Recombinant polypeptide, pharmaceutical composition and application thereof

Abstract

Provided are recombinant polypeptides, pharmaceutical compositions and uses thereof, and methods of preventing or treating cancer. The recombinant polypeptide comprises a first subunit and a second subunit, the first subunit comprises all or fragments of the amino acid sequence of programmed death ligand 1 (PD-L1), and the second subunit comprises all or fragments of the amino acid sequence of a melanoma preferential expression antigen (PRAME). The dual antigen cancer vaccine co-targeting PD-L1 and PRAME effectively prevents, ameliorates or treats cancer and / or inhibits tumor growth in the prophylactic and therapeutic aspects.

Description

References to sequence lists

[0001] This application contains a sequence list that has been filed electronically in ST.26 (xml) format and is hereby incorporated by reference in its entirety. The ST.26 copy created on June 16, 2023 is named “C903.001.PROUS.xml” and is 25.6 kilobytes in size. Technical Field

[0002] This application relates to recombinant peptides, pharmaceutical compositions, kits, methods, and uses for the prevention or treatment of cancer, or for the prevention or treatment of one or more complications of cancer treatment. Background Technology

[0003] Cancer is one of the leading causes of death worldwide. While some treatments are available, many patients do not respond to current therapies or preventative measures. Furthermore, current treatments only manage a subgroup of disease symptoms, are costly, and are not effective in the long term. Therefore, new drug candidates for the prevention or treatment of cancer are urgently needed. Summary of the Invention

[0004] This article discloses novel recombinant peptides, compositions, kits, methods and uses for the prevention or treatment of cancer; methods for preparing recombinant peptides and compositions; methods for using compositions; and intermediates used in the preparation of recombinant peptides and compositions.

[0005] In some embodiments, a recombinant polypeptide is provided, the recombinant polypeptide comprising a first subunit and a second subunit, the first subunit comprising all or a fragment of the amino acid sequence of programmed death-ligand 1 (PD-L1), and the second subunit comprising all or a fragment of the amino acid sequence of melanoma preferentially expressed antigen (PRAME).

[0006] In some embodiments, recombinant fusion protein vaccines (PD-PR and PD-PR-GM) containing PD-L1 and PRAME, with or without GM-CSF, are expressed as inclusion bodies in an E. coli-based system. After purification, the fusion protein vaccines are formulated with Toll-like receptor 9 agonist CpG oligodeoxynucleotides as adjuvants for vaccination and aluminum hydroxide.

[0007] This invention offers numerous advantages. In some embodiments, novel biantigen cancer vaccines co-targeting PD-L1 and PRAME have demonstrated tumor-inhibiting effects in both prophylactic and therapeutic syngeneic tumor mouse models. Both vaccine designs exhibit significant tumor growth inhibition in both preventative and therapeutic applications.

[0008] In some embodiments, to further investigate the tumor growth inhibitory effect of the provided recombinant peptide and fusion protein vaccine, the specific T-cell responses and antibody responses to different cancer antigens in mice vaccinated with PD-PR were examined by Elispot and ELISA, respectively. In some embodiments, the provided recombinant peptide and fusion protein vaccine can induce antigen-specific immune responses against mouse PD-L1 and human PRAME, and the immune responses can also cross-react with human PD-L1. Attached Figure Description

[0009] Figure 1 This is a schematic diagram and amino acid sequence of an exemplary PD-PR fusion protein according to an exemplary embodiment. Bold: PD-L1 sequence; Underlined: Connector sequence; Non-bold: PRAME sequence; Underlined and italicized: HisTag sequence.

[0010] Figure 2 It is based on Figure 1 A schematic diagram of the expression plasmid map of the fusion protein vaccine PD-PR, an exemplary embodiment of the present invention.

[0011] Figure 3 This is a schematic diagram and amino acid sequence of the PD-PR-GM fusion protein according to another exemplary embodiment. Bold: PD-L1 sequence; Underlined: Connector sequence; Non-bold: PRAME sequence; Italic: GM-CSF sequence; Underlined and italicized: HisTag sequence.

[0012] Figure 4 It is based on Figure 3 A schematic diagram of the expression plasmid map of the fusion protein vaccine PD-PR-GM, an exemplary embodiment of the present invention.

[0013] Figure 5 This is a schematic diagram of a plasmid map of PRAME antigen expression in H-cells according to an exemplary embodiment.

[0014] Figure 6 This is an exemplary vaccination schedule for a preventive tumor model study according to an exemplary embodiment.

[0015] Figure 7 This is an exemplary vaccination schedule for a therapeutic tumor model study according to an exemplary embodiment.

[0016] Figure 8A and Figure 8BThese are gel photographs showing, according to an exemplary embodiment, the expression of PD-PR (A) and PD-PR-GM (B) in bacterial cell lysates before and after IPTG induction, analyzed by Coomassie blue staining and Western blotting (immunoblotting) using an anti-His antibody, respectively. M: biomarker; NI: before IPTG induction; I: after IPTG induction.

[0017] Figures 9A to 9C These are gel photographs showing, according to an exemplary embodiment, the analysis of bacterial cell lysates and purified fusion protein vaccines by Western blotting using anti-PD-L1 antibody (A), anti-PRAME antibody (B), and anti-GM-CFS antibody (C), respectively. M: Marker; Lane 1: BL21 (DE3) cell lysate; Lane 2: BL21 (DE3) cell lysate expressing PD-PR; Lane 3: Rosetta (DE3) cell lysate expressing PD-PR-GM; Lane 4: Purified BSA; Lane 5: Purified PD-PR; Lane 6: Purified PD-PR-GM.

[0018] Figure 10 This is a gel photograph showing Western blot analysis of cell lysates prepared from pcDNA-PRAME-transfected mouse HCC cells expressing PRAME. Arrows indicate the electrophoretic positions of PRAME in the blot according to an exemplary embodiment. Lane-1: Marker; Lane-2H: Parental H-cell lysate; Lanes-3 to 10: Cell lysates from different pcDNA-PRAME-transfected clones; Lane-11: Cell lysate from H-clone-37.

[0019] Figure 11 This is a gel photograph showing the analysis of tumor cells and tumor tissue lysates in mouse HCC tumor tissue expressing PRAME by Western blot analysis. Arrows indicate the electrophoretic positions of PRAME in the blot according to an exemplary embodiment. M: Marker; Lane 1: A549 cell lysate; Lane 2: HepG2 cell lysate; Lane 3: Parental H-cell lysate; Lane 4: H-clone-37 cell lysate; Lane 5: Parental H-cell tumor tissue lysate; Lane 6: H-clone-37 tumor tissue lysate.

[0020] Figures 12A to 12C These are graphs showing the changes in body weight after vaccination with PD-PR (A), PD-PR-GM (B), and PBS control (C), respectively. Each line represents data for a single mouse, and the blue arrows indicate the vaccination time of the exemplary embodiment.

[0021] Figures 13A to 13DThe results of the experimental protocol for a preventive HCC tumor model study according to an exemplary embodiment are shown. A graph is plotted showing the individual tumor growth and mean tumor volume (D) of the PBS control group (A), the PD-PR vaccination group (B), and the PD-PR-GM vaccination group (C).

[0022] Figures 14A to 14E Display according to, for example Figure 7 Results of the experimental protocol for a therapeutic tumor model study, as shown in the exemplary embodiment, are presented. Individual tumor growth data and the mean tumor volume (D) for the PBS control group (A), the PD-PR vaccination group (B), and the PD-PR-GM vaccination group (C) are plotted. Survival curves after cancer cell implantation in each vaccination group are shown. Figure 14E Circle: PBS control group; Square: PD-PR vaccine group; Triangle: PD-PR-GM vaccine group.

[0023] Figure 15 This is an exemplary vaccination and sample collection schedule for an immune response study according to another exemplary embodiment.

[0024] Figure 16 This is a gel photograph showing Western blot analysis of cell lysates prepared from pcDNA-PRAME-transfected mouse melanoma cells expressing PRAME. Arrows indicate the electrophoretic positions of PRAME in the blot according to an exemplary embodiment. Lane-1: Marker; Lane-2B: Parental B16 / F10 lysate; Lane-3: Cell lysate from B16 / F10#18; Lanes-4 to 7: Cell lysates from different pcDNA-PRAME-transfected clones.

[0025] Figure 17 This is a gel photograph showing the analysis of tumor cells and tumor tissue lysates by Western blotting of mouse melanoma tumor tissue expressing PRAME. The dashed boxes indicate the electrophoretic positions of PRAME in the blot according to an exemplary embodiment. M: Marker; Lane 1: Parental B16 / F10 cell lysates; Lane 2: B16 / F10#18 cell lysates; Lanes 3-8: Parental B16 / F10 tumor tissue lysates; Lanes 9-14: B16 / F10#18 tumor tissue lysates.

