the present recombinant polypeptide may be amidated, glypiated, biotinylated, or glycosylated.
  • the present recombinant polypeptide may further comprise a third polypeptide operably linked to the N-or C-terminus of the first or the second polypeptides for various purposes.
  • the third polypeptide may serve as a reporter, and in such case, it is selected from the group consisting of, ⁇ -galactosidase (lacZ) , chloramphenicol acetyltransferase (CAT) , luciferase, green fluorescent protein (GFP) , red fluorescent protein (RFP) , cyan fluorescent protein (CFP) , and yellow fluorescent protein (YFP) .
  • the third polypeptide may serve as a selection marker, and in such case, it is selected from the group consisting of, ⁇ -lactamase, aminoglycoside-3’-phosphotransferase (APH (3’) ) , and orotidine-5’-phosphate decarboxylase (ODCase) .
  • the third polypeptide may serve as an affinity purification tag, and in such case, it is selected from the group consisting of, chitin binding protein (CBP) , maltose binding protein (MBP) , Strep-tag, glutathione-S-transferase (GST) , thioredoxin (TRX) , albumin-binding protein (ABP) , alkaline phosphatase (AP) , biotin-carboxy carrier protein (BCCP) , calmodulin binding peptide (CBP) , bacteriophage T7 epitope (T7-tag) , poly-histidine tag, FLAG-tag, Myc-tag, HA-tag, Spot-tag, NE-tag, and a combination thereof.
  • the third polypeptide is a poly-histidine tag (i.e., 6His) .
  • the present recombinant polypeptide comprises in sequence, the first, the second, and the third polypeptides, in which each polypeptide is directly connected to its neighboring peptide (s) .
  • the linker comprises a sequence of (G 4 S) 1 .
  • the linker comprises a sequence or (G 4 S) 2 .
  • the linker comprises a sequence or (G 4 S) 3 .
  • the present recombinant polypeptide has an amino acid sequence at least 85%identical to SEQ ID NO: 4, such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 4.
  • the present recombinant polypeptide has the amino acid sequence 100%identical to SEQ ID NO. 4.
  • the present recombinant polypeptide has an amino acid sequence at least 85%identical to SEQ ID NO: 5, such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 5.
  • the second polypeptide has the amino acid sequence 100%identical to SEQ ID NO. 5.
  • the present recombinant polypeptide has an amino acid sequence at least 85%identical to SEQ ID NO: 6, such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 6.
  • the present recombinant polypeptide has the amino acid sequence 100%identical to SEQ ID NO. 6.
  • the present disclosure pertains to a pharmaceutical composition for preventing and/or treating a cancer in a subject in need thereof.
  • the pharmaceutical composition comprises the present recombinant polypeptide and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be any of a liquid, gel, cream, ointment, lotion, suspension, emulsion, adhesive, amniotic membrane, skin substitute, artificial skin, or skin equivalents.
  • the pharmaceutically acceptable carrier of the present pharmaceutical composition is phosphate buffers (PBS) .
  • the pharmaceutical composition may further comprises, an antioxidant (e.g., ascorbic acid or methionine) ; a preservative (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; benzoates, sorbate and m-cresol) ; a low molecular weight (i.e., less than about 10 residues) polypeptide; a protein (e.g., serum albumin, gelatin, or immunoglobulins) ; a hydrophilic polymer (e.g., polyvinylpyrrolidone) ; an amino acid (e.g., glycine, glutamine,
  • the pharmaceutical composition described herein can be formulated in unit dosage forms, such as tablets, pills, capsules, powders, granules, gels, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation, or intrathecal or intracerebral routes.
  • the present pharmaceutical composition is formulated in a solution form.
  • the present recombinant polypeptide is present at a level of about 0.1%to 99%by weight, based on the total weight of the pharmaceutical composition. In some embodiments, the present recombinant polypeptide is present at a level of at least 1%by weight, based on the total weight of the pharmaceutical composition. In certain embodiments, the present recombinant polypeptide is present at a level of at least 5%by weight, based on the total weight of the pharmaceutical composition. In still other embodiments, the present recombinant polypeptide is present at a level of at least 10%by weight, based on the total weight of the pharmaceutical composition. In still yet other embodiments, the present recombinant polypeptide is present at a level of at least 25%by weight, based on the total weight of the pharmaceutical composition.
  • the pharmaceutical composition described herein can be prepared by any method known in the art of pharmacology.
  • such preparatory methods include bringing the present recombinant polypeptide (i.e., the “active ingredient” ) into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single-or multi-dose unit.
  • a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.
  • the present recombinant polypeptide provided herein is typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described herein will be decided by a physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the species, age, body weight, general health, sex, and diet of the subject, severity of the side effects or disorder; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs identity of the particular present recombinant polypeptide used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.
  • the present recombinant polypeptide in manufacturing a pharmaceutical composition, wherein the pharmaceutical composition is used for treating a cancer in a subject in need.
  • the present pharmaceutical composition may further comprise an anti-PD-L1 antibody.
