Peptide saporin complex for cancer treatment

Abstract

We provide novel, specific cytotoxic drug therapies with minimal side effects. [Solution] A composition for a membrane-permeable complex for transport across a lipid membrane is provided, comprising one or more molecular induction system (MGS) peptides and a cytotoxic agent. A method for treating cancer is also provided, comprising administering this composition to a cancer patient.

Description

Technical Field

[0001] Description Regarding Federal Government Funds This invention was made with government support under Grant No. 7R01CA164447-04 awarded by the National Cancer Institute of the National Institutes of Health, U.S. Department of Health and Human Services. The government has certain rights in this invention.

Background Art

[0002] The American Cancer Society estimates that this year, more than 1.6 million new cases of cancer will be diagnosed in the United States and nearly 600,000 people will die from cancer. The burden on society is enormous. The NIH estimates that cancer causes an annual burden of $216.6 billion, of which $89 billion is direct medical costs. The first-line treatment for most cancers still relies on cytotoxic therapy. These treatments require actively dividing cells and cause harmful side effects. Treatments are typically given at the maximum tolerated dose rather than the maximum effective dose. Also, these treatments are ineffective against quiescent cancer cells, stem cells, or poorly differentiated cancer cells because they are not actively dividing. As a result, recurrence occurs. Most targeted therapies that are more specific for tumor biomarkers are cell cycle inhibitors rather than cytotoxic agents. Therefore, they may slow tumor growth but do not cure the tumor. Finally, resistance to both targeted and non-targeted therapies exists clinically. Thus, there is a need for novel specific cytotoxic therapies with minimal side effects.

Summary of the Invention

[0003] Saporins are ribosome-inactivating proteins (RIP, 34KD) that lack a cell entry domain. Considering their cell entry mechanism, saporins catalytically remove a single adenine from the ribosomal RNA of the ribosome's large subunit, completely inactivating the ribosome. Therefore, if saporins can be delivered to the intracellular side, they become potent toxins that rapidly kill cells. Described herein are cancer-targeted therapies comprising one or more MGS peptides and plant toxin saporins. Depending on the embodiment, the compositions described herein can bind to cancer cells, mediate their internal migration into cancer cells, and consequently induce rapid cell death. Due to the cancer specificity of MGS peptides, these therapies have minimal effect on normal tissues.

[0004] The compositions described herein can be prepared as fusion proteins, i.e., by chemical bonding. The complex is deliverable, and MGS directs saporins to tumors, mediating their uptake by tumor cells, where they can bind to their targets. This compound offers several advantages over current treatments. For example, in some embodiments, the disclosed compositions (1) allow highly potent cytotoxic agents to be delivered into cells and exert their function there; (2) MGS can be cancer-specific, minimizing toxin uptake in other tissues; (3) because all cells, even non-dividing cells, depend on protein synthesis, the MGS peptide-saporin complex may be effective against cells resistant to common cytotoxic agents; (4) resistance is less likely to be due to the protein mechanism of action, and multidrug resistance pumps are less likely to negate activity; and (5) peptide targeting agents are small, relatively inexpensive compared to antibody targeting agents, and complex with saporins. It is possible.

[0005] Disclosed herein are one or more molecular guidance systems. This composition contains (MGS) peptide and a cytotoxic agent.

[0006] Disclosed herein are membrane-permeable complexes for transport across lipid membranes, which include: one or more molecular induction system (MGS) peptides and cytotoxic agents.

[0007] Disclosed herein is a method for targeting intracellular targets, which comprises administering one or more MGS peptides that are complexed with a cytotoxic agent, the cytotoxic agent targeting the intracellular target.

[0008] Other features and advantages of this composition and method are described in the following description, drawings, and claims.

[0009] The accompanying drawings are incorporated into this specification and constitute part thereof, illustrating several embodiments of the disclosed methods and compositions, and serving to illustrate the principles of the disclosed methods and compositions in conjunction with the description. [Brief explanation of the drawing]