[0026] Figures 18A to 18D Display according to similar Figure 6 Results of the experimental protocol for a preventive melanoma tumor model study, an exemplary embodiment of which are presented. Individual tumor growth and the mean tumor volume (D) of the adjuvant control group (A), the PD-PR vaccination group (B), and the PD-PR-GM vaccination group (C) are plotted. Detailed Implementation definition

[0027] As used herein and in the claims, the terms “comprising” (or any related form such as “comprise” and “comprises”), “including” (or any related form such as “include” or “includes”), “containing” (or any related form such as “contain” or “contains”) mean to include the following elements, but do not exclude other elements. It should be understood that for each embodiment in which the terms “comprising” (or any related form such as “comprise” and “comprises”), “including” (or any related form such as “include” or “includes”), or “containing” (or any related form such as “contain” or “contains”) are used, this disclosure / application also includes alternative embodiments in which the terms “comprising”, “including”, or “contains” are replaced with “consistently consisting of” or “consisting of”. These alternative embodiments using the terms "composed of" or "substantially composed of" are understood to be smaller-scale embodiments that "comprise", "include", or "contain" the embodiments.

[0028] For example, alternative embodiments of "a composition comprising A, B, and C" would be "a composition consisting of A, B, and C" and "a composition consisting essentially of A, B, and C". This disclosure / application includes those embodiments even if the latter two are not explicitly stated. Furthermore, it should be understood that the scope of the three embodiments listed above is different.

[0029] For clarity, “comprising,” “including,” and “containing,” as well as any related forms, are open-ended terms that allow for additional elements or features beyond the specified essential elements, while “composes of” is a closed-ended term that is limited to the elements listed in the claims and excludes any elements, steps, or ingredients not specified in the claims.

[0030] For clarity, "characterized as" or "characterized in" (along with their related forms as described above) does not limit or change the nature of whether the following list of terms is open or closed. For example, in a claim relating to "a composition comprising A, B, C and characterized in D, E, and F," elements D, E, and F remain open-ended terms, and the claim is intended to include other elements due to the use of the word "comprising" preceding the claim.

[0031] The phrase "consistently of..." limits the scope of the claim to specific materials, components, or steps ("essential elements") that do not materially affect one or more essential features of the claimed invention. In some embodiments, essential features are one or more basic and novel features of the claimed invention. For example, in some embodiments, the essential elements of the compositions disclosed herein may be one or more active ingredients, such as the recombinant peptides described herein. Even if the composition contains additional excipients, such embodiments of "consistently of compound A" still include compositions having the aforementioned additional excipients, provided that the additional excipients do not materially affect the essential features of the compound, such as the ability of the recombinant peptide to treat cancer.

[0032] As used herein, the singular forms “a / an” and “the” are intended to also include the plural forms unless the context clearly indicates otherwise. When a range is mentioned in the specification, the range is understood to include every discrete point within the range. For example, 1-7 means 1, 2, 3, 4, 5, 6, and 7.

[0033] As used herein, the term “about” is understood to mean within the normal tolerance range in this field and not exceeding ±10% of the specified value. For example only, about 50 means all values ​​from 45 to 55, inclusive. As used herein, the phrase “about” also includes the specific value; for example, about 50 includes 50.

[0034] As used herein and in the claims, an "effective amount" is an amount that effectively achieves the desired effect of at least a measurable amount. For example, the amount can effectively elicit an immune response against a pathogen carrying a polypeptide of interest, and / or it can effectively elicit a protective response against the pathogen. In some embodiments, the amount can effectively elicit an immune response against cancer or tumor.

[0035] As used herein and in the claims, “subject” means an animal, such as a mammal, including, but not limited to, primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In some embodiments, the desired subject is a human.

[0036] As used herein, the term "pharmaceutical composition" or "composition" refers to a formulation containing one or more active pharmaceutical ingredients. In some instances, a pharmaceutical composition contains one or more peptides targeting PD-L1 and PRAME, and optionally a pharmaceutically acceptable carrier. In some instances, a pharmaceutical composition is used as a medicine or vaccine capable of inducing an immune response in a subject in need. In some instances, a pharmaceutical composition is also referred to as a "vaccine," "peptide vaccine," or "peptide cancer vaccine."

[0037] As used herein, the term "PD-L1" refers to a ligand of programmed cell death protein 1, which typically contains an extracellular domain (ECD), a transmembrane domain, and an intracellular cytoplasmic domain. In some instances, the provided pharmaceutical compositions contain one or more peptides or fragments thereof that target one or more epitopes specific to one or more of these domains of PD-L1. PD-L1 proteins can be derived from animals such as humans, mice, rats, hamsters, insects, cattle, pigs, sheep, monkeys, goats, dogs, cats, and camels. In some instances, the PD-L1 protein is mouse or human PD-L1, or a functional fragment or functional homolog thereof. In some instances, the term "PD-1" refers to programmed cell death protein 1, a cell surface receptor on cells (such as T cells and B cells) that regulates immune responses through interaction with its ligands, including PD-L1.

[0038] As used herein and in the claims, the terms “prevent,” “preventing,” “preventive / preventative,” or “prevention” refer to a method of reducing the risk of the onset, recurrence, or spread of a disease or disorder or one or more symptoms thereof.

[0039] As used herein, the terms “treat,” “treating,” or “treatment” refer to the following methods of: relieving, eliminating, or reducing the symptoms of a disease or condition in a preventive and / or therapeutic manner; preventing additional symptoms; reducing or preventing the basal metabolic cause of the symptoms; inhibiting the disease or condition; halting the progression of the disease or condition; alleviating the disease or condition; causing the remission of the disease or condition; relieving the condition caused by the disease or condition; or terminating the symptoms of the disease or condition.

[0040] As used herein, the term "variant sequence" refers to a nucleic acid or polypeptide sequence that exhibits a degree of identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with a reference or wild-type nucleic acid or polypeptide sequence. For example, when compared to a reference or wild-type sequence, a variant sequence has one or more additions, deletions, insertions and / or substitutions, or other modifications (e.g., codon encoding optimization). In some instances, variant sequences also include sequences of functional homologs.

[0041] As used herein, the term "functional homolog" refers to a polypeptide that exhibits some degree of sequence identity with a reference or wild-type sequence and possesses certain aspects of the functionality of the reference or wild-type polypeptide. For example, functional homologs of PD-L1 have the ability to exhibit similar immune responses to cells expressing PD-L1 and / or to prevent or treat PD-1 or PD-L1-related diseases or conditions (such as cancer).

[0042] As used herein, the term "PRAME" refers to a melanoma preferentially expressed antigen, which is encoded by the PRAME gene and expressed by melanoma, and recognized by cytolytic T lymphocytes.

[0043] As used in this article, the term "GM-CSF" refers to granulocyte-macrophage colony-stimulating factor, a monomeric glycoprotein secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells, or fibroblasts, which functions as a cytokine.

[0044] As used herein, the term "polypeptide" or "peptide" refers to a chain of two or more amino acids linked together by peptide bonds. In some instances, a polypeptide refers to an amino acid sequence having an amino acid residue spanning a specific region of the PD-L1 protein or its functional homologs, or a variant sequence having at least 75% or higher sequence identity. In some instances, having at least 75% sequence identity means having at least about 75%, 80%, 85%, 90%, 95%, 99% sequence identity or even higher (e.g., 100%). In some instances, a polypeptide contains multiple peptides chemically linked together. In some instances, a polypeptide comprises a synthetic long peptide (SLP). In some instances, a polypeptide comprises a fusion protein resulting from the expression of two or more genes linked together, said genes originally encoding a single peptide. In some instances, a polypeptide is expressed as an inclusion body.

[0045] As used herein, the term "recombinant peptide" refers to a peptide produced through recombinant technology. In some embodiments, the recombinant peptide is heterologously expressed in a host organism.

[0046] As used herein, the term "fusion protein" refers to a protein produced by the expression of two or more peptide genes linked together, which originally encode a single peptide.

[0047] As used herein, the terms “nucleic acid system,” “nucleic acid construct,” or “vector” are used interchangeably and generally refer to genetically engineered nucleic acids or gene circuits comprising one or more nucleic acid sequences that encode one or more polypeptides or fragments thereof, or complementary sequences thereof, and optionally one or more promoters, enhancers, terminators, adapters, polyA tails, operable adapters, multiple cloning sites, markers, and / or other regulatory elements. The nucleic acid sequences and promoters can be placed in any order and can be located in the same or different molecules.