  • anti-PD-L1 antibody suitable to be employed in the present pharmaceutical composition includes, but is not limited to, atezolizumab (MPDL3280A, RO5541267, ) , avelumab durvalumab (MEDI4736, ) , anti-PD-L1 antibody (clone 10F. 9G2) or an anti-PD-L1 antibody generated from polyclonal antisera, from the standard procedures for preparing monoclonal antibodies, or from whole gene synthesis by grafting and fusing with desired DNA sequences (e.g., VH, VL, Fc, signal peptide, IRES, linker, and etc. ) into desired constructs.
  • the anti-PD-L1 antibody is clone 10F. 9G2.
  • Another aspect of the present disclosure is to provide a method for preventing and/or treating a cancer in a subject in need thereof.
  • the method comprises administering to the subject a therapeutically effective amount of the aforementioned recombinant polypeptide or the pharmaceutical composition comprising the present recombinant polypeptide.
  • the present recombinant polypeptide or the pharmaceutical composition described herein are useful in preventing and/or treating a cancer in a subject (e.g., a human patient having, suspected of having, or at risk for the cancer) .
  • the cancers treatable by the present methods include, but are not limited to, bladder cancer, biliary cancer, bone cancer, brain tumor, breast cancer, cervical cancer, colorectal cancer, colon cancer, esophageal cancer, epidermal carcinoma, gastric cancer, gastrointestinal stromal tumor (GIST) , glioma, hematopoietic tumors of lymphoid lineage, hepatic cancer, non-Hodgkin’s lymphoma, Kaposi’s sarcoma, leukemia, lung cancer, lymphoma, intestinal cancer, melanoma, myeloid leukemia, pancreatic cancer, prostate cancer, retinoblastoma, ovary cancer, renal cell carcinoma, spleen cancer, squam
  • the present recombinant polypeptide or the pharmaceutical composition can be administered by a suitable route as known to those skilled in the art, including oral, intracranial, intraspinal, intrathecal, intramedullar, intracerebral, intracerebroventricular, intravenous, intraarterial, intracardial, intracutaneous, subcutaneous, transdermal, intraperitoneal, or intramuscular administration.
  • a suitable route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in circulation) , and/or the condition of the subject (e.g., whether the subject is able to tolerate intraperitoneal administration or intravenous (iv. ) administration) .
  • the exact amount of the present recombinant polypeptide or pharmaceutical composition required to achieve an effective amount will vary from subject to subject, depending on factors as described above.
  • a therapeutically effective amount may be included in a single dose (e.g., single intraperitoneal injection dose, or single intravenous injection dose) or multiple doses (e.g., multiple intraperitoneal injection doses, or multiple intravenous injection doses) .
  • the frequency of administering the multiple doses to the subject is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every other week, one dose monthly or one dose every other month.
  • the frequency of administering the multiple doses to the subject is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject is one dose every other day (or three times per week) . In certain embodiments, the frequency of administering the multiple doses to the subject is one dose every third day (or twice per week) . In certain embodiments, when multiple doses are administered to a subject, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject.
  • the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject. In a specific embodiment, the duration between the first dose and last dose of the multiple doses is three weeks.
  • a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between about 8 ng and 80 ⁇ g, inclusive, of the present recombinant polypeptide or the pharmaceutical composition described herein, for example, about 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710,
  • a dose described herein includes independently between 40 ng and 8 ⁇ g, inclusive, of the present recombinant polypeptide or the pharmaceutical composition. In certain embodiments, a dose described herein includes independently between 80 ng and 800 ng, inclusive, of the present recombinant polypeptide or the pharmaceutical composition. In certain embodiments, a dose described herein includes independently between 200 ng and 500 ng, inclusive, of the present recombinant polypeptide or the pharmaceutical composition. In certain embodiments, a dose described herein includes independently between 270 ng and 410 ng, inclusive, of the present recombinant polypeptide or the pharmaceutical composition.
  • the method may further comprise administering to the subject one or more additional therapy prior to, concurrently with or after administering the present recombinant polypeptide or the pharmaceutical composition to the subject.
  • the additional therapeutic (s) is/are useful in preventing and/or treating cancers, such as a surgery, a radiotherapy, a chemotherapy, an immunotherapy, a hormone therapy, a targeted therapy, a thermal therapy, or a combination therapy thereof.
  • combination use of the present pharmaceutical composition and the additional therapy exhibits a synergistic effect on the treatment of cancers.
  • different therapies or therapy may be administered to the cancer patients at different time intervals via different routes, according to the dose and/or on the time schedule determined for that therapies or therapy.
  • certain therapy for immunotherapies such as antibodies, interferons, and interleukins
  • these drugs are often given at intervals of one, two, three, or four weeks.
  • the anti-PD-L1 antibody (clone 10F. 9G2) is given every third day (or twice per week) , ip.
  • the present pharmaceutical composition is often administered every other day (or three times per week) , ip..
