Fusion protein for inducing immune response and treating cancer

Abstract

The present invention relates to fusion proteins for use in the induction of immune responses and in the treatment of cancer, and in particular as immunopotentiators and for use in the induction of humoral immune responses and cell-mediated immune responses.

Description

Technical Field

[0001] The present invention relates to a fusion protein for inducing an immune response and treating cancer, and particularly to a fusion protein as an immune enhancer and for inducing a humoral immune response and a cell-mediated immune response. Background Art

[0002] Traditional chemical and physical therapies have limitations in treating cancer, including tumor recurrence and the development of drug resistance in cancer cells. These problems can lead to increased mortality and treatment failure, mainly due to non-specific toxicity and the overexpression of ATP-binding cassette (ABC) transporters in tumor cells. ABC transporters function in pumping chemotherapeutic drugs from the cytoplasm to the extracellular space, making it difficult for the drugs to effectively target tumor cells.

[0003] However, although surgical resection of solid tumors often results in permanent scarring and cosmetic damage to organs and tissues, it is associated with increased survival rates. In addition, small populations of tumor cells may remain after surgery, so there is still a risk of undetected micrometastases, which can lead to cancer recurrence.

[0004] Targeted cancer therapy is an immunotherapy that focuses on blocking specific receptors expressed by cancer cells or delivering therapeutic drugs or toxins directly to the cancer site. The latter approach involves using conjugate proteins composed of specific domains, such as monoclonal antibodies, Fabs, Fvs, or receptor ligands, conjugated to cytotoxic effector molecules from bacteria, plants, animals, or insects. These conjugates are called immunotoxins (ITs). The development of immunotoxins was inspired by the concept of the "magic bullet", where a lethal agent is chemically or genetically fused to a site-specific moiety to ensure specificity for cancer cells. In the construction of immunotoxins, bacterial or plant toxins can be used. These toxins are capable of inducing apoptosis and blocking the protein synthesis machinery due to their ADP-ribosyltransferase activity, thereby blocking the elongation of peptide chains.

[0005] There is still a need for improved cancer therapies to achieve better clinical outcomes and produce fewer side effects. Summary of the Invention

[0006] In some embodiments, the present disclosure provides a fusion protein. The fusion protein comprises an antigen-presenting cell (APC) binding peptide or a CD91 receptor binding peptide, a translocation peptide, a linker peptide, and one or more repeated cysteine-rich peptides. In one embodiment, the fusion protein may further comprise a retention signal domain selected from an endoplasmic reticulum (ER) retention sequence, a Golgi retention sequence, and a proteasome targeting sequence.

[0007] In one embodiment, the fusion protein sequentially comprises, from the N-terminus to the C-terminus: an APC-binding peptide or a CD91 receptor-binding peptide, a translocation peptide, a linker peptide, one or more repeated cysteine-rich peptides, and a retention signal domain.

[0008] In some embodiments, the APC-binding peptide or the CD91 receptor-binding peptide has an amino acid sequence having at least 90% or at least 95% identity with SEQ ID NO:1. In some embodiments, the APC-binding peptide or the CD91 receptor-binding peptide has an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identity with SEQ ID NO:1. In one embodiment, the APC-binding peptide or the CD91 receptor-binding peptide has the amino acid sequence of SEQ ID NO:1.

[0009] In some embodiments, the translocation peptide has an amino acid sequence having at least 90% or at least 95% identity with SEQ ID NO:2. In some embodiments, the translocation peptide has an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identity with SEQ ID NO:2. In one embodiment, the translocation peptide has the amino acid sequence of SEQ ID NO:2.

[0010] In some embodiments, the cysteine-rich peptide comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity with the amino acid sequence of PCCGCCGCGC (SEQ ID NO:4). In some embodiments, the cysteine-rich peptide comprises an amino acid sequence having about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% (about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%) or about 100% identity with the amino acid sequence of PCCGCCGCGC (SEQ ID NO:4). In some embodiments, the cysteine-rich peptide comprises an amino acid sequence having one or more amino acid residue differences from the amino acid sequence of PCCGCCGCGC (SEQ ID NO:4). In some embodiments, the cysteine-rich peptide comprises an amino acid sequence having 1, 2, 3, 4, 5 or 6 amino acid residue differences from the amino acid sequence of PCCGCCGCGC (SEQ ID NO:4). In one embodiment, the cysteine-rich peptide comprises the amino acid sequence of PCCGCCGCGC (SEQ ID NO:4).