[0010] [Figure 1] This study demonstrates that HCC15.2 is specific to cancer cells and can interlocate into cancer cells. [Figure 2] This demonstrates that HCC15.2 binds to all subtypes of NSCLC. [Figure 3] This indicates that peptide internalization is receptor-mediated. [Figure 4] This study demonstrates that tetramerization of sequence number 1 does not significantly improve internal translocation compared to the monomer of sequence number 1. [Figure 5] This study demonstrates that cleavage at the N-terminus and C-terminus reveals the minimal binding sequences (FHAVPQSFYTAP, SEQ ID NO: 1; FHAVPQSFYTA, SEQ ID NO: 2; and FHAVPQSFYT, SEQ ID NO: 3). [Figure 6]This study demonstrates that acetylation improves the binding of cleavage-type peptides (SEQ ID NO: 3, FHAVPQSFYTAP, SEQ ID NO: 1). [Figure 7] This demonstrates that peptides coexist with lysosomes. [Figure 8] This shows that HCC15.2 accumulates in lysosomes over time. [Figure 9] The in vitro drug data for HCC15.2 complexed with saporin are shown. [Figure 10] The HCC15.2 saporin complex reduces tumor growth in a human NSCLC xenograft model. [Figure 11] This shows the columnar sections obtained from flow cytometry experiments and the quantification of those sections. [Figure 12] This bar graph shows that HCC15.2 binds to other cancer cell lines. [Figure 13] This is a line graph showing the results of flow cytometry to measure KD. [Figure 14] This study demonstrates that tetramerization does not significantly improve the maximum bonded half. [Figure 15] This demonstrates that cleavage at the N-terminus and C-terminus reveals the minimal binding sequence. The upper panel shows the following sequences from top to bottom: SEQ ID NOs: 1, 2, 3, 78, and 79. The lower panel shows the following sequences from top to bottom: SEQ ID NOs: 1, 2, 3, 78, 79, and 80. [Figure 16] This shows that HCC15.2 accumulates in lysosomes over time. [Figure 17] This study demonstrates that HCC15.2-targeted NIR dyes accumulate in tumors in vivo. [Figure 18] This demonstrates that HCC15.2-targeted dyes are retained in tumors over time. [Figure 19] We demonstrate that tumors show clear accumulation of HCC15.2-targeted dye ex vivo. [Figure 20] The results of ex vivo imaging of organs are shown. [Figure 21] HCC15.2 is shown to direct saporin towards cancer cells in vitro. [Figure 22] It is an example of a cancer cell killing assay in which HCC15.2 is shown to direct saporin towards cancer cells in vitro. [Figure 23] HCC15.2 directed towards saporin is shown to significantly slow tumor growth. [Figure 24] It is a table listing various MGSs, indications, target cell types, intracellular locations, and payloads to be delivered. [Figure 25] For the selected MGS peptides, it shows the sequences (in order from top to bottom: SEQ ID NOs: 22, 39, 40, 23, 47, 25, 26, 27, 28, and 29), intracellular locations, valencies, cancer specificities, and binding profile information. [Figure 26] It is a table showing further characterization of the selected MGS peptides. The starting sequences are, from top to bottom: SEQ ID NOs: 7, 5, 36, 32, and 1. The current sequences are, from top to bottom: SEQ ID NOs: 81, 82, 83, 84, and 80. [Figure 27] It shows the co-localization of the 15-mer of H1299.3 with autophagosomes (upper panel), and the results after administration in vivo. [Figure 28] It is a bar graph showing the cancer cell binding of 1299.3Ac-15-mer compared to normal cell binding. [Figure 29] It is a table showing saporin delivery in vitro using other peptides (except HCC15.2, unoptimized parent peptides). [Figure 30] It shows examples of multimeric MGS peptides and the experimental results using the multimeric MGS peptide (SEQ ID NO 22). MODE FOR CARRYING OUT THE INVENTION

[0011] The methods and compositions disclosed can be more readily understood by referring to the following detailed description of specific embodiments and the examples contained herein, as well as by referring to the drawings and the descriptions before and after them.

[0012] Before disclosing and describing the compositions and methods, it should be noted that, unless otherwise specified, they are not limited to specific synthesis methods or specific reagents, and are therefore, of course, modifiable. Similarly, the terms used herein are intended solely to describe specific embodiments and not to limit them. Any methods and materials similar to or equivalent to those described herein may be used in the execution and testing of the present invention, but illustrative methods and materials are described herein.

[0013] Furthermore, naturally, unless explicitly specified otherwise, no method described herein is intended in any way to be deemed to require that the steps of that method be performed in a particular order. Accordingly, if a method claim does not actually enumerate the order in which the steps of that method should be followed, or if it is not specifically specified in the claim or specification that the steps are limited to a specific order, no order is intended in any way to be inferred. This applies to all possible unexpressed interpretive basis, including logical matters relating to the arrangement of the flow of steps or operations, obvious meanings arising from grammatical construction or punctuation, and the number or types of embodiments described in the specification.

[0014] All publications referenced herein are incorporated herein by reference in relation to the content of which such publications are cited in order to disclose or explain methods and / or materials. The publications described herein merely indicate that they were disclosed prior to the filing date of this application. Nothing in this specification should be considered an endorsement that the present invention is not considered to precede such publications on the grounds of prior art. Furthermore, the dates of publications presented herein may differ from the actual publication dates, which may need to be verified.

[0015] definition As used herein and in the appended claims, the singular forms "a," "an," and "the" also include plural references unless explicitly stated in the context.

[0016] Where used herein, the word "or" means any one member of a particular enumeration, as well as any combination of multiple members of that enumeration. For example, a reference to "a MGS peptide" includes cases where there are multiple such MGS peptides, and a reference to "the MGS peptide" refers to one or more MGS peptides and their equivalents known to those skilled in the art.

[0017] Throughout this specification and the claims, the word “comprise” and its variations, e.g., “comprising” and “comprises,” mean “including, but not limited to,” and are not intended to exclude, for example, other additional components, elements, integers, or processes. More specifically, in any method described as including one or more processes or operations, each process is specifically intended to include those listed (unless the process includes a limiting term such as “consisting of”), and this means that each process is not intended to exclude, for example, other additional components, elements, integers, or processes not listed in the process.