  • Non-limiting examples of the chemotherapeutic agent include, but are not limited to, actinomycin D, aminoglutethimide, amsacrin, anastrozol, anthracycline, bexaroten, bleomycin, buselerin, busulfan, camptothecin derivates, capecitabin, carboplatin, carmustine, chlorambucil, cisplatin, cladribin, cyclophosphamide, cytarabin, cytosinarabinoside, dacarbacin, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, estramustine, etoposid, exemestan, fludarabin, fluorouracil, formestan, gemcitabin, goselerin, hycamtin, idarubicin, ifosfamid, imatinib, irinotecan, letrozol, le
  • Non-limiting examples of the immunotherapeutic agent include, but are not limited to, such as an anti-PD-1 antibody (e.g., pembrolizumab nivolumab ) , an anti-PD-L1 antibody (e.g., atezolizumab (MPDL3280A, RO5541267, ) , avelumab durvalumab (MEDI4736, ) , clone 10F.
  • an anti-PD-1 antibody e.g., pembrolizumab nivolumab
  • an anti-PD-L1 antibody e.g., atezolizumab (MPDL3280A, RO5541267, )
  • avelumab durvalumab e.
  • an anti-CTLA-4 antibody e.g., ipilimumab tremelimumab (CP-675, 206)
  • an anti-CTLA-4 antibody e.g., ipilimumab tremelimumab (CP-675, 206)
  • riluzole trigriluzole
  • IFN- ⁇ IL-2
  • IL-15 IL-15
  • IL-23 M-CSF
  • GM-CSF tumor necrosis factor
  • lipid A CpG, CD80, CD86, and ICAM-1.
  • the immunotherapeutic agent is anti-PD-L1 antibody (e.g., clone 10F. 9G2) .
  • hormone therapeutic agent examples include, but are not limited to, such as prednisone, methylprednisolone dexamethasone tamoxifen leuprolide and cyproterone acetate
  • Non-limiting examples of the targeted therapeutic agent include, but are not limited to, such as gefitinib (ZD1839) , erlotinib HC1 (Tarceva TM ) , CL-387, 785, EKB-569, HKI-272, trastuzumab pyrazolopyridine, benzoxepin, adenine derivatives, and 2-carboxamide cycloamino urea derivatives.
  • the subject treatable by the present pharmaceutical composition is a mammal.
  • the subject is a mouse.
  • the subject is a human.
  • kits for use in treating any of the cancers described herein may comprise the present recombinant polypeptide or pharmaceutical composition.
  • the kit may further comprise one or more additional pharmaceutical agents as described herein.
  • Mouse T-cell lymphoma cell line CTLL-2 and mouse colon cancer cell line CT26 were grown in the base medium of Roswell Park Memorial Institute (RPMI) -1640, while mouse melanoma cell line B16F10 were grown in the base medium of Dulbecco’s Modified Eagle’s Medium (DMEM) . All the base media were supplemented with 10%fetal bovine serum (FBS) , 2 mM L-glutamine, 100 units/ml penicillin and 100 ⁇ g/ml streptomycin, 1mM sodium pyruvate, and 10%T-STIM with Con A, and the cells were grown at 37°C with a humidified atmosphere of 5%CO 2 .
  • FBS fetal bovine serum
  • 2 mM L-glutamine 100 units/ml penicillin and 100 ⁇ g/ml streptomycin
  • 1mM sodium pyruvate 1mM sodium pyruvate
  • 10%T-STIM with Con A were grown at 37°C with a humidified atmosphere
  • mice Male BALB/c and C57BL/6J mice about 5 or 6 weeks of age were used in the present study. All animals were maintained in the animal facility with controlled temperature (20-24°C) , humidity (50-80%) and a 12 h/12 h light/dark cycle with food and water provided ad libitum.
  • Nucleic acid sequences for expressing the recombinant polypeptides of human IL-15-6His (SEQ ID NO: 4) , human IL-15-ABD (SEQ ID NO: 5) , and human IL-15-ABP (SEQ ID NO: 6) were inserted into an Escherichia coli (E. coli) expression plasmid pET56.
  • the constructs were then delivered into E. coli Rosetta gamiB (DE3) cells. After picking up E. coli colonies that could successfully express the recombinant polypeptides, they were scaled up to obtain a great amount of the recombinant polypeptides via the standard E. coli culture procedure. The procedures for protein recovery from inclusion bodies of E.
  • IL-15-6His was used in all the Examples within the present disclosure as the IL-15 control. Therefore, for convenience, the terms “IL-15, ” “IL-15-ABD, ” and “IL-15-ABP” are used to represent human IL-15-6His, human IL-15-ABD, and human IL-15-ABP independently in the present disclosure.
  • IL-15 (3.33 ⁇ g/dose)
  • IL-15-ABD 5 ⁇ g/dose
  • IL-15-ABP 4.5 ⁇ g/dose
  • mice were intraperitoneally injected into mice, and blood from the mice were harvested at 0, 15, 30, 45, 60, 90, 120, 240, 480, 1,440, 2,160 and 3,120 min post-injection.
  • concentration of IL-15 in serum was quantified by IL-15 Duoset ELISA kit (R&D system) .