[0048] As used herein and in the claims, the term "pharmaceutically acceptable carrier" or "carrier" refers to a molecule or substance, such as a protein, that serves as a medium or mediator for delivering a drug or active ingredient in a pharmaceutical composition. In some instances, a pharmaceutically acceptable carrier is conjugated to one or more active ingredients, such as peptides as described herein.

[0049] As used herein, the term "adjuvant" refers to a molecule or substance that is mixed with an active ingredient when administered to a subject in need to enhance their immune response.

[0050] As used herein, the term "checkpoint inhibitor" refers to a drug or compound that binds to or targets inhibitory immune checkpoint molecules and blocks their activity.

[0051] As used herein, the term "cancer" refers to a group of diseases involving the abnormal growth of cells that can invade or spread to other parts of the body, such as, but not limited to, the skin, breast, prostate, lungs, liver, kidneys, pancreas, stomach, or intestines. In some instances, cancer can include non-metastatic or metastatic tumors, such as, but not limited to, melanoma, lymphoma, leukemia, fibrosarcoma, etc.

[0052] As used herein, the term "linker" refers to an amino acid sequence of two or more amino acids that can operatively link the components of a fusion protein directly or indirectly. Linker lengths can range from, for example, 2 to 100 amino acids, such as those between 2 and 50 amino acids, for example, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, the linker sequence is or contains three repeating sequences (GGGGS x 3) having four glycine residues and one serine residue. Other suitable linker sequences may be used instead.

[0053] In some embodiments, a pharmaceutically acceptable carrier is selected from the group consisting of keyhole hemocyanin (KLH), bovine serum albumin (BSA), human serum albumin (HSA), fetal bovine serum (FBS), liposomes, cyclodextrin, polyethylene glycol (PEG), nanoparticles, microspheres, hydrogels, and combinations thereof. In some embodiments, a pharmaceutically acceptable carrier is or contains keyhole hemocyanin (KLH).

[0054] In some embodiments, the composition further comprises one or more excipients, adjuvants, other checkpoint inhibitors, vaccines or therapeutics, and combinations thereof.

[0055] In some embodiments, the checkpoint inhibitor targets one or more checkpoint proteins selected from the group consisting of: PDL1, PD1, cytotoxic T-lymphocyte antigen 4 (CTLA4), T-cell immune receptor having Ig and ITIM domains (TIGIT), T-cell immunoglobulin and mucin domain 3 (Tim3), lymphocyte activation gene 3 (LAG3), B7 homolog 3 (B7-H3), B7 homolog 4 (B7-H4), and combinations thereof. Checkpoint inhibitors targeting one or more other checkpoint proteins known in the art may also be added to the composition.

[0056] In some embodiments, the adjuvant is selected from the group consisting of: CpG ODN 1826 (CpG), aluminum hydroxide gel (Alum), aluminum phosphate, incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA), NH2, MF59, MVF, Montanide ISA TM AS01, AS03, AS04, GM-CSF, cytokines, MPL, Matrix-M, pan-DR epitopes (PADRE), and combinations thereof. In some embodiments, the adjuvant comprises or is substantially composed of CpG ODN 1826 and Alum. In some embodiments, the adjuvant comprises or is substantially composed of CFA and IFA. In some embodiments, the adjuvant is CpG ODN 1826 (CpG) and aluminum hydroxide gel (Alum).

[0057] In some embodiments, the composition further comprises a second active ingredient, such as an immunostimulatory substance. For example, the second active ingredient includes checkpoint inhibitors, other vaccines or therapeutics, interleukins, and / or chemotherapeutic agents.

[0058] In some embodiments, a nucleic acid system is provided comprising one or more nucleic acid sequences of the peptides described herein. In some embodiments, the peptides are heterologously expressed in a host organism such as, but not limited to, bacteria (e.g., *Escherichia coli*), yeast (e.g., *Saccharomyces cerevisiae*), and mammalian cells (e.g., *Chinese hamster ovary* cells). In some other embodiments, the nucleic acid sequences are cloned into one or more plasmids and transformed into a host organism (such as *E. coli*); the peptides are then expressed and purified. In some other embodiments, the nucleic acid sequences are sequences encoding corresponding amino acid sequences, derivative sequences thereof, partial sequences thereof, degenerate sequences thereof, codon-optimized sequences thereof, and / or sequences to which they hybridize under stringent conditions.

[0059] In some embodiments, the PD-L1 protein is derived from animals selected from the group consisting of: humans, mice, rats, hamsters, insects, cattle, pigs, sheep, monkeys, goats, dogs, cats, and camels. In some embodiments, PD-L1 is derived from any animal or mammal that expresses the protein. In some embodiments, PD-L1 is derived from mice or humans.

[0060] In some embodiments, the first subunit has at least about 75%, 80%, 85%, 90%, or 95% sequence identity with all or a fragment of NP_068693.1, or a functional homolog thereof. In some embodiments, the first subunit has at least about 75%, 80%, 85%, 90%, or 95% sequence identity with all or a fragment of NP_001254635.1, NP_001300958.1, or NP_054862.1, or a functional homolog thereof.

[0061] In some embodiments, the first subunit contains all or fragments of one or more domain sequences of PD-L1, wherein the domain is an extracellular domain (ECD), a transmembrane domain, and / or an intracellular cytoplasmic domain, or a combination thereof.

[0062] In some embodiments, PRAME is derived from animals selected from the group consisting of: humans, mice, rats, hamsters, insects, cattle, pigs, sheep, monkeys, goats, dogs, cats, and camels. In some embodiments, PRAME is derived from any animal or mammal that expresses the protein. In some embodiments, PRAME is derived from mice or humans.

[0063] In some embodiments, the second subunit has at least about 75%, 80%, 85%, 90%, or 95% sequence identity with all or a fragment of NP_001278644.1, or a functional homolog thereof. In some embodiments, the second subunit has at least about 75%, 80%, 85%, 90%, or 95% sequence identity with all or a fragment of NP_001278644.1, NP_001278645.1, NP_001278646.1, NP_001278648.1, or NP_001305055, or a functional homolog thereof.

[0064] In some embodiments, GM-CSF is derived from animals selected from the group consisting of: humans, mice, rats, hamsters, insects, cattle, pigs, sheep, monkeys, goats, dogs, cats, and camels. In some embodiments, GM-CSF is derived from any animal or mammal that expresses the protein. In some embodiments, GM-CSF is derived from mice or humans.

[0065] In some embodiments, the third subunit has at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with all or a fragment of NP_034099.2 or its functional homologs.

[0066] In some embodiments, the third subunit has at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with all or a fragment of NP_000749.2 or its functional homologs.

[0067] In some embodiments, a recombinant polypeptide is provided, which is substantially encoded by any of the recombinant nucleic acid systems as described herein.

[0068] In some embodiments, a vaccine is provided comprising one or more of the following: a pharmaceutical composition as described herein, a polypeptide expressed by a recombinant nucleic acid system, or a recombinant polypeptide.

[0069] In some embodiments, a kit is provided that includes one or more of a pharmaceutical composition, a recombinant nucleic acid system, or a recombinant peptide as described herein.

[0070] In some embodiments, a method is provided for preventing or treating a disease or symptom associated with the expression of PD-1, PD-L1, and / or PRAME in a subject of need, the method comprising the step of administering to the subject an effective amount of a pharmaceutical composition, recombinant nucleic acid, or recombinant peptide as described herein.

[0071] In some embodiments, the recombinant peptide or pharmaceutical composition as described herein is provided for use in the manufacture of a medicament for the prevention or treatment of a disease or symptom associated with the expression of PD-1, PD-L1, and / or PRAME in a subject of need. In some embodiments, the disease or symptom is cancer.

[0072] In some embodiments, recombinant peptides or pharmaceutical compositions as described herein are provided for use in the prevention or treatment of a disease or symptom associated with the expression of PD-1, PD-L1, and / or PRAME in a subject of need. In some embodiments, the disease or symptom is cancer.

[0073] In some embodiments, administration is performed via subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, or a combination thereof.

[0074] In some embodiments, the method further includes one or more of the following steps: radiotherapy, chemotherapy, surgery, immunotherapy, targeted therapy, hormone therapy, immune checkpoint inhibitor therapy, and / or stem cell transplantation.

[0075] In some embodiments, the disease or symptom is selected from the group consisting of: hepatitis, inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), type 1 diabetes mellitus (T1D), systemic vasculitis, myositis, autoimmune diseases, autoimmune encephalomyelitis, autoimmune hepatitis, Behcet's disease, myasthenia gravis, autoimmune uveitis, Sjögren's syndrome, ankylosing spondylitis, stroke, Alzheimer's disease, multiple sclerosis, cancer, and combinations thereof.