[0018] A range may be expressed herein as from one specific value to and / or from another specific value to the same specific value. Where such a range is expressed, further aspects include from one specific value to and / or from another specific value. Similarly, where a value is expressed as an approximation by the use of the antecedents "about" or "about," the specific value naturally forms further aspects. Furthermore, each limit value of a range is meaningful both in relation to the limit value of the other and independently of the limit value of the other. Also naturally, there may be multiple values ​​disclosed herein, and each value is also disclosed herein as an "about" specific value in addition to its specific value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Also naturally, each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0019] Where used herein, the terms “optional” or “optional” mean that the event or situation described thereafter may or may not occur, and that this description includes both cases in which the event or situation occurs and cases in which it does not occur.

[0020] Where used herein, the term “subject” refers to the target of administration, e.g., humans. That is, the subjects of the disclosed methods may be vertebrates, e.g., mammals, fish, birds, reptiles, or amphibians. The term “subject” also includes domestic animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, fruit flies, etc.). In one embodiment, the subject is a mammal. In another embodiment, the subject is a human. This term does not imply a specific age or sex; that is, adult, child, adolescent, and neonatal subjects. This is intended to include, as well as, the fetus, regardless of whether it is male or female.

[0021] Where used herein, the term “patient” refers to a subject suffering from a disease or disorder. The term “patient” includes human and veterinary subjects. Depending on the aspect of the disclosed method, the “patient” may be, for example, diagnosed with an autoimmune disorder requiring treatment prior to the administration step.

[0022] Where used herein, the term “amino acid sequence” refers to a list of abbreviations, letters, symbols, or words representing amino acid residues. The amino acid abbreviations used herein are conventional single-letter amino acid codes, represented as follows: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine.

[0023] As used herein, "polypeptide" refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is composed of a sequence of amino acids. The term "polypeptide" encompasses both natural and synthetic molecules.

[0024] Furthermore, as used herein, the term "polypeptide" refers to amino acids linked to each other by peptide bonds or modified peptide bonds, such as peptide isosters, and this term may include modified amino acids other than the 20 amino acids encoded by genes. Polypeptides can be modified by natural processes, such as post-translational processing, or by chemical modification techniques well known in the art. Modifications can occur at any part of the polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. The same type of modification can be present at multiple sites in a given polypeptide, to the same or different degrees. Similarly, a given polypeptide can have multiple types of modifications. Modifications include, without particular limitation, tRNA-mediated amino acid addition to proteins such as acetylation, acylation, ADP-ribosylation, amidation, covalent crosslinking or cyclization, covalent addition of flavin, covalent addition of heme moiety, covalent addition of nucleotide or nucleotide derivative, covalent addition of lipid or lipid derivative, covalent addition of phosphatidylinositol, disulfide bond formation, demethylation, formation of cysteine ​​or pyroglutamic acid, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenization, sulfation, and arginylation. (See Proteins - Structure and Molecular Properties 2nd Ed., TE Creighton, WH Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, BC Johnson, Ed., Academic Press, New York, pp. 1-12 (1983)).

[0025] When used herein, the term "nucleic acid" refers to a natural or synthetic oligonucleotide or polynucleotide that can hybridize with a complementary nucleic acid by Watson-Crick base pairing, regardless of whether it is DNA, RNA, or a DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense. The nucleic acids of the present invention may also include nucleotide analogs (e.g., BrdU) and non-phosphodiester nucleoside bonds (e.g., peptide nucleic acid (PNA) or thiodiester bonds). In particular, nucleic acids are And, without any particular restriction, we can list DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0026] As used herein, “Sample” means an animal; animal tissue or organ; cells (whether internal to the subject, taken directly from the subject, or derived from cells or cultured cell lines maintained in culture); cell lysates (or lysate fractions) or cell extracts; or a solution containing one or more molecules (e.g., polypeptides or nucleic acids) derived from cells or cellular material that are assayed as described herein. Samples may also be any bodily fluids or excretions containing cells or cellular components (e.g., blood, urine, feces, saliva, tears, bile, etc.).

[0027] As used herein, “adjust” means to change by increasing or decreasing.

[0028] Where used herein, the “effective dose” of a compound means an amount of the compound sufficient to provide the desired effect. The exact required amount will vary from subject to subject, depending on the species, age, and overall condition of the subject, the severity of the disease being treated (or the underlying genetic defect), the specific compound used, and the mode of administration. In other words, it is impossible to specify an exact “effective dose.” However, a suitable “effective dose” can be determined by those skilled in the art using only standard experimental methods.

[0029] As used herein, “isolated polypeptide” or “purified polypeptide” means a polypeptide (or fragment thereof) that is substantially free of materials typically associated with polypeptides in nature. Polypeptides or fragments thereof of the present invention can be obtained, for example, by extraction from natural sources (e.g., mammalian cells), by expression of recombinant nucleic acids encoding the polypeptide (e.g., in cells or in cell-free translation systems), or by chemical synthesis of polypeptides. Polypeptide fragments can also be obtained by any of these methods, or by cleavage of full-length proteins and / or polypeptides.