  • the binding of the present recombinant polypeptides (including IL-15, IL-15-ABD and IL-15-ABP) to albumin from human, dog, and mouse were confirmed by enzyme-linked immunosorbent assay (ELISA) .
  • the albumins from the indicated species were coated on a 96-well microplate in an ELISA coating buffer (137 mM NaCl, 2.7 mM KCl, 10mM Na 2 HPO 4 and 1.8 mM KH 2 PO 4 ) to a final concentration of 300 ng/ml, and incubated overnight at 4°C. The wells were washed, and subsequently blocked with 3%skimmed milk in PBS for 2 hrs at 25°C.
  • the indicated concentration of the present recombinant polypeptide were added into the wells, and incubated for 2 hrs at 25°C.
  • the microplates were then washed, and incubated with an anti-6His antibody conjugated with HRP (1: 2000 dilution) for 1 hr at 25°C.
  • the HRP activity was detected using 3, 3’, 5, 5’-tetramethylbenzidine (TMB) , and the reaction was stopped with 0.5 M H 2 SO 4 .
  • the microplates were then subjected to a microplate reader to measure the absorbance of 450 nm.
  • the cells were washed twice with FACS buffer (0.8%bovine serum albumin in PBS, pH 7.2) , and then incubated with anti-STAT5 pY694 antibody (ThermoFisher) (1: 50 dilution in FACS buffer) for 2 hrs at RT.
  • the cells were washed, and a goat anti-rabbit Fc antibody conjugated FITC (Jackson) was added into the cells.
  • the mean fluorescence intensity (MFI) of the cells was determined by using a flow cytometer (CytoFLEX, Beckman-Coulter) .
  • B16F10/luc2 a melanoma cell line with bioluminescence
  • a vector containing CMV-luciferase2 (pGL4.50 [luc2/CMV] ) were used for this purpose.
  • B16F10 cells were transfected with 5 ⁇ g of the foregoing vector. After one day of incubation, hygromycin B 200 ⁇ g/ml was added to the culture, and Luc2 expression cells (i. e., B16F10/luc2) were obtained after incubation for another two weeks.
  • mice were anaesthetised with 1-2%isoflurane during surgery.
  • the mice were treated twice per week with PBS, wild-type IL-15 (600,000 IU) , or IL-15-ABP (600,000 IU) ; or control (0.1%DMSO in H 2 O) , IL-15 (5 ⁇ g/treat) , or IL-15-ABD (5 ⁇ g/treat) via intraperitoneal injection for 3 weeks.
  • Tumor size were measured 3 times per week using calipers, and the tumor volume was calculated by the formula: (0.523) ⁇ L ⁇ W 2 .
  • mice were then randomly divided into four groups. Treatments were given as follows: (1) control group (0.1%DMSO in H 2 O, ip. injection) ; (2) IL-15-ABD group (IL-15-ABD (5 ⁇ g/treat) three times per week, ip. injection) ; (3) anti-PD-L1 group (the anti-PD-L1 antibody (clone 10F. 9G2) (100 ⁇ g/treat) twice per week, ip.
  • control group (0.1%DMSO in H 2 O, ip. injection
  • IL-15-ABD group IL-15-ABD (5 ⁇ g/treat) three times per week, ip. injection
  • anti-PD-L1 group the anti-PD-L1 antibody (clone 10F. 9G2) (100 ⁇ g/treat) twice per week, ip.
  • combination group IL-15-ABD (5 ⁇ g/treat) plus the anti-PD-L1 antibody (clone 10F. 9G2; 100 ⁇ g/treat) , each amount was as described above) .
  • the tumors from each group were removed on day 21, and photographed. The tumor volume was evaluated by caliper 3 times per week.
  • mice 7. Evaluation of the immunosuppressive cells (T reg cells and myeloid-derived suppressor cells (MDSCs) ) in mice
  • TDLNs tumor-draining lymph nodes
  • spleens spleens
  • bone marrow of femur bones of the mice were obtained on day 21, and the RBCs were lysed with ACK buffer.
  • the cells containing T reg cells (CD4 + /CD25 + /FOXP3 + ) or MDSCs (CD11b + /GR-1 + ) were stained with anti-FOXP3-Alexa Fluor 488/anti-CD4-PerCP-Cy TM 5.5/anti-CD25-PE antibodies or anti-CD11b-FITC/anti-Gr-1-PE antibodies (BD Bioscience) , respectively, and the percentages of T reg cells or MDSCs within the cells were confirmed using flow cytometry The resulting data were analyzed by the FlowJo software (Tree Star) .
  • CD8 + T cells For evaluation of CD8 + T cells, procedures were as follows. Three to four weeks after tumor inoculation, the mice were euthanized, and the tumors, ascites, TDLNs and spleens were collected. For isolating the leukocytes from tumors, the tumor tissues were put in the presence of 2.5 mg/ml collagenase IV (Worthington) for 20 min, and then centrifuged using a discontinuous percoll gradient (GE Healthcare) . The CD8 + T cells within the leukocytes were isolated using anti-CD8 microbeads (Miltenyi Biotec) and a MACS column. The percentage of the CD8 + T cells within the leukocytes was >5%.