[0011] In some embodiments, the cysteine-rich peptide comprises from 2 to 15 repeats of the amino acid sequence PCCGCCGCGC (SEQ ID NO:4). In some embodiments, the cysteine-rich peptide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 repeats of the amino acid sequence PCCGCCGCGC (SEQ ID NO:4). In some embodiments, the cysteine-rich peptide comprises 7 repeats of the amino acid sequence PCCGCCGCGC (SEQ ID NO:4) or consists of 7 repeats of the amino acid sequence PCCGCCGCGC (SEQ ID NO:4), i.e., (PCCGCCGCGC)7 (SEQ ID NO:5).

[0012] In some embodiments, the cysteine-rich peptide bears an antigen attached to at least one cysteine residue in the cysteine-rich peptide. In embodiments where the cysteine-rich peptide comprises the amino acid sequence PCCGCCGCGC (SEQ ID NO:4), one or more antigens are attached to one or more cysteine residues in the cysteine-rich peptide. For example, 1, 2, 3, 4, 5 or 6 antigens may be attached to 1, 2, 3, 4, 5 or 6 cysteine residues in the cysteine-rich peptide, respectively. In some embodiments, these antigens are the same as or different from each other.

[0013] Any antigen (especially an antigen with low immunogenicity) can be attached to multiple cysteine residues in the cysteine-rich peptide. Exemplary antigens include but are not limited to: glycotopes (e.g., Tn, sTn, GM2 and GM3), glycolipids (e.g., Globo H and SSEA3) and glycosphingolipids (e.g., B-I and B-II). The antigen can also be a steroid hormone, such as a glucocorticoid (e.g., prednisone, dexamethasone or triamcinolone), a mineralocorticoid (e.g., fludrocortisone), vitamin D (e.g., dihydrotachysterol), an androgen (e.g., oxandrolone or nandrolone), an estrogen (e.g., diethylstilbestrol) or a progestogen (e.g., norethindrone or medroxyprogesterone acetate). Androgens and estrogens are collectively referred to as sex hormones.

[0014] In some embodiments, the antigen is a carbohydrate antigen. In some embodiments, the antigen is a cancer-associated antigen. In some embodiments, the antigen comprises one or more antigens selected from the group consisting of: T, Tn, sialyl Tn (sTn), GM2, GM3, phosphoserine, phosphothreonine, sialic acid, N-acetylglucosamine (GlcNAc), Globo H, Lewis x oligosaccharide, Lewis y oligosaccharide, and steroid hormones.

[0015] In one embodiment, the antigen comprises one or more cancer-associated antigens selected from: Tn antigen (GalNAcα1-O-Ser / Thr), T antigen (Galβ1-3GalNAcα1-O-Ser / Thr), sialyl Tn (sTn) antigen, or a combination of the foregoing. The structures of the Tn antigen, T antigen, and sTn antigen are respectively as Figures 1A to 1C shown.

[0016] In some embodiments, the antigen is linked to a cysteine residue in the cysteine-rich peptide via a linker, such as a maleimide linker capable of reacting with amino groups and sulfhydryl groups. Examples of maleimide linkers can be N-ε-maleimidocaproic acid, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-ε-maleimidocaproyloxy succinimide ester (EMCS), or succinimidyl 6-[β-maleimidopropionamido]-hexanoate (SMPH).

[0017] In some embodiments, the fusion protein described herein further comprises a linker peptide located between the translocation peptide and the cysteine-rich peptide. The linker peptide has an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO:3. In some embodiments, the linker peptide has an amino acid sequence that is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO:3. In one embodiment, the linker peptide has the amino acid sequence of SEQ ID NO:3.