[0030] As used herein, “isolated nucleic acid” or “purified nucleic acid” means DNA from the natural genome of the organism from which the DNA of the present invention is derived, excluding genes adjacent to that gene. Therefore, this term includes, for example, recombinant DNA incorporated into a vector, such as a self-replicating plasmid or virus; or DNA incorporated into the genomic DNA of a prokaryotic or eukaryotic organism (e.g., a transgene); or DNA existing as an isolated molecule (e.g., by PCR, restriction endonuclease digestion, or chemical or in vitro). This term includes cDNA or genomes or cDNA fragments produced by in vitro synthesis. This term also includes recombinant DNA, which is part of a hybrid gene encoding additional polypeptide sequences. The term “isolated nucleic acid” also refers to RNA, e.g., mRNA molecules encoded by isolated DNA molecules, or chemically synthesized mRNA molecules, or mRNA molecules isolated from or substantially free from at least some kind of cellular component, e.g., other types of RNA molecules or polypeptide molecules.

[0031] As used herein, “to treat” means administering the compounds or molecules of the present invention to a subject having cancer, for example, a human or other mammal (e.g., an animal model), for the purpose of preventing or delaying the worsening of the effects of the disease or symptoms, or partially or completely reversing the effects of the disease.

[0032] As used herein, “prevent” means minimizing the chances of cancer developing in a person who is highly susceptible to developing cancer.

[0033] As used herein, “specifically binds” means that an antibody recognizes and physically interacts with its congener antigen or target (e.g., a disclosed synthetic MGS sequence), but does not specifically recognize or interact with other antigens or targets; such antibodies may be polyclonal or monoclonal antibodies, and they are produced by techniques well known in the art.

[0034] Where used herein, “probe,” “primer,” or “oligonucleotide” means a single-stranded DNA or RNA molecule of a defined sequence that can base-pair with a second DNA or RNA molecule containing a complementary sequence (“target”). The stability of the resulting hybrid depends on the degree of base-pairing that occurs. The degree of base-pairing is influenced by parameters such as the degree of complementarity between the probe molecule and the target molecule, and the degree of stringency of the hybridization conditions. The degree of hybridization stringency is influenced by parameters such as temperature, salt concentration, and concentration of organic molecules such as formamide, and is determined by methods known to those skilled in the art. Probes or primers specific to nucleic acids (e.g., genes and / or mRNA) capable of encoding the disclosed MGS sequence have at least 80% to 90% sequence complementarity, preferably at least 91% to 95% sequence complementarity, more preferably at least 96% to 99% sequence complementarity, and particularly preferably 100% sequence complementarity with the region of the nucleic acid capable of encoding the disclosed MGS sequence with which they hybridize. Probes, primers, and oligonucleotides can be labeled detectably with radioactive or non-radioactive labels by methods well known to those skilled in the art. Probes, primers, and oligonucleotides are used in methods involving nucleic acid hybridization, such as: nucleic acid sequencing, reverse transcription and / or nucleic acid amplification by polymerase chain reaction, single-stranded higher-order polymorphism (SSCP) analysis, restriction enzyme fragment polymorphism (RFLP) analysis, Southern hybridization, Northern hybridization, in situ hybridization, and electrophoretic mobility shift analysis (EMSA).

[0035] As used herein, “specifically hybridizes” means that a probe, primer, or oligonucleotide recognizes a substantially complementary nucleic acid (e.g., a nucleic acid capable of encoding the disclosed MGS sequence) under high stringent conditions and physically interacts with it (i.e., forms base pairs) but does not substantially form base pairs with other nucleic acids.

[0036] As used herein, “high stringent conditions” refers to 0.5M NaHPO4 This refers to conditions that enable hybridization comparable to that achieved using a DNA probe of at least 40 nucleotides in length, in a buffer containing 7% SDS, 1 mM EDTA, and 1% BSA (fraction V) at 65°C, or in a buffer containing 48% formamide, 4.8 × SSC, 0.2 M Tris-Cl (pH 7.6), 1 × Denhart solution, 10% dextran sulfate, and 0.1% SDS at 42°C. Other conditions for highly stringent hybridization, e.g., for PCR, Northern, Southern, or in situ hybridization, and for DNA sequencing, are well known to those skilled in the field of molecular biology. (See, for example, F. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998).

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the field to which the disclosed methods and compositions belong. Any methods and materials similar to or equivalent to those described herein may be used to perform or test the methods and compositions, but any particularly useful methods, apparatus, etc. The materials and other details are as described herein. Publications cited herein and the materials for which they are cited are specifically incorporated herein by reference. Nothing in this specification should be considered an endorsement that the present invention is not considered to precede such publications on the grounds of prior art. No reference constitutes an endorsement of prior art. The descriptions of references are intended to describe what their authors claim, and the applicant has the right to object to the accuracy and appropriateness of the cited documents. Obviously, although multiple publications are mentioned herein, such references do not constitute an endorsement that any of those documents form part of the common understanding in the art.