  • the CD8 + T cells within the TDLNs and the spleens were characterized by anti-CD8-FITC staining.
  • the cells were further subjected to anti-IL-2-PerCP/Cy5.5 or anti-IFN- ⁇ -PE antibodies (BD Bioscience) staining.
  • the activation of CD8 + T cells were determined by the expression of intracellular IL-2 and IFN- ⁇ using flow cytometry.
  • the total number of the infiltrating CD8 + T cells in tumors per gram was calculated by multiplying the percentage of the CD8 + T cells by the total number of the lymphocytes, and dividing the resulting number by 100 and the weight of the tumors.
  • NK cells For evaluation of NK cells, procedures were as follows. The spleens of the mice were collected on day 21 after tumor inoculation, and lysed by ACK buffer to purify the splenocytes. The NK cells within the spleens were characterized by subjected to anti-CD3-FITC/anti-CD49b-PE/anti-CD335-PerCP-Cy5.5 antibodies staining for BALB/c, and anti-CD3-FITC/anti-CD49b-PE/anti-NK1.1-PerCP-Cy5.5 antibodies staining for C57BL/6.
  • CD3 - /CD49b + , CD3 - /CD335 + , CD3 - /CD49b + /CD335 + , as well as CD3 - /CD49b + , CD3 - /NK1.1 + , and CD3 - /CD49b + /NK1.1 + were recognized as NK cell populations.
  • the percentages of these cells types were acquired by flow cytometry and data was analyzed by FlowJo software.
  • mice bearing B16F10/luc2 tumors of each group were intraperitoneally injected with 200 ⁇ l of 150 mg/kg D-luciferin in PBS, and anesthetized with 1-2%isoflurane 10 min before imaging. Mice were then subjected onto the imaging platform, and continuously exposed to 1-2%isoflurane throughout the acquisition time.
  • the luc2 signal was collected by a bioluminescence imaging instrument (IVIS50 Imaging System; Xenogen) once per week, with the acquisition time as 1 min. Regions of interest (ROI) were around the tumors, and quantified with the Living Image software as photons/s/cm 2 /sr.
  • Formalin-fixed and paraffin-embedded tissues from mice were subjected to IHC staining as follows. Sections of paraffin-embedded tumor tissue on slides were deparaffinized in xylene, rehydrated with serial decreasing concentrations of ethanol (0, 30, 70, 100%H 2 O) , and then incubated in 3%H 2 O 2 for 10 min. After washing, the slides were blocked with 5%normal goat serum for 5 min, and followed by incubation with the indicated primary antibodies in a dilution of 1: 100-500 at 4°C overnight. Finally , the slides were counterstained with hematoxylin. At least three slides from each group were used for IHC staining. Images from the slides were acquired by a light microscope (Nikon ECLIPSE Ti-U) at 200 ⁇ magnification, and quantified by ImageJ software.
  • Mouse sera were collected, and secreted proteins IL-15 and VEGF were assayed by ELISA following the manufacturer’s instruction for each corresponding ELISA assay (Elabscience) .
  • the ELISA readings were determined at OD 450 using a microplate reader (SpectraMax iD3 microplate reader; Molecular Devices) .
  • FIG. 1A The structures of the present recombinant polypeptides including IL-15, IL-15-ABD, and IL-15-ABP, are depicted in FIG. 1A; while the pharmacokinetics of each recombinant polypeptide are depicted in in FIGs 1B and 1C.
  • IL-15-ABD lasted for about 3,000 min in circulation, with the concentration of IL-15 at 3,000 min being about 10,000 pg/ml (FIG 1B) .
  • the overall half-life of IL-15-ABD was calculated to be about 552.7 min.
  • IL-15-ABP and IL-15 they respectively lasted for about 1,440 min and 240 min in circulation (FIG 1C) .
  • the overall half-life of IL-15-ABD and IL-15 was independently about 560 min and 44 min.
  • the overall pharmacokinetics data were shown in Table 1.
  • each recombinant polypeptide was incubated with the sera containing albumin from human, dog, or mouse, and the results are depicted in FIGs. 2A to 2C, respectively.
  • both IL-15-ABD and IL-15-ABP showed a binding activity to serum albumin, albeit in different level.
  • IL-15-ABD in various concentrations consistently exhibited a greater level of binding to serum albumin than that of IL-15-ABP.
  • the binding ability of IL-15-ABD in concentrations of 100, 200, or 300 nM to human serum albumin is higher than that of IL-15-ABP in the same concentrations.
  • the K d for IL-15-ABD to human serum albumin was about 3.040 nM, while that of IL-15-ABP was about 169.3 nM. Similar results were found in the dog serum albumin, and in the mouse serum albumin.
  • the K d for IL-15-ABD to dog serum albumin was about 2.252 nM, while that of IL-15-ABP was 78.44 nM.
  • the K d for IL-15-ABD to mouse serum albumin was about 2.793 nM, and that of IL-15-ABP was about 33.73 nM.