[0018] In some embodiments, the retention signal domain comprises an amino acid sequence selected from KDEL, KKMP, KKTN, XDEL, XXEL or KKXX, where X can represent any amino acid. Examples of the amino acid sequence of the retention signal domain include but are not limited to: KDEL, HDEF, HDEL, RDEF, RDEL, WDEL, YDEL, HEEF, HEEL, KEEL, REEL, KAEL, KCEL, KFEL, KGEL, KHEL, KLEL, KNEL, KQEL, KREL, KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL, KDEF, KKEL, HADL, HAEL, HIEL, HNEL, HTEL, KTEL, HVEL, NDEL, QDEL, REDL, RDEL, RNEL, RTDL, RTEL, SDEL, TDEL, SKEL or REDLK. In certain embodiments, the ER retention sequence contained in the fusion protein described herein comprises the amino acid sequence of KDEL (SEQ ID NO:6), RDEL (SEQ ID NO:7) or REDLK (SEQ ID NO:8). In some embodiments, the ER retention sequence can be a multiple repeat amino acid sequence of KDEL (SEQ ID NO:6), RDEL (SEQ ID NO:7) or REDLK (SEQ ID NO:8). For example, the ER retention sequence can be a 2-, 3- or 4-repeat amino acid sequence of KDEL (SEQ ID NO:6), RDEL (SEQ ID NO:7) or REDLK (SEQ ID NO:8). In some embodiments, the ER retention sequence consists of the amino acid sequence of KDEL (SEQ ID NO:6), RDEL (SEQ ID NO:7) or REDLK (SEQ ID NO:8). In one embodiment, the ER retention sequence comprises the amino acid sequence of REDLK (SEQ ID NO:8) or consists of the amino acid sequence of REDLK (SEQ ID NO:8).

[0019] In some embodiments, the fusion protein described herein comprises an amino acid sequence having at least 90% or at least 95% identity with SEQ ID NO:9. In some embodiments, the fusion protein comprises an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identity with SEQ ID NO:9. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:9. In one embodiment, the fusion protein consists of the amino acid sequence of SEQ ID NO:9.

[0020] In some embodiments, the fusion proteins described herein do not contain the amino acid sequence of SEQ ID NO:10 or a fragment thereof. The amino acid sequence of SEQ ID NO:10 is shown below. GDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQ DLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSL TLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK(SEQ ID NO:10)

[0021] In some embodiments, the present disclosure also provides an isolated nucleic acid. The isolated nucleic acid encodes the fusion protein described above.

[0022] In some embodiments, the present disclosure also provides a vector. The vector contains the isolated nucleic acid described above.

[0023] In some embodiments, the present disclosure also provides a host cell. The host cell contains the isolated nucleic acid or vector described above.

[0024] In some embodiments, the present disclosure also provides a pharmaceutical composition. The pharmaceutical composition contains the fusion protein described above and a pharmaceutically acceptable carrier.

[0025] In some embodiments, the present disclosure also provides a kit. The kit contains the fusion protein described herein or the pharmaceutical composition described herein. In some embodiments, the kit further includes instructions for use.

[0026] In some embodiments, the present disclosure also provides a method for treating cancer. The method includes administering an effective amount of the fusion protein described herein or the pharmaceutical composition described herein to an individual in need thereof.

[0027] In some embodiments, the cancer can be any cancer that expresses Tn antigen, sTn antigen, and / or T antigen.

[0028] In some embodiments, the cancer is selected from the group consisting of: breast cancer, liver cancer, colorectal cancer, bladder cancer, cervical cancer, leukemia, lung cancer, central nervous system cancer, melanoma, ovarian cancer, gastric cancer, pancreatic cancer, head and neck cancer, kidney cancer, and prostate cancer

[0029] In some embodiments, the present disclosure also provides methods for treating a viral infection in an individual in need thereof. The methods comprise administering to the individual in need thereof an effective amount of the fusion protein described herein or the pharmaceutical composition described herein.

[0030] In some embodiments, the present disclosure also provides methods for inducing an immune response in an individual. The methods comprise administering to the individual in need thereof an effective amount of the fusion protein described herein or the pharmaceutical composition described herein.

[0031] In some embodiments, the immune response is an adaptive immune response. In one embodiment, the immune response is a humoral immune response. In one embodiment, the immune response is a cell-mediated immune response. In one embodiment, the immune response is a combination of a humoral immune response and a cell-mediated immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] This application includes at least one color drawing. The Patent and Trademark Office will provide a copy of the patent or patent application publication containing color drawings if a request is received and the necessary fee is paid. The following drawings form a part of this specification and are included to further illustrate certain embodiments of the present disclosure, which may be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein. Exemplary embodiments are shown in the reference drawings. The embodiments and drawings disclosed herein are illustrative only and not restrictive.

[0033] Figures 1A to 1C Respectively show the chemical structures of Tn antigen, T antigen, and sTn antigen.