[0038] Disclosed are the components used to prepare the disclosed compositions, as well as the compositions themselves used in the methods disclosed herein. While these and other materials are disclosed herein, naturally, when combinations, subsets, interactions, groups, etc., of these materials are disclosed, each is specifically intended and described herein, even if the specific references to each individual and collectively of various combinations and sequences of these compounds are not explicitly disclosed. That is, if a certain class consisting of molecules A, B, and C, and a certain class consisting of molecules D, E, and F are disclosed, and for example, the combination AD is disclosed, then, individually and collectively, it is intended that the combinations AE, AF, BD, BE, BF, CD, CE, and CF are disclosed, even if they are not individually mentioned. Similarly, any subsets or combinations thereof are also disclosed; for example, the subgroups AE, BF, and CE would be considered disclosed. This concept applies to all aspects of this application, including, but not limited to, the steps in the methods for preparing and using the disclosed compositions. In other words, if there are various additional steps that can be performed, each of those additional steps can naturally be performed in any particular embodiment or combination of embodiments of the disclosed method.

[0039] Also disclosed are the components to be used in the preparation of the disclosed compositions, as well as the compositions themselves to be used in the methods disclosed herein. While these and other materials are disclosed herein, naturally, when combinations, subsets, interactions, groups, etc., of these materials are disclosed, specific references to each of the various combinations and sequences of these compounds, both individually and collectively, are not explicitly disclosed, but each is specifically intended and described herein.

[0040] Disclosed herein is a cancer-targeted therapy comprising a peptide complex containing the peptide sequence FHAVPQSFYT (SEQ ID NO: 3) complexed with a saporin, a plant toxin. The saporin is a ribosome-inactivating protein (RIP, 34kD) that lacks a cell entry domain. Considering its cell entry mechanism, the saporin inactivates ribosomes by catalytically removing a single adenine from the ribosomal RNA of the ribosome's large subunit. Therefore, if the saporin could be delivered to the intracellular side, it would be a potent toxin that rapidly kills cells. As described herein, this challenge can be solved by complexing a saporin with a peptide that is selective for cancer cells, binds to cancer cells, mediates the internal translocation of the toxin into the cancer cells, and consequently leads to rapid cell death. Due to the cancer specificity of the MGS peptide complex disclosed herein, this therapy has minimal effect on normal tissues.

[0041] Disclosed herein are anticancer therapies applicable to lung cancer, breast cancer, colorectal cancer, ovarian cancer, and pancreatic cancer. The compounds, compositions, or peptide conjugates disclosed herein are targeted cytotoxic therapies that may have broad applications in the treatment of cell tumors.

[0042] The compositions or complexes disclosed herein (e.g., MGS peptide-saporin complexes) can be prepared as fusion proteins, i.e., by chemical bonding. The complexes are deliverable, and the MGS peptide directs a toxin (e.g., a saporin) towards tumor or cancer cells, mediating the uptake of the saporin into the tumor cells, where it can bind to its target.

[0043] The delivery of the compositions disclosed herein allows for the specific delivery of saporins to cancer cells, targeting them while avoiding or minimizing saporin uptake by normal cells, for example, when one or more molecular induction system (MGS) peptides are coupled to or complexed with saporins. The MGS peptides disclosed herein have low nanomolar affinity to a subset of epithelial-derived cancers. Because these MGS peptides are small, they can be easily complexed with saporins or expressed as fusion proteins. With approximately 1.6 million new diagnoses and nearly 600,000 deaths per year in the United States, resulting in an annual healthcare cost burden of approximately $89 billion, the complexes disclosed herein offer novel cell tumor therapies that have the potential to overcome the limitations of current cancer treatments.

[0044] Peptide HCC15.2 can be cleaved by removing one or more amino acids. Furthermore, a PEG linker can be added to the monomeric peptide, allowing for acetylation of the amino terminus.

[0045] composition MGS peptides. Disclosed herein are molecularly derived system peptides (MGS) or tumor-targeting peptides. These peptides can selectively bind to tumors, including malignant tumors. Examples of MGS peptides usable or modifiable in the disclosed compositions include, but are not limited to, one or more MGS peptides disclosed in McGuire et al., Sci Rep. 2014 Mar 27;4:4480. Examples of MGS peptides usable in the disclosed compositions and methods include, but are not limited to, the MGS sequences shown in Table 1 and Figures 24 and 25. Table 1. Peptide sequences [Table 1] TIFF2026062852000003.tif179168 TIFF2026062852000004.tif178168

[0046] In one embodiment, the composition comprises one or more molecular induction system (MGS) peptides and a cytotoxic agent. In another embodiment, a membrane-permeable complex for transport across a lipid membrane may contain one or more molecular induction system (MGS) peptides and a cytotoxic agent.