  • the binding data were shown in Table 2.
  • Example 1.1 To investigate if the recombinant polypeptide of Example 1.1 still possesses the biological activity of its natural counterpart, the ability of the recombinant polypeptide of Example 1.1 to stimulate cell growth was tested. As depicted in FIG. 2D, IL-2 was adopted to serve as a positive stimulation that induced cell growth, and the relationship between IL-2 in various concentrations and the corresponding cell growth (represented as OD 450 value) had been plotted as a standard curve for reference.
  • IL-15 5 ng/ml
  • IL-15-ABP 5 ng/ml
  • the abilities of IL-15 and IL-15-ABP (5 ng/ml) in stimulating cell growth corresponded to 8.1 U/ng and 5.2 U/ng, respectively, which is an indication that IL-15-ABP could stimulate cell growth as its natural counterpart does.
  • Example 1.1 has prolonged half-life while retaining its binding specificity and biological activity.
  • Example 2 Effect of the recombinant polypeptide of Example 1.1 on tumor growth and metastasis
  • Example 1.1 The ability of the recombinant polypeptide of Example 1.1 to inhibit tumor growth and metastasis were investigated in the present Example using both melanoma and colon cancer animal models.
  • IL-15-ABP In melanoma model, IL-15-ABP exhibited apparent inhibition on tumor growth, as compared with PBS control and IL-15 (FIG. 3A) , in which the tumor size of the IL-15-ABP group was around 500 mm 3 , while that of PBS group and IL-15 group both were around 1,000 mm 3 . Namely, the tumor size of the IL-15-ABP group was about half of that of both PBS and IL-15 groups.
  • IL-15 both in dose of 60,000 IU and 200,000 IU
  • IL-15-ABP 60,000 IU
  • IL-15-ABP 200,000 IU
  • mice On D28, mice were sacrificed, and the tumor weight of the tumors within the intestines was measured. As shown in FIG. 3D, tumors in the PBS groups were about 6 g in weight, while that in the IL-15-ABP group were about 0.5 g in weight.
  • the body weight (BW) and tumor weight (TW) of the test animals were recorded and the ratio of TW/BW in each group was calculated (Table 3) .
  • Mice in the PBS group in general possessed larger tumor with TW/BW ratio ranged from 21.21 to 31.03, except one mouse had a smaller tumor (TW/BW ratio was about 7.66) .
  • mice in the IL-15-ABP group had smaller tumor, in which the TW/BW ratio fell in the range of 0.00-0.21, except one mouse whose TW/BW ratio was about 10.07, still significantly lower than that in the PBS group.
  • the recombinant polypeptide of Example 1.1 can inhibit the growth and metastasis of melanoma.
  • Example 1.1 suppressed growth and metastasis of colon cancers
  • mice bearing CT26 tumor cells were given specified treatments, and the results indicated that tumor volume from the IL-15-ABD group was smaller than that from the control or IL-15 group since day 9 after treatment (FIG. 4A) . Also, tumor weight measured on day 18 after treatment showed that the mice in IL-15-ABD group had the lowest tumor weight, which was about one third or one half to that of the control or the IL-15 group, independently (FIG. 4B) .
  • Example 1.1 the ability of the recombinant polypeptide of Example 1.1 in inhibiting colon cancer metastasis was also examined.
  • the numbers of the tumor lumps in the lung of the mice administered with specified treatments were counted, and the results are illustrated in FIG. 4C, in which the numbers of the tumor lumps in the IL-15-ABD group was obviously lower than that of the PBS and the IL-15 groups.
  • the numbers of the tumor lumps in the lung, colon, and liver of the mice were counted as well, and the results were illustrated in FIG. 4D. Similar to the results of FIG. 4C, the result of FIG. 4D also confirmed that, as compared with the PBS or IL-15 groups, the number of tumor lumps was obviously lower in the IL-15-ABD group.
  • these data suggest that IL-15-ABD is more effective in suppressing colon cancer growth and metastasis as compared with that of IL-15.
  • Example 1.1 To delineate the underlying mechanisms that the recombinant polypeptide of Example 1.1 has exerted to cause tumor growth suppression, especially via immune regulation, the changes in immune cell population were investigated.
  • one of the immunosuppressive cells, MDSCs, as recognized by CD11b + /GR-1 + was examined by flow cytometry. As illustrated in FIGs. 5A to 5B, percentage of the CD11b + /GR-1 + cell populations decreased in the IL-15-ABD group both in bone marrow and in spleen, as compared with that of the control and IL-15 groups.
  • Another immunosuppressive cell, T reg cell, as recognized by CD4 + /CD25 + /FOXP3 + was also examined.
  • the CD4 + /CD25 + /FOXP3 + cell populations prominently fell down in the IL-15-ABD group both in TDLN and in spleen, as compared with that of the control and IL-15 groups (FIGs. 5C to 5D) . Further, it has been noticed that the population of T reg cells in the IL-15-ABD group decreased to a level that reached one half of the control group, and was even lower than that of the IL-15 group as well.