[0034] Figure 2 Show the internalization of the fusion protein into NIH 3T3 cells. The fusion protein comprises domains Ia, Ib, and II of Pseudomonas endotoxin (PE) (PE(ΔIII)) and the ER retention sequence of REDLK. mStrawberry (mSB) red fluorescence is used as an indicator to confirm the internalization ability of the fusion protein.

[0035] Figure 3Shown is the LDH release of MDA-MB-231 cells expressing Tn antigen (target cells, T) after co-culture with T cells (effector cells, E) isolated from the spleens of BALB / c mice, where the BALB / c mice were immunized with a fusion protein comprising domains Ia, Ib and II of Pseudomonas endotoxin (PE) A (PE(ΔIII)) and the ER retention sequence of REDLK. The T cells and MDA-MB-231 cells were co-cultured at 37 °C for 18 hours at different T:E ratios. The amount of LDH released by damaged cells was detected to represent the ability of T cells to kill target cells. Cultures with a T:E ratio of 1:100 showed significant cytotoxicity, as indicated by the highest amount of LDH released by MDA-MB-231 cells (p = 0.004).

[0036] Figure 4 Shown is the monitoring of mouse serum antibody production in the immunization experiment. Polycysteine or polycysteine conjugated to Tn was used as a substrate to detect serum samples extracted from mice immunized with a fusion protein (PE(ΔIII), Tn antigen and REDLK sequence) or immunized with adjuvant alone. Detailed Description

[0037] Cross-Reference to Related Applications This application claims the priority of U.S. Provisional Patent Application No. 63 / 623,781, filed on January 22, 2024, the content of which is incorporated herein by reference in its entirety.

[0038] Sequence Listing This application contains a sequence listing, which has been submitted electronically in.xml format and is hereby incorporated by reference in its entirety herein. The.xml copy was created on January 9, 2025, named "US15429-SequenceListing.xml", and is 11 kb in size.

[0039] It should be understood that the present invention is not limited to the specific materials or methods described herein. It should also be understood that the methods described herein are only for describing specific embodiments and are not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0040] Definitions

[0041] It must be noted that, unless otherwise expressly indicated, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents. Thus, for example, "a biomarker" includes mixtures of two or more.

[0042] Unless otherwise indicated, the term "comprise" or "comprising" as used in the text of this specification will be understood to include the stated component or integer or group of components or integers, but not to exclude any other component or integer or group of components or integers.

[0043] As used in this application, terms such as "about" and "approximately" are used as synonyms. Any numerical values used in this application, whether or not accompanied by the word "about" / "approximately", are intended to cover any normal fluctuations understood by those of ordinary skill in the relevant technical field. In certain embodiments, unless otherwise stated or apparent from the context (unless the numerical value would exceed 100% of the possible value), "about" and "approximately" refer to a numerical value (whether greater or less) within a range of 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the stated reference value.

[0044] As used interchangeably herein, the terms "polynucleotide" and "nucleic acid" refer to polymeric forms consisting of more than about 100 nucleotides, which can be ribonucleotides or deoxyribonucleotides. Thus, these terms include, but are not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemical or biochemical modified, unnatural or derivatized nucleobases. "Oligonucleotide" generally refers to a polynucleotide of single-stranded or double-stranded DNA or RNA consisting of about 5 to about 100 nucleotides. However, for the purposes of this disclosure, there is no upper limit to the length of oligonucleotides. Oligonucleotides are also referred to as "oligomers" or "oligomers", and can be isolated from genes or chemically synthesized by known techniques. The terms "polynucleotide" and "nucleic acid" should be understood to include single-stranded (e.g., sense or antisense strands) and double-stranded polynucleotides applicable to the stated embodiments.

[0045] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures, as well as vectors integrated into the genome of a host cell into which they have been introduced. Certain vectors are capable of driving the expression of nucleic acids operably linked thereto. Such vectors are referred to herein as "expression vectors".

[0046] As used herein, the terms "treat", "treatment" and "treating" refer to a method for obtaining a beneficial or desired result, such as a clinical result. For the purposes of this invention, a beneficial or desired result may include inhibiting or arresting the initiation or progression of a disease; ameliorating the symptoms of a disease or reducing its development; or a combination thereof.