[0076] In some embodiments, the cancer is selected from the group consisting of: liver cancer, skin cancer, colon cancer, bladder cancer, lung cancer, head and neck cancer, gastric cancer, B-cell lymphoma, T-cell lymphoma, pancreatic cancer, bladder cancer, brain cancer, monocytic leukemia, B-cell leukemia, T-cell leukemia, breast cancer, melanoma, small cell lung cancer, non-small cell lung cancer, lung cancer, Hodgkin's lymphoma, kidney cancer, breast cancer, ovarian cancer, esophageal cancer, testicular cancer, colorectal cancer, thyroid cancer, prostate cancer, gastric cancer, cervical cancer, and combinations thereof. In some embodiments, the cancer is liver cancer and / or melanoma.

[0077] In some embodiments, the recombinant peptides described herein have antitumor immune responses and / or specificity against major histocompatibility complex class I (MHCI) or major histocompatibility complex class II (MHCII) domains. Numbered Examples Group 1

[0078] Example 1. A recombinant polypeptide comprising a first subunit and a second subunit, the first subunit comprising all or a fragment of the amino acid sequence of programmed death-ligand 1 (PD-L1), and the second subunit comprising all or a fragment of the amino acid sequence of melanoma preferentially expressed antigen (PRAME).

[0079] Example 2. The recombinant polypeptide as described in Example 1, wherein the first subunit is located upstream of the second subunit.

[0080] Example 3. The recombinant polypeptide as described in any of the preceding examples further comprises a first linker, wherein the first linker is located between the first subunit and the second subunit.

[0081] Example 4. The recombinant polypeptide as described in any of the preceding examples further comprises a third subunit, said third subunit comprising all or a fragment of the amino acid sequence of GM-CSF.

[0082] Example 5. The recombinant polypeptide as described in any of the preceding examples further comprises a second connector, wherein the second connector is located between the second subunit and the third subunit.

[0083] Example 6. The recombinant polypeptide as described in Example 5, wherein the second subunit is located downstream of the first subunit and upstream of the third subunit.

[0084] Example 7. A recombinant polypeptide as described in any of the preceding examples, wherein the first subunit comprises all or a fragment of a mouse PD-L1 polypeptide.

[0085] Example 8. The recombinant polypeptide as described in Example 7, wherein the PD-L1 polypeptide comprises an amino acid sequence, or a fragment or variant thereof, having at least about 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO.:5.

[0086] Example 9. A recombinant polypeptide as described in any of the preceding examples, wherein the second subunit comprises all or a fragment of a human PRAME polypeptide.

[0087] Example 10. The recombinant polypeptide as described in Example 9, wherein the FRAME polypeptide comprises an amino acid sequence, or a fragment or variant thereof, having at least about 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO.:6.

[0088] Example 11. A recombinant polypeptide as described in any of the preceding examples, wherein the third subunit comprises all or a fragment of a mouse GM-CSF polypeptide.

[0089] Example 12. The recombinant polypeptide as described in Example 11, wherein the GM-CSF polypeptide comprises an amino acid sequence, or a fragment or variant thereof, having at least about 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO.:7.

[0090] Example 13. A recombinant polypeptide as described in any of the preceding examples, wherein the first adapter and / or the second adapter comprises an amino acid sequence, or a fragment or variant thereof, having at least about 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO.:8.

[0091] Example 14. A recombinant polypeptide as described in Example 1, wherein the polypeptide is expressed from the nucleic acid sequence of SEQ ID NO.:2 or SEQ ID NO.:4.

[0092] Example 15. The recombinant polypeptide as described in Example 14, wherein the polypeptide is expressed as an inclusion body in Escherichia coli.

[0093] Example 16. A recombinant polypeptide comprising an amino acid sequence, or a fragment or variant thereof, having at least about 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO.:1 or SEQ ID NO.:3.

[0094] Example 17. A pharmaceutical composition comprising a recombinant polypeptide as described in any of the preceding examples and optionally a pharmaceutically acceptable carrier, diluent, and / or adjuvant.

[0095] Example 18. A pharmaceutical composition as described in Example 17, wherein the adjuvant is a Toll-like receptor 9 agonist CpG oligodeoxynucleotide, aluminum hydroxide, or a combination thereof.

[0096] Example 19. A method for preventing or treating cancer by administering to a subject in need a recombinant polypeptide as described in any one of Examples 1-16, or a pharmaceutical composition as described in claim 17 or claim 18.

[0097] Example 20. Use of the recombinant polypeptide as described in any one of Examples 1-16, or the pharmaceutical composition as described in claim 17 or claim 18, for the manufacture of a medicament for the prevention or treatment of cancer.

[0098] Example 21. A recombinant polypeptide as described in any one of Examples 1-16 or a pharmaceutical composition as described in any one of Claims 17-18, for use in the prevention or treatment of cancer. Group 2

[0099] Example 1. A recombinant polypeptide comprising: a first subunit comprising all or a fragment of the amino acid sequence of programmed death-ligand 1 (PD-L1), or a variant sequence thereof having at least 75% sequence identity; and a second subunit comprising all or a fragment of the amino acid sequence of melanoma preferentially expressed antigen (PRAME), or a variant sequence thereof having at least 75% sequence identity.

[0100] Example 2. The recombinant polypeptide as described in Example 1, wherein the first subunit is located upstream of the second subunit.

[0101] Example 3. The recombinant polypeptide as described in any of the preceding examples further comprises a first linker, wherein the first linker is located between the first subunit and the second subunit.

[0102] Example 4. The recombinant polypeptide as described in any of the preceding examples further comprises a third subunit, said third subunit comprising all or a fragment of the amino acid sequence of GM-CSF, or a variant sequence thereof having at least 75% sequence identity.

[0103] Example 5. The recombinant polypeptide as described in Example 4, further comprising a second connector, wherein the second connector is located between the second subunit and the third subunit.

[0104] Example 6. The recombinant polypeptide as described in Example 5, wherein the second subunit is located downstream of the first subunit and upstream of the third subunit.

[0105] Example 7. The recombinant polypeptide as described in any of the preceding examples, wherein the PD-L1 is derived from an animal selected from the group consisting of: human, mouse, rat, hamster, insect, cow, pig, sheep, monkey, goat, dog, cat, and camel.

[0106] Example 8. A recombinant polypeptide as described in any of the preceding examples, wherein the first subunit comprises an amino acid sequence, or a fragment or variant thereof, having at least about 75%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO.:5.

[0107] Example 9. The recombinant polypeptide as described in any of the preceding examples, wherein the PRAME is derived from an animal selected from the group consisting of: human, mouse, rat, hamster, insect, cow, pig, sheep, monkey, goat, dog, cat, and camel.

[0108] Example 10. A recombinant polypeptide as described in any of the preceding examples, wherein the second subunit comprises an amino acid sequence, or a fragment or variant thereof, having at least about 75%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO.:6.

[0109] Example 11. The recombinant polypeptide as described in any of the preceding examples, wherein the GM-CSF is derived from an animal selected from the group consisting of: human, mouse, rat, hamster, insect, cow, pig, sheep, monkey, goat, dog, cat, and camel.

[0110] Example 12. A recombinant polypeptide as described in any one of Examples 4-11, wherein the third subunit comprises an amino acid sequence, or a fragment or variant thereof, having at least about 75%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO.:7.

[0111] Example 13. A recombinant polypeptide as described in any of the preceding examples, wherein the first adapter and / or the second adapter comprises an amino acid sequence, or a fragment or variant thereof, having at least about 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO.:8.

[0112] Example 14. A recombinant polypeptide as described in Example 1, wherein the polypeptide is expressed from the nucleic acid sequence of SEQ ID NO.:2 or SEQ ID NO.:4.

[0113] Example 15. A recombinant polypeptide as described in any of the preceding examples, wherein the polypeptide is expressed as an inclusion body in Escherichia coli.

[0114] Example 16. A recombinant polypeptide comprising an amino acid sequence, or a fragment or variant thereof, having at least about 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO.:1 or SEQ ID NO.:3.

[0115] Example 17. A pharmaceutical composition comprising a recombinant polypeptide as described in any of the preceding examples and optionally a pharmaceutically acceptable carrier, diluent, and / or adjuvant.

[0116] Example 18. A pharmaceutical composition as described in Example 17, wherein the adjuvant is a Toll-like receptor 9 agonist CpG oligodeoxynucleotide, aluminum hydroxide, or a combination thereof.

[0117] Example 19. A recombinant nucleic acid system comprising at least one nucleic acid sequence encoding any one of the recombinant polypeptides described in the foregoing examples.