[0047] In some embodiments, one or more MGS peptides can be any of the MGS peptides disclosed herein. In some embodiments, one or more MGS peptides include SEQ ID NOs: 1, 2, 3, 34, 35, 36, 37, 38, 39, 40, 41, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84, or a combination thereof. In some embodiments, one or more MGS peptides include SEQ ID NOs: 1, 2, or 3. In some embodiments, the cytotoxic agent can be a saporin or a biologically active variant thereof. In one embodiment, one or more MGS peptides can be SEQ ID NO: 3, and the cytotoxic agent can be a saporin. In one embodiment, the composition may contain one or more MGS peptides, for example, depending on the embodiment. In some embodiments, the composition may contain one, two, three, four, or five MGS peptides. In some embodiments, one or more MGS peptides may form a tetrameric scaffold protein. In some embodiments, one or more MGS peptides disclosed herein may be cleaved. In some embodiments, one or more MGS peptides may be modified. In some embodiments, one or more MGS peptides may be acetylated at the N-terminus. Figure 26 presents an example of selected MGS peptides, which have been further characterized. In some embodiments, one or more MGS peptides may be chemically bonded to a cytotoxic agent. In some embodiments, the chemical bond may be polyethylene glycol (PEG). In some embodiments, the number of PEG units may be one, two, three, four, five, six, seven, eight, nine, ten, eleven, or more. In some embodiments, the number of PEG units may be sufficient to detach one or more MGS peptides from the cytotoxic agent and prevent any static interactions between the one or more MGS peptides and the cytotoxic agent. For example, disclosed herein are compositions comprising a chemical bond, wherein the chemical bond is PEG, and the PEG comprises 11 PEG units. In one embodiment, one or more MGS peptides comprise SEQ ID NO: 3, which can be acetylated at its N-terminus and chemically bonded to PEG; and the cytotoxic agent can be a saporin, which can be chemically bonded to PEG.

[0048] Cytotoxic agents. A wide variety of toxic (e.g., cytotoxic) agents can be included in the disclosed compositions. Cytotoxic agents can form covalent bonds or fusion proteins with one or more MGS peptides disclosed herein. Cytotoxic agents can be proteins. In some embodiments, cytotoxic agents can be bacterial or phytotoxins. In some embodiments, cytotoxic agents can be phytotoxins. In some embodiments, cytotoxic agents can be saporins or their biological variants. Cytotoxic agents are modifiable. In some embodiments, cytotoxic agents are fragments of bacterial or phytotoxins.

[0049] The methods for preparing the complex or fusion proteins are known to those skilled in the art and can be carried out using known techniques. In one embodiment, the MGS peptide is complexed with a cytotoxic agent (e.g., saporin) using polyethylene glycol. Chemically bonding the MGS peptides disclosed herein using optimal PEG units is within the capabilities of those skilled in the art.

[0050] Labels. Also described herein are compositions comprising one or more molecular induction system (MGS) peptides and labels. For example, compositions disclosed herein may include detectable labels. Such detectable labels may include, but are not limited to, tag sequences designed for the detection (e.g., purification or localization) of expressed polypeptides or sequences. Examples of tag sequences include green fluorescent protein, glutathione S-transferase, polyhistidine, c-myc, hemagglutinin, or Flag® tags, which can be fused to encoded nucleic acids. Such detectable labels may include, but are not limited to, fluorescent agents, enzymatic labels, and radioisotopes.

[0051] Pharmaceutical composition Disclosed herein are pharmaceutical compositions comprising one or more compositions disclosed herein and the pharmaceutically acceptable carriers described above. Depending on the embodiment, the MSG peptide may be SEQ ID NO: 3, the cytotoxic agent may be a saporin or a biological variant thereof, and the pharmaceutical composition is formulated for intravenous administration. The compositions of this disclosure also contain a therapeutically effective amount of the cytotoxic agent as described herein. The compositions may be formulated for administration by any of the various routes of administration and may contain one or more physiologically acceptable excipients, the excipients of which are modifiable depending on the route of administration. In this context, the term “excipient” means any compound or substance, including those that may also be called “carrier” or “diluent.” The preparation of pharmaceutically and physiologically acceptable compositions is routinely studied in the art, and therefore, those skilled in the art can consult numerous sources as guidance, if necessary.

[0052] Pharmaceutical compositions as disclosed herein can be prepared for oral or parenteral administration. Pharmaceutical compositions prepared for parenteral administration include those prepared for intravenous (or intra-arterial), intramuscular, subcutaneous, intraperitoneal, transmucosal (e.g., intranasal, vaginal, or rectal), or transdermal (e.g., topical) administration. Aerosol inhalation is also available for delivering the fusion protein. That is, compositions can be prepared for parenteral administration comprising a fusion protein dissolved or dispersed in an acceptable carrier, such as aqueous carriers, e.g., water, buffered water, saline, buffered saline (e.g., PBS), etc. One or more excipients may be used to approximate physiological conditions, e.g., pH adjusters and buffers, osmotic regulators, wetting agents, surfactants, etc. If the composition contains solid components (for example, if they are for oral administration), one or more excipients may act as binders or fillers (e.g., for formulations such as tablets and capsules). When a composition is formulated for application to the surface of the skin or mucous membrane, one or more of the excipients may be solvents or emulsifiers for formulations such as creams and ointments.