[0047] The term "sequence identity" refers to the percentage of identical bases or amino acids between two polynucleotide or polypeptide sequences, where the bases or amino acids are in the same relative positions. Thus, one polynucleotide or polypeptide sequence has a certain percentage of sequence identity with another polynucleotide or polypeptide sequence. When performing a sequence comparison, usually one sequence is a reference sequence to which the test sequence is compared. The term "reference sequence" refers to the molecule used for comparison with the test sequence.

[0048] As used herein, the term "effective amount" refers to the minimum amount of an agent or composition required to effect a particular physiological effect.

[0049] As used interchangeably herein, the terms "individual", "subject", "host", and "patient" refer to a mammal, including but not limited to rodents (e.g., rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, suines, caprines), etc. Specifically, the individual is vaccinated.

[0050] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government of the United States or listed in the U.S. Pharmacopeia, European Pharmacopeia, or other generally recognized pharmacopeias for use in mammals, and more specifically in humans.

[0051] Fusion protein

[0052] The disclosure described herein is based in part on the following discovery: The fusion protein can induce a strong immune response and thus can be used to treat cancer (e.g., cancer expressing Tn antigen, T antigen, and / or sTn antigen) or viral infection. This therapeutic benefit is due to the activation of MHC class I and MHC class II antigen presentation pathways after the fusion protein is internalized into target cells (such as tumor cells). In the MHC class I antigen presentation pathway, the fusion protein is broken down by the proteasome into smaller peptide fragments, which bind to class I MHC molecules in the endoplasmic reticulum (ER) and are then presented on the cell surface to CD8+ T cells, thereby inducing a strong cell-mediated immunity. Cell-mediated immunity helps kill infected cells or tumor cells and is thus important for the treatment of cancer and viral infection. In the MHC class II antigen presentation pathway, the fusion protein is degraded into small peptides in the endolysosome, and then these peptides bind to class II MHC molecules to form a complex. The complex is transported to the cell surface of antigen-presenting cells (APCs), allowing the antigen to be presented to CD4+ T cells, thereby triggering a humoral immune response. By simultaneously triggering cell-mediated and humoral immune responses, the fusion protein described herein can effectively treat cancer or viral infection.

[0053] Thus, the fusion protein sequentially includes, from the N-terminus to the C-terminus: an APC-binding peptide or a CD91 receptor-binding peptide, a translocation peptide, a linker peptide, a cysteine-rich peptide, and an ER retention sequence.

[0054] In one embodiment, an amino acid sequence having at least 90% or at least 95% identity with SEQ ID NO:1 can be used as the APC-binding peptide or the CD91 receptor-binding peptide described herein. In one embodiment, the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 90% identity with SEQ ID NO:2 can be used as the translocation peptide described herein. In one embodiment, the amino acid sequence of SEQ ID NO:3 or an amino acid sequence having at least 90% identity with SEQ ID NO:3 can be used as the linker peptide described herein.

[0055] Variants having sequence identity with an APC-binding peptide or a CD91 receptor-binding peptide, a translocation peptide, a linker peptide, a cysteine-rich peptide, and an ER retention sequence can also be used in the present disclosure. The sequence alignment methods for comparing or determining the percentage of sequence identity are well-known methods. The optimal alignment of sequences can be carried out by a variety of methods, for example, using the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443 (1970)), using the similarity search method of Pearson and Lipman (Proc. Nat’l. Acad. Sci. USA 85:2444 (1988)), or through computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see Brent et al., Current Protocols in Molecular Biology (2003)), or using algorithms well-known in the art, including the BLAST and BLAST 2.0 algorithms, which are described in the journal articles of Altschul et al. (Nuc. Acids Res. 25:3389-3402 (1977)) and Altschul et al. (J. Mol. Biol. 215:403-410 (1990)), respectively. The software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information of the United States.

[0056] In one embodiment, in the cysteine-rich peptide carrying an antigen, the cysteine-rich peptide comprises the amino acid sequence of PCCGCCGCGC (SEQ ID NO: 4).