[0118] Example 20. The system as described in Example 19, comprising a nucleic acid sequence of SEQ ID NO.:2 or SEQ ID NO.:4.

[0119] Example 21. A method for preventing or treating cancer T by administering to a subject in need a recombinant polypeptide as described in any one of Examples 1-16, or a pharmaceutical composition as described in any one of Examples 17-18, or a polypeptide expressed by a recombinant nucleic acid system as described in any one of Examples 19-20.

[0120] Example 22. The method as described in Example 21, wherein the cancer is selected from the group consisting of: liver cancer, skin cancer, colon cancer, bladder cancer, lung cancer, head and neck cancer, gastric cancer, B-cell lymphoma, T-cell lymphoma, pancreatic cancer, bladder cancer, brain cancer, monocytic leukemia, B-cell leukemia, T-cell leukemia, breast cancer, melanoma, small cell lung cancer, non-small cell lung cancer, lung cancer, Hodgkin lymphoma, kidney cancer, breast cancer, ovarian cancer, esophageal cancer, testicular cancer, colorectal cancer, thyroid cancer, prostate cancer, gastric cancer, cervical cancer, and combinations thereof.

[0121] Example 23. Use of the recombinant polypeptide as described in any one of Examples 1-16, or the pharmaceutical composition as described in any one of Examples 17-18, or the polypeptide expressed by the recombinant nucleic acid system as described in any one of Examples 19-20, for the manufacture of a medicament for the prevention or treatment of cancer.

[0122] Example 24. A recombinant polypeptide as described in any one of Examples 1-16, or a pharmaceutical composition as described in any one of Examples 17-18, or a polypeptide expressed by a recombinant nucleic acid system as described in any one of Examples 19-20, for use in the prevention or treatment of cancer. Example

[0123] This document provides examples of certain embodiments of this disclosure that are described in more detail. The examples provided herein are for illustrative purposes only and are not intended to limit the scope of the invention in any way. All references set forth below and elsewhere in this application are hereby incorporated herein by reference. Example 1 1. Materials and Methods 1.1 Construction of expression vectors for fusion protein vaccine design 1.1.1 Construction of expression vector for PDL1-PRAME (PD-PR)

[0124] Mouse PD-L1 and human PRAME sequences were used in the design of the fusion protein vaccine. The selected mouse PD-L1 sequence is a partial sequence (with 221 amino acid residues) of NCBI reference sequence NP_068693.1 and is shown in SEQ ID NO.:5, and the human PRAME sequence is a partial sequence (with 509 amino acid residues) of NP_001278644.1 and is shown in SEQ ID NO.:6. An adapter sequence (SEQ ID NO.:8) with three repeating sequences (GGGGS x 3) having four glycine and one serine residue was placed between the PD-L1 and PRAME sequences. The structural diagram and amino acid sequence of the PD-PR fusion protein are shown in [images / details]. Figure 1 and SEQ ID NO.:1.

[0125] The DNA sequence encoding the PD-PR fusion protein vaccine was optimized to utilize E. coli codons, as shown in SEQ ID NO.:2. Based on the optimized codons, a PD-PR DNA fragment was synthesized and cloned into a pET-22b(+) expression vector with NdeI and XhoI sites to generate a pPD-PR expression plasmid. Figure 2 Therefore, the PD-PR fusion protein contains a HisTag at the C-terminus for purification and is expressed under the control of the T7 promoter. After cloning, the inserted sequence was confirmed by sequencing. 1.1.2 Construction of expression vector for PDL1-PRAME-GMCSF (PD-PR-GM)

[0126] The mouse PD-L1, human PRAME, and mouse GM-CSF sequences were used in the fusion protein vaccine design. The selected mouse PD-L1 sequence is a partial sequence of NP_068693.1 (containing 221 amino acid residues) and is shown in SEQ ID NO.:5; the human PRAME sequence is a partial sequence of NP_001278644.1 (containing 509 amino acid residues) and is shown in SEQ ID NO.:6; and the mouse GM-CSF sequence is a partial sequence of NP_034099.2 (containing 124 amino acid residues) and is shown in SEQ ID NO.:7. These three protein fragments are linked together with a (GGGGS X3) linker and are shown in SEQ ID NO.:8. The structural diagram and amino acid sequence of the PD-PR-GM fusion protein vaccine design are shown in [the diagrams provided]. Figure 3 and SEQ ID NO.:3.

[0127] The DNA sequence encoding the PD-PR-GM fusion protein vaccine design was optimized to utilize E. coli codons, as shown in SEQ ID NO.:4. Based on the optimized codons, the DNA fragment of the fusion protein was synthesized and cloned into the pET-22b(+) expression vector with NdeI and XhoI sites to generate the PD-PR-GM expression plasmid. Figure 4 Therefore, the PD-PR-GM fusion protein contains a HisTag at the C-terminus for purification and is expressed under the control of the T7 promoter. After cloning, the inserted sequence was confirmed by sequencing. 1.2 Expression and purification of fusion protein vaccine design 1.2.1 Expression and purification of PD-PR

[0128] Rosetta (DE3) cells were transformed with the expression plasmid pPD-PR and cultured at 37°C (180 rpm) with 0.1 mg / mL ampicillin. When the OD600 nm of the cell culture reached 0.3–0.6, the cell culture was mixed with glycerol and stored at -80°C until use. To express the PD-PR fusion protein, 1 mL of frozen glycerol cell culture was added to 0.5 L LB and cultured at 37°C (150 rpm) until the OD600 nm reached 0.2–0.4. Then, an aliquot of 25 mL of culture was added to 0.5 L LB containing 1% glucose in a 2 L flask and incubated at 37°C with 0.1 mg / mL ampicillin. When the OD600 nm of the cell culture reached approximately 0.4, 1 mM IPTG was added to induce protein expression, followed by incubation at 37°C for 4 hours. After incubation, cells were harvested and lysed in HT homogenization buffer using a Freund's crusher at 25 kpsi. The inclusion body precipitate was then dissolved in extraction buffer (6 M GdnHCl and 20 mM β-ME in HT buffer) and clarified by centrifugation at 119,000 g for 40 min. The supernatant was loaded onto an IMAC column containing 10 mL of Ni-NTA resin to purify the PD-PR fusion protein. After loading, the column was washed with the same buffer containing 6 M GdnHCl / HT buffer and 20 mM imidazole, followed by 150 volumes of 10 mM Na₂HPO₄ containing 0.1% Triton X-114 (pH 9.3) to remove endotoxins. Finally, the column was washed with 10 mM Na₂HPO₄ to remove residual detergent, and the PD-PR fusion protein vaccine was eluted with 10 mM Na₂HPO₄ containing 300 mM imidazole. 1.2.2 Expression and purification of PD-PR-GM

[0129] E. coli BL21(DE3) was transformed with pPD-PR-GM and cultured at 37°C (180 rpm) with 0.1 mg / mL ampicillin. When the OD600nm of the cell culture reached 0.3–0.6, the cell culture was mixed with glycerol and stored at -80°C until use. To express the PD-PR-GM fusion protein vaccine, 1 mL of frozen cell culture was added to 0.5 L LB and cultured at 37°C (150 rpm) until the OD600nm reached 0.3–0.6. An aliquot of 25 mL of culture was added to 0.5 L LB containing 1% glucose in a 2 L flask and incubated at 37°C with 0.1 mg / mL ampicillin. When the OD600nm of the cell culture reached approximately 0.8, 1 mM IPTG was added to induce protein expression, followed by incubation at 37°C for 4 hours. After incubation, cultured cells were harvested and lysed in HT homogenization buffer using a Freund's crusher at 25 kpsi. The inclusion body precipitate was then dissolved in extraction buffer (6 M GdnHCl and 20 M β-ME in HT buffer) and further clarified by centrifugation at 119,000 g for 40 min. The supernatant was loaded onto an IMAC column containing 10 mL Ni-NTA resin to purify the fusion protein. The column was washed with the same buffer containing 6 M GdnHCl / HT buffer and 20 mM imidazole, followed by 150 volumes of 10 mM Na₂HPO₄ containing 0.1% Triton X-114 (pH 9.3) to remove endotoxins. Finally, the column was washed with 10 mM Na₂HPO₄ to remove residual detergent. After washing, the fusion protein was eluted with 10 mM Na₂HPO₄ containing 300 mM imidazole. 1.3 Identification of fusion proteins