  • CD8 + T cell two immune cells, including CD8 + T cells and NK cells, which represent adaptive and innate immunity, were also examined.
  • CD8 + T cell it was found that the overall population of CD8 + T cells increased in the IL-15-ABD group both in TDLN and in spleen, and the increase was about two-folds as compared to that of the control group (FIGs. 5E to 5F) .
  • NK cells as recognized by CD3 - /CD49b + , CD3 - /CD335 + , and CD3 - /CD49b + /CD335 + , were also effectively induced in the IL-15-ABD group; numbers of the CD3 - /CD49b + , CD3 - /CD335 + , and CD3 - /CD49b + /CD335 + cell population were all greatly elevated in the IL-15-ABD group than those in the control or IL-15 groups (FIG. 5G) . Decrease in the mice body weight was found in the IL-15 group (FIG. 5H) , suggesting that long-term treatment of IL-15 may cause adverse effects on normal physiology.
  • Example 3 The combination therapy exhibited an improved effect on treating cancers
  • Example 1.1 Whether the effect the recombinant polypeptide of Example 1.1 on a subject may be improved by co-administering to the subject an additional therapy (e.g., immunotherapy) was examined in this Example.
  • an additional therapy e.g., immunotherapy
  • an anti-PD-L1 antibody was used with the recombinant polypeptide of Example 1.1 in colon cancer and melanoma animal models. Results are provided in FIGs. 6A to 6J.
  • FIGs. 6A to 6B indicated that as compared with the control group, the administration of IL-15-ABD or the anit-PDL1 antibody efficiently suppressed the tumor growth in both the colon cancer model (FIG. 6A) and in the melanoma model (FIG. 6B) . It is noted that, in both animal models, the growth of tumor was almost completely suppressed in the combination treatment group (i.e., animals receiving both IL-15-ABD and the anit-PDL1 antibody) (FIGs. 6A to 6B) . Consistent results were observed in the tumor weight measurements on day 18, in which co-administration of IL-15-ABD and anti-PD-L1 antibody almost completely suppressed the tumor growth (FIGs. 6C to 6D) .
  • Example 1.1 To study the possible immune responses involved in tumor suppression caused by the recombinant polypeptide of Example 1.1 or the combination therapy, changes in the populations of immune cells (such as CD8 + T cells and NK cells) and immunosuppressive cells (such as T reg cells and MDSCs) , were investigated in this Example.
  • immune cells such as CD8 + T cells and NK cells
  • immunosuppressive cells such as T reg cells and MDSCs
  • CD8 + T cells which express IFN- ⁇ and IL-2 (as marked as CD8 + /IFN- ⁇ + or CD8 + /IL-2 + cells, respectively) .
  • CD8 + /IFN- ⁇ + or CD8 + /IL-2 + cells were determined by flow cytometry.
  • Cells were extracted from TDLN and spleen of the mice treated with specified treatments, and then subjected to flow cytometry to analyze the percentage of the CD8 + /IFN- ⁇ + or CD8 + /IL-2 + cell populations within the samples.
  • the percentage of the CD8 + /IFN- ⁇ + T cells in both the TDLN and spleen of the mice in the IL-15-ABD group was higher than those in the control group, while the percentage of the CD8 + /IFN- ⁇ + T cells was even higher in the combination therapy group (FIGs. 7A to 7B) .
  • the percentage of the CD8 + /IL-2 + T cells in both the TDLN and spleen of the mice in the IL-15-ABD group was higher than that in the control group, and the percentage was even higher in the combination therapy group (FIGs. 7C to 7D) .
  • mice treated with specified treatments in both the colon cancer and melanoma models were also examined.
  • NK cell markers i.e., CD3 - /CD49b + , CD3 - /CD335 + , and CD3 - /CD49b + /CD335 +
  • results are depicted in FIG. 8A.
  • treatment of IL-15-ABD significantly induced the population of the NK cells in the spleen of the mice, while co-administration of IL-15-ABD and the anti-PD-L1 antibody further enhanced the induction effect of IL-15-ABD on NK cell populations (FIG. 8A) .
  • NK cell markers i.e., CD3 - /CD49b + , CD3 - /NK1.1 + , and CD3 - /CD49b + /NK1.1 + .
  • IL-15-ABD treatment increased the number of the CD3 - /CD49b + , CD3 - /NK1.1 + , and CD3 - /CD49b + /NK1.1 + NK cells in the spleen of the mice as compared with those of the control group, and the co-administration of IL-15-ABD and the anti-PD-L1 antibody further enhanced the induction effect of IL-15-ABD on NK cell populations (FIG. 8B) .
  • NK cells The functionality of these NK cells was also tested by measuring the level of IFN- ⁇ expressed by these functional NK cells. Results are provided in FIGs. 8C to 8F. It was found that the amount of the CD3 - /CD335 + /IFN- ⁇ + and CD3 - /NK1.1 + /IFN- ⁇ + cells significantly increased in the combination treatment group in both models, indicating the activity of IFN- ⁇ was prominently induced in the combination treatment group (FIGs. 8C to 8D) . In addition, the protein expression levels of CD49b and IFN- ⁇ in both colon cancer and melanoma were further confirmed by IHC staining. According to FIGs. 8E to 8F, the protein expression levels of CD49b and IFN- ⁇ increased in the treatment groups.