[0057] In one embodiment, any endoplasmic reticulum (ER) retention sequence can be used in the fusion protein. The endoplasmic reticulum (ER) retention sequence plays an important role in controlling the surface expression of ion channels. It serves as a quality control mechanism that only allows correctly assembled ion channels (where the retention signal is sterically hidden) to be transported to the cell surface. KDEL is a common sequence for ER retention. Thus, an ER retention sequence similar to the KDEL sequence can be used in the present disclosure. In one embodiment of the fusion protein described herein, the simultaneous presence of a linker peptide (e.g., having the amino acid sequence of SEQ ID NO:3) and an APC presenting cell-binding peptide or a CD91 receptor-binding peptide (e.g., having the amino acid sequence of SEQ ID NO:1) helps to stabilize the folding of the fusion protein and promotes the recognition of target cells and stronger binding between the target cell surface receptor and the APC presenting cell-binding peptide or the CD91 receptor-binding peptide, thereby inducing more efficient internalization of the fusion protein.

[0058] A method for preparing a cysteine-rich peptide with an antigen by a linker is described in the article by Chiang, Hsiao-Ling et al. ("A novel synthetic bipartite carrier protein for developing glycotope-based vaccines." Vaccine vol.30, 52 (2012): 7573-81. doi:10.1016 / j.vaccine.2012.10.041).

[0059] In some embodiments, the fusion protein described herein may sequentially include the peptide sequences shown in Table 1 from the N-terminus to the C-terminus.

[0060] Table 1

[0061] In addition, an antigen of interest (e.g., a cancer-associated carbohydrate antigen as described herein) is linked to a cysteine residue in a cysteine-rich peptide by the method described in the article by Chiang, Hsiao-Ling et al. ("A novel synthetic bipartite carrier protein for developing glycotope-based vaccines." Vaccine vol.30, 52 (2012): 7573-81. doi:10.1016 / j.vaccine.2012.10.041). As an example, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) can be used as a crosslinking agent to link the antigen to the cysteine residue in the cysteine-rich peptide.

[0062] In some embodiments, the fusion protein described herein comprises the following sequence or consists of the following sequence. MAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGN DALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKP SNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAIS HAGVSVVMAQTQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWE GKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQP RGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQP EQARLALTLAAAESERFVRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDA LLERNYPTGAEFLGDGELGGGGSGSPCCGCCGCGCPCCGCCGCGCPCCGCCGCGCP CCGCCGCGCPCCGCCGCGCPCCGCCGCGCPCCGCCGCGCKLAAASGHHHHHHGR EDLK (SEQ ID NO:9)

[0063] The design of fusion proteins containing endotoxin A domains Ia, Ib, and II within the genus Pseudomonas, cysteine-rich peptides bearing antigens (e.g., Tn antigen, T antigen, and / or sTn antigen), and ER retention peptides can successfully induce strong adaptive immune responses in individuals with cancer, including both humoral immune responses and cell-mediated immune responses, thereby reducing the risk of cancer metastasis and recurrence in the individual.

[0064] Pharmaceutical Compositions and Methods of Administration

[0065] The fusion proteins and the compositions described herein can be used as vaccines and / or antigenic compositions to induce protective immune responses in vertebrates. The present disclosure provides pharmaceutical compositions comprising the fusion proteins. The pharmaceutical compositions of the present disclosure are formulated with suitable diluents, carriers, excipients, and other agents to provide improved transfer, delivery, tolerability, etc. These compositions can be formulated for specific uses, such as for veterinary use or for pharmaceutical use in humans. The form of the composition and the excipients, diluents, and / or carriers used will depend on the intended use of the fusion protein and, for therapeutic uses, also on the mode of administration. A variety of suitable formulations can be found in formularies well known to all pharmaceutical chemists, including Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (e.g., LIPOFECTIN.TM., Life Technologies, Carlsbad, Calif.), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0066] The fusion proteins and pharmaceutical compositions described herein can be administered to a patient (e.g., a human patient with cancer) in a variety of dosage forms to treat cancer or induce an immune response.

[0067] Examples of cancers include, but are not limited to, breast cancer, liver cancer, colorectal cancer, bladder cancer, cervical cancer, leukemia, lung cancer, central nervous system cancer, melanoma, ovarian cancer, gastric cancer, pancreatic cancer, head and neck cancer, kidney cancer, and prostate cancer.

[0068] Cancer can be treated by an immune response, particularly an adaptive immune response. Exemplars of an adaptive immune response include, but are not limited to, a humoral immune response, a cell-mediated immune response, or a combination of the foregoing.

[0069] The fusion proteins and compositions described herein can be administered by a variety of routes, such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intraocularly, or parenterally. The most appropriate route of administration in any particular case will depend on the fusion protein being administered, the patient, the method of drug formulation, the method of administration, the age, weight, sex of the patient, the severity of the disease being treated, the diet and excretion rate of the patient.