[0130] Purified fusion protein vaccines were analyzed by 8% SDS-PAGE followed by Coomassie blue staining, or by Western blotting (using anti-PDL1, anti-PRAME, anti-GM-CSF, and anti-His antibodies for different parts of the fusion protein vaccine design). Appropriate species-specific horseradish peroxidase (HRP)-conjugated anti-IgG was used as the secondary antibody for signal detection. HRP activity was visualized using Clarity™ Western ECL substrate. The purity of the purified protein was estimated by quantifying the Coomassie blue staining intensity using ImageJ software. 1.4 Establishment of a mouse tumor model expressing PRAME antigen 1.4.1 Cell Culture and PRAME Transfection Transfecting mouse hepatocellular carcinoma cells to express PRAME

[0131] The mouse hepatocellular carcinoma (HCC) cell line (H-cells) was derived from a mouse model of spontaneous HCC induced by oncogenes, achieved by introducing AKT and NRAS, along with open reading frames (ORFs) encoding alternative tumor antigens and luciferases, into the chromosomes of hepatocytes using a hydrodynamic injection and transposon system, as described in Front. Cell Dev. Biol. [Frontiers in Cell and Developmental Biology] (2022) 10:821224. H-cells were cultured at 37°C and 5% CO2 in DMEM medium supplemented with 10% (v / v) heat-inactivated fetal bovine serum, non-essential amino acids, sodium pyruvate (1 mM), HEPES (10 mM), and penicillin / streptomycin (50 units / mL). To express human PRAME antigen in H-cells, the human PRAME sequence (NP_001278644.1) was optimized to use mouse codons. It was synthesized, sequenced, and cloned into a pcDNA 3.0 expression vector containing HindIII and ApaI sites to generate the pcDNA-PRAME plasmid for PRAME antigen expression. Figure 5 H-cells were transfected using pcDNA-PRAME transfection reagent via PolyJet™ transfection. Briefly, the transfection mixture was prepared by mixing 3 μg plasmid DNA and 4 μL PolyJet™ reagent in 200 μL serum-free DMEM and incubating at room temperature for 15 minutes. The H-cell pellet was resuspended using the transfection mixture and then incubated at 37°C for 20 minutes. The transfected cells were plated in 24-well plates and allowed to recover for 12 hours. Complete medium containing 0.5 μg / mL G418 was then added to the cells for selection. Following G418 selection, Western blot analysis was performed on surviving cells (382 clones) targeting PRAME antigen expression. 1.4.2 Tumor Model Establishment and Characterization Establishment of a mouse hepatocellular carcinoma model expressing PRAME

[0132] From 1.4.1, a clone expressing PRAME (H-clone-37) was selected and implanted into mice for tumor development. Female C57BL / 6 mice (5–12 weeks old) were purchased from the Laboratory Animal Center in Taipei, Taiwan. All animals were housed and bred at the Laboratory Animal Center of the National Health Research Institutes in Taiwan. All animal studies were approved and conducted in accordance with the guidelines of the Animal Committee of the National Health Research Institutes. H-clone-37 cells were cultured in G418, harvested, and washed with PBS. 5 × 10⁶ cells were subcutaneously seeded in 100 µL PBS and 20 µL of matrix gel on the left ventral side of the mice. 5H-cells. Expression of the PRAME antigen in tumor tissue was detected by Western blotting using an anti-PRAME antibody. Anti-GAPDH blotting was used as an internal control for all Western blotting analyses in this study. 1.5 Preventive Studies on Fusion Protein Vaccine Design In mouse HCC model

[0133] To test the efficacy of PD-PR and PD-PR-GM antitumor vaccines in a mouse HCC model for prophylaxis, 4–6-week-old female C57BL / 6 mice (n = 10 / group) were immunized with PD-PR and PD-PR-GM (30 μg / 0.2 mL / dose) formulated with 300 μg / dose Al(OH)3 and 30 μg / dose CpG1018 (GeneDireX; a synthetic CpG-B oligonucleotide with a thiophosphate backbone and the sequence 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO.:8)) via subcutaneous injection in the back. Mice injected with PBS alone served as controls. All animals were immunized on days (-21) and (-7) prior to cancer cell engraftment. On day (0), H-clone-37 cells (1 × 10⁻⁶ cells) in 100 μL PBS plus 20 µL of matrix gel were injected. 5 The tumor was subcutaneously injected into the left inguinal region of the animal. Body weight was measured, and tumor size was measured using calipers three times weekly. Tumor volume was estimated using the formula V = length × width × width / 2. The Kruskal-Wallis test and Dunn's multiple comparison test were used to determine the statistical significance of differences in tumor volume. Figure 6 As shown, booster doses of the vaccine were administered on day (+1) and day (+8). 1.6 Research on therapeutic tumor models for fusion protein vaccine design

[0134] To test the antitumor efficacy of PD-PR and PD-PR-GM in treatment, on day (0), 1 × 10⁻⁶ ppm of 100 μL PBS was added to 20 μL of matrix gel. 5H-clone-37 cells were implanted into the left inguinal region. Mice (n = 10 / group) were immunized by subcutaneous injection into the back on days (+1), (+8), (+15), (+22), and (+29). PD-PR (30 μg / 0.2 mL / dose) and PD-PR-GM (30 μg / 0.2 mL / dose) were formulated with 300 μg / dose Al(OH)3 and 30 μg / dose CpG1018 (GeneDireX; a synthetic CpG-B oligonucleotide with a thiophosphate backbone and the sequence 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO.:8)). Figure 7 Mice injected with PBS alone served as controls. Body weight and tumor size were measured three times weekly, and tumor volume was estimated using the formula V = length × width × width / 2. Statistical significance of differences in tumor volume was determined using the Kruskal-Wallis test and the Dunn multiple comparison test, and differences in survival were determined using the log-rank test. 2-Results 2.1 Expression and purification of fusion protein vaccine design

[0135] Expression of PD-PR and PD-PR-GM in *E. coli* was examined by 8% SDS-PAGE followed by Coomassie blue staining and Western blotting using anti-HisTag antibody. Following IPTG induction, the correctly sized fusion protein vaccine was expressed at significant levels. Figure 8A and Figure 8B ).

[0136] As described in the Materials and Methods section, the identity of the purified fusion protein vaccines was analyzed. The yields of PD-PR and PD-PR-GM were 3 mg / L LB and 2 mg / L LB, respectively. After purification, the purity of the purified fusion protein was greater than 90%. Western blot analysis using anti-PD-L1 and anti-PRAME antibodies indicated that both fusion protein vaccines contained PD-L1 and PRAME as designed. Further analysis using anti-GM-CFS antibody confirmed that the PD-PR-GM fusion protein vaccine contained GM-CFS (… Figures 9A to 9C ). 2.2 Establishment of a mouse HCC model expressing PRAME antigen.

[0137] H-cells were transfected with the pcDNA-PRAME plasmid, and clones surviving after G418 selection were further analyzed for PRAME expression. Of the more than 100 clones tested, H-clone-37 showed positive expression of the PRAME antigen. Figure 10 ), and its expression level was comparable to that of A549 human tumor cells ( Figure 11 H-clone-37 was selected and subcutaneously implanted into C57BL / 6 mice for solid tumor development. Tumor tissue derived from H-clone-37 cells also expressed the PRAME antigen (…). Figure 11 The results indicate that the development of H-clone-37 tumors in C57BL / 6 mice can be used as an syngeneic mouse tumor model to study the efficacy of PD-PR and PD-PR-GM cancer vaccines in vivo. 2.3 Transient and reversible changes in body weight after vaccination

[0138] Animals vaccinated with PD-PR and PD-PR-GM experienced a transient weight loss of less than 5% after each dose, which returned to pre-vaccination levels in approximately 3–5 days. Figures 12A to 12C Furthermore, none of the mice exhibited abnormal behavior or decreased activity after vaccination. These results indicate that there was no significant acute toxicity caused by the fusion protein vaccine design. 2.4 PD-PR and PD-PR-GM significantly inhibit tumor growth in prevention.

[0139] In a prophylactic HCC tumor model study, mice immunized with PD-PR and PD-PR-GM formulated with Al(OH)3-CpG showed reduced tumor volume before cancer cell engraftment, compared to mice immunized with PBS alone. On day 19, compared to the PD-PR group (302.8 mm), the tumor volume was significantly smaller. 3 76.4% tumor growth inhibition (p < 0.01) and PD-PR-GM group (518.9 mm) 3 The tumor growth was inhibited by 59.5% (p < 0.05) compared to the control group, where the tumor volume reached 1281.8 mm. 3 These results indicate that immunization designed using PD-PR and PD-PR-GM fusion protein vaccines exerts anti-tumor activity in prevention. Figures 13A to 13D ). 2.4 PD-PR and PD-PR-GM significantly inhibit tumor growth in treatment.