  • VEGF as an inflammation indicator
  • FIG. 8G the secreted level of VEGF in mouse serum was considerably lower in the combination therapy group, suggesting the combination therapy may help decrease inflammation within the mice.
  • the secreted level of IL-15 was effectively induced by the combination therapy (FIG. 8H) .
  • T reg cell populations (as marked as CD4 + /CD25 + /FOXP3 + cells) in both tumor models were determined.
  • the population of MDSCs (as marked as CD11b + /Gr-1 + cells) within the bone marrow and spleen of the mice in both tumor models were also analysed by flow cytometry.

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Abstract

L'invention concerne un polypeptide recombiné comprenant un premier polypeptide et un second polypeptide lié de manière fonctionnelle à l'extrémité N-terminale ou C-terminale du premier polypeptide, le premier polypeptide ayant une séquence d'acides aminés d'au moins 85 % d'identité à IL-15, et le second polypeptide ayant une séquence d'acides aminés d'au moins 85 % d'identité au domaine de liaison à l'albumine (ABD) ou un peptide de liaison à l'albumine (ABP). En outre, l'invention concerne des compositions pharmaceutiques comprenant ledit polypeptide recombiné pour la prévention et/ou le traitement d'un cancer chez un sujet en ayant besoin. L'invention concerne également des méthodes de prévention et/ou de traitement du cancer à l'aide de ladite composition pharmaceutique.
PCT/CN2020/109183 2019-08-16 2020-08-14 Polypeptides recombinés et leurs utilisations WO2021031998A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101492508A (zh) * 2009-01-24 2009-07-29 温州医学院 链亲和素/白细胞介素15融合蛋白
WO2013192546A1 (fr) * 2012-06-22 2013-12-27 Cytomx Therapeutics, Inc. Anticorps activables ayant des fragments stériques ne se liant pas et leurs procédés d'utilisation
WO2015109124A2 (fr) * 2014-01-15 2015-07-23 Kadmon Corporation, Llc Agents immunomodulateurs
WO2017132555A1 (fr) * 2016-01-29 2017-08-03 Vedantra Pharmaceuticals, Inc. Polypeptides alk et leurs procédés d'utilisation
WO2018151868A2 (fr) * 2017-02-16 2018-08-23 Sonnet Bio Therapeutics Protéines de fusion à domaine de liaison à l'albumine

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101492508A (zh) * 2009-01-24 2009-07-29 温州医学院 链亲和素/白细胞介素15融合蛋白
WO2013192546A1 (fr) * 2012-06-22 2013-12-27 Cytomx Therapeutics, Inc. Anticorps activables ayant des fragments stériques ne se liant pas et leurs procédés d'utilisation
WO2015109124A2 (fr) * 2014-01-15 2015-07-23 Kadmon Corporation, Llc Agents immunomodulateurs
WO2017132555A1 (fr) * 2016-01-29 2017-08-03 Vedantra Pharmaceuticals, Inc. Polypeptides alk et leurs procédés d'utilisation
WO2018151868A2 (fr) * 2017-02-16 2018-08-23 Sonnet Bio Therapeutics Protéines de fusion à domaine de liaison à l'albumine

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN QI-XIN , CHEN LING-NA , CHEN ZHENG-HUA: "Gene Cloning and Construction of Prokaryotic and Plant Expression Vectors of IL15/PAP Fusion Protein", JOURNAL OF HENAN AGRICULTURAL UNIVERSITY, vol. 40, no. 3, 30 June 2006 (2006-06-30), pages 301 - 306, XP055782357, ISSN: 1000-2340, DOI: 10.16445/j.cnki.1000-2340.2006.08.019 *
HOPP J; HORNIG N; ZETTLITZ K A; SCHWARZ A; FUSS N; MULLER D; KONTERMANN R E: "The effects of affinity and valency of an albumin-binding domain (ABD) on the half-life of a single-chain diabody-ABD fusion protein.", PROTEIN ENGINEERING DESIGN AND SELECTION, vol. 23, no. 11, 1 November 2010 (2010-11-01), pages 827 - 834, XP055054496, ISSN: 1741-0126, DOI: 10.1093/protein/gzq058 *
HUANG HAOMIN; LUO YUYING; BARADEI HANNA; LIU SHAN; HAENSSEN KENESHIA K; SANGLIKAR SUPRIYA; KUMAR SENTHIL; CINI JOHN: "A novel strategy to produce high level and high purity of bioactive IL15 fusion proteins from mammalian cells.", PROTEIN EXPRESSION AND PURIFICATION, vol. 148, 26 March 2018 (2018-03-26), pages 30 - 39, XP085392323, ISSN: 1046-5928, DOI: 10.1016/j.pep.2018.03.010 *

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