[0070] The effective amount of a particular agent can be expressed in a variety of ways depending on the nature of the agent, such as mass / volume, cell number / volume, particle / volume, (agent mass) / (individual mass), cell number / (individual mass), or particle / (individual mass). The effective amount of a particular agent can also be expressed as the maximum half-maximal effective concentration (EC50), which represents the concentration of the agent that results in a physiological response intensity that is between the reference level and the maximum response level.

[0071] The following examples are provided to illustrate certain embodiments of the invention and to assist those of ordinary skill in the art to which the invention pertains in practicing the invention. These examples should in no way be construed as limiting the scope of the invention in any way. Without further elaboration, it is believed that those of ordinary skill in the art to which the invention pertains can make full use of the invention based on the embodiments described in the specification. All publications cited herein are incorporated herein by reference in their entirety.

[0072] Examples

[0073] Example 1: Fusion protein-induced cell-mediated immune response

[0074] To evaluate whether the fusion protein can be internalized into the cytoplasm, mStrawberry (mSB) red fluorescence was used as an indicator and fused to the fusion protein with or without the PE domain (domains Ia, Ib, and II) and the ER retention sequence REDLK. The recombinant mSB fluorescent protein was cultured with NIH 3T3 cells at 37 °C for 30 minutes. Subsequently, the cells were washed with an acidic phosphate-buffered saline (PBS) solution to remove the excess recombinant fluorescent protein. Images were acquired using an Olympus microscope equipped with an Olympus DP70 digital camera; original magnification 10x; scale bar = 200 μm.

[0075] As Figure 2As shown, the mSB fluorescent protein with a PE domain and an ER retention sequence REDLK can enter cells. In contrast, no fluorescence was observed in the construct group without the PE domain and the ER retention sequence REDLK. These results indicate the importance of the PE domain and the ER retention sequence REDLK in antigen internalization into cells.

[0076] Example 2: Cell-mediated immunity induced by fusion protein

[0077] The LDH release assay was performed to confirm the ability of the fusion protein to induce T cell cytotoxicity. BALB / c mice were immunized by intramuscular injection of the fusion protein (PE(△III)+Cys42Tn+REDLK) or the adjuvant CpG1018 once every two weeks for a total of four times (on days 1, 15, 29, and 43 respectively). That is, for the mice receiving the fusion protein, 50 μL of the fusion protein (at a concentration of 0.4 mg / mL) and the adjuvant CpG1018 were administered each time. As a control group, another group of mice received only the adjuvant CpG1018.

[0078] On day 49 after immunization, T cells (effector cells, E) were isolated from the spleen and co-cultured with MDA-MB-231 cells (target cells, T) expressing the Tn antigen at different T:E ratios at 37 °C for 18 hours. That is, 50 μL of the cell suspension (MDA-MB-231 cells) was added to each well of a flat-bottom 96-well culture plate to reach 6250 cells per well. Then, 50 μL of the T cell suspension (isolated from the mouse spleen) was further added at different T:E ratios (T:MDA-MB-231 cells; E:T cells; T:E = 1:3, 1:10, 1:30, and 1:100). The co-cultures were incubated at 37 °C for 18 hours. Then, the amount of LDH released from the damaged cells was detected to determine the cytotoxicity of the T cells obtained from the fusion protein-treated mice.

[0079] The percentage of cytotoxicity was calculated based on the following equation. Test group: LDH released from MDA-MB-231 cells treated with the fusion protein High control group: Total LDH in MDA-MB-231 cells not treated with the fusion protein Low control group: Spontaneously released LDH from MDA-MB-231 cells not treated with the fusion protein

[0080] Figure 3Data showed that a T:E ratio of 1:100 led to a significant increase in the cytotoxicity of T cells from mice receiving the fusion protein against MDA-MB-231 cells expressing Tn antigen compared to mice receiving only adjuvant (p = 0.004), indicating that the fusion protein was able to induce a strong cell-mediated immune response.

[0081] Example 3: Humoral immune response induced by fusion protein

[0082] To evaluate whether the fusion protein could induce a humoral immune response, sera were collected from the mice receiving the fusion protein or adjuvant in Example 2 to monitor antibody production against the Tn antigen. Polycysteine or polycysteine conjugated to Tn was used as a substrate to detect serum samples obtained from mice immunized with the fusion protein or adjuvant alone.