[0140] In a therapeutic tumor model study, vaccination with a fusion protein cancer vaccine was initiated after cancer cell implantation. Tumor growth was monitored three times weekly, and mice immunized with PD-PR and PD-PR-GM formulated with Al(OH)3-CpG showed reduced tumor volume compared to mice immunized with PBS alone. On day 23, the tumor volume in the control group reached 2250.5 mm. 3 Vaccination with PD-PR resulted in a 43.4% inhibition of tumor growth, p < 0.01 (1273.4 mm). 3 Compared to the control group's 2250.5 mm3 Furthermore, PD-PR-GM inhibited tumor growth by 40.7%, p < 0.05 (1333.6 mm). 3 Compared to the control group's 2250.5 mm 3 59.5%. Vaccination using vaccines designed with both fusion proteins also significantly prolonged survival in animals after cancer cell engraftment (p < 0.01). These results indicate that immunization using vaccines designed with PD-PR and PD-PR-GM fusion proteins exerts anti-tumor activity in treatment. Figures 14A to 14E ). 3-Summary

[0141] The experimental results shown in this invention demonstrate that using fusion protein vaccines to design co-targeting tumor antigens PD-L1 and PRAME can exert tumor growth inhibition effects in both prevention and treatment. Example 2 4. Materials and Methods 4.1 Establishment of a mouse tumor model expressing PRAME antigen

[0142] In this example, another mouse tumor model (melanoma) expressing FRAME was established using a method similar to that described in 1.4 of Example 1, but instead of the mouse tumor model (HCC), the melanoma cell line B16F10 was used.

[0143] 4.1.1 Cell Culture and PRAME Transfection

[0144] Transfect mouse melanoma cells to express PRAME

[0145] B16F10 is a mouse melanoma cell line used in cancer research, such as evaluating anticancer drugs, immunotherapy, and other treatment strategies. It was originally derived from melanoma tumors in C57BL / 6 mice. These cells exhibit a spindle-shaped and epithelial-like morphology and have been extensively characterized for their tumorigenicity and metastatic potential. To express PRAME in mouse melanoma cells, the pcDNA-PRAME plasmid (as shown in Example 1) was transfected using a PolyJet™ transfection reagent specific to H-cells. Figure 5 Transfected B16F10. After G418 selection, Western blot analysis was performed on surviving cell clones targeting PRAME antigen expression. 4.1.2 Tumor Model Establishment and Characterization Establishment of a mouse melanoma model expressing PRAME

[0146] From 4.1.1, a clone expressing PRAME (B16F10#18) was selected and inserted into mice for tumor development. Mice were subcutaneously inoculated with 3 × 10⁻⁶ PBS in 100 μL of PBS in the left inguinal region. 4 Individual cells. Regarding the melanoma model, Western blotting was used to detect the expression of PRAME antigen in tumor tissue. 4.2 Preventive Studies on Fusion Protein Vaccine Design In a mouse melanoma model

[0147] To test the efficacy of antitumor vaccines in PD-PR and PD-PR-GM mouse melanoma models for prevention, 4-6 week old female C57BL / 6 mice (n = 10 / group) were immunized with 300 μg / dose Al(OH)3 and 30 μg / dose CpG via subcutaneous injection in the back. 1018 PD-PR and PD-PR-GM (30 μg / 0.2 mL / dose) were formulated together with a synthetic CpG-B oligonucleotide having a thiophosphate backbone and the sequence 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO.:8). Mice injected alone with Al(OH)3-CpG adjuvant served as controls. All animals were immunized on days (-21) and (-7) prior to cancer cell engraftment. On day (0), B16F10#18 cells (3 × 10⁻⁶ cells) were placed in 100 μL PBS with 20 µL of matrix gel. 4 The inoculation was administered subcutaneously to the left inguinal region of the animal. Body weight and tumor size were measured similarly on days (+1) and (+8), as described above. Figure 6 The vaccination schedule shown in the mouse HCC model was similar, with booster doses administered, but using a mouse melanoma model and clone B16F10#18. 4.3 Research on therapeutic tumor models for fusion protein vaccine design

[0148] To test the efficacy of PD-PR and PD-PR-GM as antitumor vaccines in a mouse melanoma model for therapeutic purposes, mouse melanoma cells expressing human PRAME were subcutaneously implanted into mice. Following melanoma cell implantation, mice were immunized on days (+1), (+8), (+15), (+22), and (+29) by subcutaneous injection into the back with PD-PR (30 μg / 0.2 mL / dose) and PD-PR-GM (30 μg / 0.2 mL / dose), formulated with 300 μg / dose Al(OH)3 and 30 μg / dose CpG1018 (GeneDireX; a synthetic CpG-B oligonucleotide with a phosphate thioester backbone and the sequence 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO.:8)). Mice injected with PBS or adjuvant alone served as controls. Body weight and tumor size were measured three times weekly, and tumor volume was estimated using the formula V = length × width × width / 2. Statistical significance of differences in tumor volume was determined using the Kruskal-Wallis test and the Dunn multiple comparison test, and differences in survival were determined using the log-rank test. In some instances, fusion protein design was able to therapeutically inhibit the growth of PRAME-positive melanoma tumors in mice. 5-Results 5.1 Establishment of a mouse melanoma model expressing PRAME antigen

[0149] B16F10 cells were transfected with the pcDNA-PRAME plasmid, and clones surviving after G418 selection were further analyzed for PRAME expression. Among the more than 100 clones tested, B16F10#18 showed positive expression of the PRAME antigen. Figure 16 ), and its expression level was comparable to that of A549 human tumor cells ( Figure 17 B16F10#18 was selected and subcutaneously implanted into C57BL / 6 mice for solid tumor development. Tumor tissue derived from B16F10#18 also expressed the PRAME antigen (…). Figure 17 The results indicate that the development of B16F10#18 melanoma in C57BL / 6 mice can be used as an syngeneic mouse tumor model to study the efficacy of PD-PR and PD-PR-GM cancer vaccines in vivo. 5.2 PD-PR and PD-PR-GM significantly inhibit tumor growth in prevention.

[0150] In a prophylactic study of the B16F10#18 melanoma model with high PRAME expression levels, vaccination with PD-PR and PD-PR-GM, formulated with 300 μg / dose Al(OH)3 and 30 μg / dose CpG1018, showed more significant tumor growth inhibition compared to vaccination in the HCC model. On day 16, compared to the PD-PR group (96 mm... 3 91.5% tumor growth inhibition (p < 0.01) and PD-PR-GM group (59 mm) 3 In contrast, the control group treated with Al(OH)3-CpG adjuvant showed 94.7% tumor growth inhibition (p < 0.001), with a tumor volume reaching 1130 mmHg. 3 These findings suggest that immunization designed using PD-PR and PD-PR-GM fusion protein vaccines exhibits surprising antitumor activity in prevention, and that this effect may be related to the expression level of PRAME in tumors. Figures 18A to 18D ).

[0151] In some implementations, tumor growth inhibition produced by administration of the PD-PR fusion protein is superior to tumor growth inhibition produced by administration of recombinant PD-L1 protein or recombinant PRAME protein (whether alone or in combination).

[0152] In some cases, PD-PR and PD-PR-GM have significantly inhibited tumor growth in treatment. Example 3 7.1 Antigen-specific immune response induced by PD-PR fusion protein vaccine 7.1.1 Immunizing mice with PD-PR fusion protein vaccine

[0153] To test the antigen-specific immune response induced by the PD-PR fusion protein vaccine, PD-PR (30 μg), 300 μg / dose Al(OH)3, and 30 μg / dose CpG were administered on days (0), (+14), and (+28). 1018 PD-PR (30 μg) was prepared together with 300 μg / dose Al(OH)3 and 30 μg / dose CpG. 1018 The co-prepared PD-PR (60 μg) was used to subcutaneously immunize mice (n = 4 / group). Figure 15 Mice injected with PBS alone served as controls. Serum samples were collected on days -1, +7, +21, and -35 for antigen-specific antibody response studies. Spleen cells were collected on day +35 for antigen-specific T cell response studies. 7.1.2 ELISA assay for antigen-specific antibody response

[0154] Diluted serum samples collected in 2.7.1 were added to ELISA wells pre-coated with recombinant mouse PD-L1 ECD protein, recombinant human PD-L1 ECD protein, and recombinant human PRAME protein to investigate antigen-specific antibody responses to these three antigens before and after vaccination with the PD-PR fusion protein. 7.1.3 Elispot assay for antigen-specific T cell response

[0155] Spleen cells collected in 3.7.1 were added to Elispot wells pre-coated with an anti-mouse IFN-γ capture antibody. An overlapping peptide pool of mouse PD-L1, human PD-L1, and human PRAME was added to the Elispot wells and incubated with the spleen cells for 24–48 hours to achieve antigen-specific T cell activation. IFN-γ secreted by activated T cells was captured and detected using an anti-mouse IFN-γ detection antibody to quantify the activated antigen-specific T cell response. 7.2 Antigen-specific immune response generated by PD-PR fusion protein vaccination

[0156] PD-PR fusion protein was administered to mice, such as Figure 15 As shown, samples were collected for antigen-specific immune response studies.