[0053] The pharmaceutical compositions are sterile, or can be sterilized by conventional sterilization techniques, or can be sterile filtered. Aqueous solutions can be packaged for ready use or lyophilized, and lyophilized formulations are included in this disclosure, which can be mixed with a sterile aqueous carrier before administration. The pH of the pharmaceutical compositions is typically 3 to 11 (e.g., about 5 to 9) or 6 to 8 (e.g., about 7 to 8). The resulting solid compositions can be packaged in multiple single-dose units, each unit containing a fixed amount of one or more of the above-mentioned agents in a sealed package, for example, a tablet or capsule. The solid compositions can also be packaged in containers for flexible quantities, for example, in a squeeze tube designed for topical creams or ointments.

[0054] Treatment method Disclosed herein are treatments for cancer patients, the methods comprising: (a) identifying a subject in need of treatment; and (b) administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising one or more molecular induction system (MGS) peptides, a cytotoxic agent, and a pharmaceutically acceptable carrier. The MGS peptide may be any of the MGS peptides disclosed herein. The cytotoxic agent may be a saporin or a biological variant thereof.

[0055] Disclosed herein are methods for targeting intracellular targets. These methods may include administering one or more MGS peptides that are complexed with a cytotoxic agent. The cytotoxic agent can target intracellular targets. In some embodiments, the intracellular target may be a lysosome, Golgi apparatus, endoplasmic reticulum, cytoplasm, or nucleus.

[0056] In one embodiment, a person skilled in the art can determine an effective dose, effective schedule, or effective route of administration of the disclosed composition or disclosed fusion protein or disclosed fusion protein so as to induce targeting to an intracellular target such that the intracellular target can be inactivated.

[0057] In one embodiment of any of the methods disclosed herein, the composition, complex, or fusion protein described herein may be used in combination with one or more further treatments. This is possible. In some embodiments, a composition, complex, or fusion protein can be administered alone or in combination with other biological activators in a composition suitable for administration to a subject. In some embodiments, a method, composition, complex, or fusion protein relating to the treatment of a subject having cancer or at risk of developing cancer, disclosed herein, can be used in combination with, for example, a therapeutically effective dose of radiotherapy, immunotherapy, or chemotherapy, or a combination thereof. The combination therapy can be administered as a co-formulation or separately. If administered separately, the combination therapy can be administered simultaneously or sequentially. The formulations can be prepared using the methods of the art.

[0058] The above-described pharmaceutical compositions may be formulated to contain therapeutically effective amounts of the compositions, complexes, or fusion proteins disclosed herein. Therapeutic administration includes prophylactic use. Based on genetic testing and other diagnostic methods, a physician may choose prophylactic administration if, in examination of a patient under their care, the patient has a clinically established predisposition or elevated susceptibility (in some cases, a significantly elevated susceptibility) to one or more autoimmune diseases, or if the patient has a clinically established predisposition or elevated susceptibility (in some cases, a significantly elevated susceptibility) to cancer.

[0059] The pharmaceutical compositions described herein can be administered to a subject (e.g., a human subject or human patient) in an amount sufficient to delay, reduce, or preferably prevent the onset of a clinical disease. Therefore, in some embodiments, the subject is a human subject. In therapeutic use, the composition is administered to a subject (e.g., a human subject) that already has or has been diagnosed with an autoimmune disease in an amount sufficient to at least partially improve the signs or symptoms, or to inhibit (and preferably suppress) the progression of the symptoms, their complications, and their consequences. An amount sufficient to achieve this is defined as a “therapeutic dose.” A therapeutic dose of a pharmaceutical composition may be an amount that achieves a cure, but the outcome is only one of several achievable outcomes. As described above, therapeutic doses include amounts that provide a treatment to delay, interfere with, or prevent the onset or progression of cancer, or amounts that induce remission of an autoimmune disease or the symptoms of an autoimmune disease. Recovery may be accelerated in the treated individual.

[0060] Depending on the circumstances, the cancer may be primary, secondary, refractory, or recurrent. In some circumstances, the cancer may be lung cancer, breast cancer, colorectal cancer, ovarian cancer, or pancreatic cancer.

[0061] The effective dose for this use may depend on the severity of the cancer, as well as the patient's weight and overall condition and health. Suitable regimens for initial and booster doses are represented by an initial dose followed by repeated doses of one or more subsequent doses spaced at intervals of hours, days, weeks, or months.

[0062] The total effective amount of the complex or fusion protein in the pharmaceutical composition disclosed herein can be administered to mammals as a single dose, either by bolus or infusion over a relatively short period of time, or using a divided therapy protocol that administers multiple doses over a longer period of time (e.g., every 4-6, 8-12, 14-16, or 18-24 hours, or every 2-4 days, every 1-2 weeks, or once a month). Alternatively, continuous intravenous infusion sufficient to maintain a therapeutically effective blood concentration is also within the scope of this disclosure.