[0083] As Figure 4 verified by the Western blot assay in

[0084] Various reference data, such as patents, patent applications, and publications, are cited herein, and their contents are incorporated herein by reference in their entirety. In addition, all reference data mentioned herein are specifically incorporated by reference to disclose and describe the methods and / or materials related to the cited publications.

Claims

1. A fusion protein that sequentially includes from the N-terminus to the C-terminus: An antigen-presenting cell-binding peptide or a CD91 receptor-binding peptide, wherein the antigen-presenting cell-binding peptide or the CD91 receptor-binding peptide has the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 90% identity with SEQ ID NO:1; A translocation peptide, wherein the translocation peptide has the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 90% identity with SEQ ID NO:2; A peptide, wherein the peptide has the amino acid sequence of SEQ ID NO:3 or an amino acid sequence having at least 90% identity with SEQ ID NO:3; One or more repeated cysteine-rich peptides carrying an antigen, wherein the cysteine-rich peptide comprises the amino acid sequence of PCCGCCGCGC (SEQ ID NO:4); and optionally A retention signal domain, wherein the retention signal domain is selected from an endoplasmic reticulum retention sequence, a Golgi retention sequence, and a proteasome localization sequence.

2. The fusion protein according to claim 1, wherein the cysteine-rich peptide comprises 2 to 15 repeats of the amino acid sequence PCCGCCGCGC (SEQ ID NO:4).

3. The fusion protein according to claim 2, wherein the cysteine-rich peptide comprises 7 repeats of the amino acid sequence PCCGCCGCGC (SEQ ID NO:4).

4. The fusion protein according to claim 1, wherein the antigen is linked to at least one cysteine residue in the cysteine-rich peptide.

5. The fusion protein according to claim 4, wherein the antigen is a carbohydrate antigen.

6. The fusion protein according to claim 4, wherein the antigen comprises a cancer-related antigen.

7. The fusion protein according to claim 4, wherein the antigen comprises one or more antigens selected from the group consisting of: T, sTn, Tn, GM2, GM3, phosphoserine, phosphothreonine, sialic acid, N-acetylglucosamine, Globo H, Lewis oligosaccharide x, Lewis oligosaccharide y, and steroid hormones.

8. The fusion protein according to claim 7, wherein the antigen comprises a T antigen, an sTn antigen, a Tn antigen, or a combination thereof.

9. The fusion protein according to claim 1, wherein the endoplasmic reticulum retention sequence comprises the amino acid sequence of KDEL (SEQ ID NO:6), RDEL (SEQ ID NO:7), or REDLK (SEQ ID NO:8).

10. The fusion protein according to claim 9, wherein the endoplasmic reticulum retention sequence comprises the amino acid sequence of REDLK (SEQ ID NO:8).

11. The fusion protein according to claim 1, wherein the fusion protein sequentially includes from the N-terminus to the C-terminus: The APC-binding peptide or the CD91 receptor-binding peptide; The translocation peptide; The peptide; The cysteine-rich peptide; and The endoplasmic reticulum retention sequence.

12. The fusion protein according to claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:

9.

13. An isolated nucleic acid encoding the fusion protein as claimed in claim 1.

14. A host cell comprising the isolated nucleic acid as claimed in claim 13.

15. A pharmaceutical composition comprising the fusion protein as claimed in claim 1 and a pharmaceutically acceptable carrier.

16. A method of treating cancer in an individual in need thereof, comprising administering to the individual an effective amount of the fusion protein as claimed in claim 1.

17. The method as claimed in claim 16, wherein the cancer is selected from the group consisting of breast cancer, liver cancer, colorectal cancer, bladder cancer, cervical cancer, leukemia, lung cancer, central nervous system cancer, melanoma, ovarian cancer, gastric cancer, pancreatic cancer, head and neck cancer, kidney cancer and prostate cancer.

18. A method of inducing an immune response in an individual, comprising administering to the individual an effective amount of the fusion protein as claimed in claim 1.

19. The method as claimed in claim 18, wherein the immune response is an adaptive immune response.

20. The method as claimed in claim 19, wherein the adaptive immune response is a humoral immune response, a cell-mediated immune response or a combination of the foregoing.

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