[0157] In some instances, serum collected from mice vaccinated with the PD-PR fusion protein showed antigen-specific antibody responses against mouse PD-L1, human PD-L1, and human PRAME compared to serum collected before vaccination, and antibody response levels increased with time to vaccination. In some instances, antibody responses generated in mice vaccinated with the PD-PR fusion protein formulated with adjuvant were stronger than those generated in mice vaccinated with the unadjuvanted PD-PR fusion protein. In some instances, in Elispot assays, spleen cells collected after PD-PR vaccination showed positive IFN-γ responses upon stimulation with mouse PD-L1 peptide, human PD-L1 peptide, or human PRAME peptide compared to spleen cells collected from mice administered PBS. In some instances, mice vaccinated with the PD-PR fusion protein formulated with adjuvant showed stronger antigen-specific T-cell responses against mouse PD-L1, human PD-L1, and human PRAME compared to mice vaccinated with the unadjuvanted PD-PR fusion protein. 8. Summary

[0158] The experimental results shown in the exemplary embodiments demonstrate that the design of co-targeting tumor antigens PD-L1 and PRAME using the fusion protein vaccine described herein exhibits an unexpected synergistic effect in inducing antigen-specific immune responses and exerts tumor growth inhibition effects in both prevention and treatment, with the tumor growth inhibition effect increasing with the protein level of PRAME in the tumor.

[0159] Exemplary embodiments of the present invention have thus been fully described. Although the description refers to specific embodiments, those skilled in the art will understand that the invention can be practiced with variations of these specific details. Therefore, the invention should not be construed as being limited to the embodiments set forth herein.

[0160] For example, in some embodiments, the PD-L1 sequence in the PD-PR fusion protein is located upstream of the PRAME sequence and linked by the adapter sequence GGGGS X 3, but other sequence arrangements can be used. In one embodiment, the PRAME sequence is located upstream of the PD-L1 sequence.

[0161] For example, in some embodiments, the PRAME sequence in the PD-PR-GM fusion protein is located downstream of the PD-L1 sequence and upstream of the GM-CSF sequence, linked by the adapter sequence GGGGS x 3. However, other sequence arrangements can be used, for example, with GM-CSF located between PD-L1 and PRAME. In one embodiment, the PRAME sequence is located upstream of the PD-L1 sequence. The same or different first and second adapters can be used instead of the adapter sequence GGGGS x 3.

[0162] For example, in some embodiments, a fusion protein of PD-PR or PD-PR-GM is provided, but in other embodiments, PD-L1, PRAME, and / or GM-SF may be prepared individually and provided as a mixture rather than a fusion protein. In other words, in some embodiments, the pharmaceutical composition comprises a mixture of peptides containing all or fragments of the amino acid sequences of PD-L1, PRAME, and / or GM-SF.

[0163] For example, in some embodiments, the fusion protein or polypeptide contains a HisTag at the C-terminus for purification and is expressed in E. coli using the pET-22b(+) expression vector, but other tag sequences (such as glutathione S-transferase and FLAG tag (DYKDDDDK)), other host organisms (such as yeast cells, insect cells and mammalian cells) and other expression vectors or systems (such as the Expi293 expression system) can be used.

Claims

1. A recombinant polypeptide comprising: The first subunit comprises all or a fragment of the amino acid sequence of programmed death ligand 1 PD-L1, or a variant sequence thereof having at least 75% sequence identity; and The second subunit contains all or a fragment of the amino acid sequence of the melanoma preferentially expressed antigen PRAME, or a variant sequence thereof having at least 75% sequence identity.

2. The recombinant polypeptide of claim 1, wherein the first subunit is located upstream of the second subunit.

3. The recombinant polypeptide as claimed in any of the preceding claims, further comprising a first linker, wherein the first linker is located between the first subunit and the second subunit.

4. The recombinant polypeptide as described in any of the preceding claims, further comprising a third subunit, said third subunit comprising all or a fragment of the amino acid sequence of GM-CSF, or a variant sequence thereof having at least 75% sequence identity.

5. The recombinant polypeptide of claim 4, further comprising a second connector, wherein the second connector is located between the second subunit and the third subunit.

6. The recombinant polypeptide of claim 5, wherein the second subunit is located downstream of the first subunit and upstream of the third subunit.

7. The recombinant polypeptide as claimed in any of the preceding claims, wherein the PD-L1 is derived from an animal selected from the group consisting of: human, mouse, rat, hamster, insect, cow, pig, sheep, monkey, goat, dog, cat and camel.

8. The recombinant polypeptide as claimed in any of the preceding claims, wherein the first subunit comprises an amino acid sequence, or a fragment or variant thereof, having at least about 75%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO.:

5.

9. The recombinant polypeptide as claimed in any of the preceding claims, wherein the PRAME is derived from an animal selected from the group consisting of: human, mouse, rat, hamster, insect, cow, pig, sheep, monkey, goat, dog, cat and camel.

10. The recombinant polypeptide as claimed in any of the preceding claims, wherein the second subunit comprises an amino acid sequence, or a fragment or variant thereof, having at least about 75%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO.:

6.

11. The recombinant polypeptide as claimed in any of the preceding claims, wherein the GM-CSF is derived from an animal selected from the group consisting of: human, mouse, rat, hamster, insect, cow, pig, sheep, monkey, goat, dog, cat and camel.

12. The recombinant polypeptide of any one of claims 4-11, wherein the third subunit comprises an amino acid sequence, or a fragment or variant thereof, having at least about 75%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO.:

7.

13. The recombinant polypeptide of any of the preceding claims, wherein the first adapter and / or the second adapter comprises an amino acid sequence, or a fragment or variant thereof, having at least about 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO.:

8.

14. The recombinant polypeptide of claim 1, wherein the polypeptide is expressed from the nucleic acid sequence of SEQ ID NO.:2 or SEQ ID NO.:

4.

15. The recombinant polypeptide as claimed in any of the preceding claims, wherein the polypeptide is expressed as an inclusion body in Escherichia coli.

16. A recombinant polypeptide comprising an amino acid sequence, or a fragment or variant thereof, having at least about 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO.:1 or SEQ ID NO.:

3.

17. A pharmaceutical composition comprising a recombinant polypeptide as described in any one of the preceding claims and optionally a pharmaceutically acceptable carrier, diluent, and / or adjuvant.

18. The pharmaceutical composition of claim 17, wherein the adjuvant is a Toll-like receptor 9 agonist CpG oligodeoxynucleotide, aluminum hydroxide, or a combination thereof.

19. A recombinant nucleic acid system comprising at least one nucleic acid sequence encoding any one of the recombinant polypeptides as described in the preceding claims.

20. The system of claim 19, comprising the nucleic acid sequence of SEQ ID NO.:2 or SEQ ID NO.:

4.

21. A method for preventing or treating cancer by administering to a subject in need a recombinant polypeptide as described in any one of claims 1-16, a pharmaceutical composition as described in any one of claims 17-18, or a polypeptide expressed by a recombinant nucleic acid system as described in any one of claims 19-20.

22. The method of claim 21, wherein the cancer is selected from the group consisting of: liver cancer, skin cancer, colon cancer, bladder cancer, lung cancer, head and neck cancer, gastric cancer, B-cell lymphoma, T-cell lymphoma, pancreatic cancer, bladder cancer, brain cancer, monocytic leukemia, B-cell leukemia, T-cell leukemia, breast cancer, melanoma, small cell lung cancer, non-small cell lung cancer, lung cancer, Hodgkin's lymphoma, kidney cancer, breast cancer, ovarian cancer, esophageal cancer, testicular cancer, colorectal cancer, thyroid cancer, prostate cancer, gastric cancer, cervical cancer, and combinations thereof.

23. Use of any recombinant polypeptide as described in any one of claims 1-16, or a pharmaceutical composition as described in any one of claims 17-18, or a polypeptide expressed by a recombinant nucleic acid system as described in any one of claims 19-20, for the manufacture of a medicament for the prevention or treatment of cancer.

24. The recombinant polypeptide as described in any one of claims 1-16, or the pharmaceutical composition as described in any one of claims 17-18, or the polypeptide expressed by the recombinant nucleic acid system as described in any one of claims 19-20, for use in the prevention or treatment of cancer.

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