[0063] The therapeutically effective amount of toxin (or cytotoxic agent) present in the compositions described herein and used in the manner disclosed herein for application to mammals (e.g., humans) should be determined taking into account individual differences in age, weight, and other general conditions (as described above). Those skilled in the art can determine this. Since the compositions, complexes, and fusion proteins of this disclosure may be stable in serum and bloodstream, in more specific cases, the dosage of a composition, complex, or fusion protein containing any of the individual components may be lower (or higher) than the effective dose when any of the individual components are unbound. Accordingly, in some embodiments, when a toxin is administered as part of a complex or fusion protein, it may have increased efficacy or reduced side effects compared to when the toxin is administered alone or not as part of a complex or fusion protein.

[0064] vector Disclosed are vectors comprising nucleic acid sequences encoding one or more of the disclosed compositions. Depending on the embodiment, the vector may comprise only nucleic acid sequences capable of encoding one or more of the disclosed MGS peptides.

[0065] kit The materials described above, as well as other materials, can be packaged together in any suitable combination as a kit useful for carrying out or assisting in carrying out the disclosed method. It is convenient if the kit components in a given kit are designed and adapted to be used together in the disclosed method. For example, what is disclosed is a kit comprising one or more of the disclosed compositions.

[0066] Depending on the embodiment, the kit may include MGS peptides, cytotoxic agents, and instructions for complex formation.

[0067] Depending on the embodiment, the kit may include a cell line containing nucleic acid sequences encoding one or more MGS peptides. [Examples]

[0068] Example 1: HCC15.2 exhibits specificity towards cancer cells and internally migrates into cancer cells. For peptides to be clinically useful reagents, it may be important to distinguish between normal and cancer cells. Figure 1 shows that the MGS peptide, HCC15.2, accumulates in cancer cells and is specific to them compared to controls. HCC15.2 binding was analyzed by flow cytometry (see Figure 1). HCC15.2 does not bind to normal HBEC cells but does bind to the LC cell line H1299. Binding was sequence-dependent and not specific to a single histopathological class (see Figure 2).

[0069] MGS peptides were synthesized using a Prelude synthesizer (Protein Technologies Inc.) with standard solid-phase FMOC techniques. The MGS peptides were purified to >95% purity using a C18 preparative column by reverse-phase HPLC and confirmed by MALDI-TOF. The optimized HCC15.2 peptide sequences were Ac-FHAVPQSFYT-PEG11-biotin with a molecular weight of 2394 Da, and Ac-FHAVPQSFYT-PEG11-C with a molecular weight of 1940 Da. Tetrameric HCC15.2 was synthesized using a previously published protocol. Stock solutions were prepared in PBS (pH 7.4), and the concentration was measured by absorbance at 280 nM using a Nanodrop 2000 (Thermo Fisher Scientific).

[0070] Flow cytometry. Biotinylated peptides were complexed with streptavidin-R-phycoerythrin (1:1) under RT for 30 minutes. The remaining binding site on streptavidin was quenched with RPMI 1640, and the solution was diluted to 25 nM. Tumor cells were then subjected to the cytometry. Cells were grown to a 90% concentration in a 12-well plate and then incubated with 500 μl of peptide dye complex at 37°C for 1 hour. After 1 hour, the peptide was removed, and the cells were washed three times with PBS for 5 minutes each, rinsed twice with acid, and rinsed once with PBS. 300 μl of trypsin was added until the cells floated to the surface, and then 700 μl of RPMI + 5% FBS was added to inactivate the trypsin. The cells were transferred to a flow tube and placed on ice in the dark. Flow cytometry was performed using BD FACSCelesta, and the data were analyzed with Flowing software. A region containing <5% of the cells in the negative control was established, and the McGuire score for each sample was calculated by multiplying the percentage of positive cells by the fluorescence intensity.

[0071] For the flow cytometry experiment, cells are treated with a dye-containing peptide (e.g., MGS peptide) for 1 hour. If the peptide enters the cell(s), the dye will also enter the cell(s) (see Figure 11). Next, the cells are washed, placed in a test tube, and flow cytometry is performed. A flow cytometer can measure the brightness of more than 10,000 individual cells.

[0072] Confocal microscopy was performed. A plasmid containing a GFP-labeled organelle-specific marker was purchased from Addgene and inserted into H1299 cells by electroporation. After G418 sorting, GFP-labeled tumor cells were seeded onto 8-well chamber slides the day before the experiment. Biotinylated peptides were complexed with streptavidin-AlexaFluor 555 (1:1) at RT for 30 minutes, quenched with RPMI, and then added to the wells at 50 nM. After 1 hour incubation, the cells were washed three times with PBS for 5 minutes each, rinsed twice with acid, and rinsed once with PBS. The cells were fixed in 2% formaldehyde for 10 minutes, washed with PBS, stained with DAPI added to mounting media, and covered with coverslips. Microscopic observation was performed using a Zeiss LSM 700 with a Pln Apo 63× / 1.4 oil DIC III objective lens. Compressed images were obtained using the maximum intensity projection method of ImageJ software.

[0073] For microscopic observation experiments, cells are treated with a dye-containing peptide (e.g., MGS peptide) for 1 hour. If the peptide enters the cell(s), the dye will also enter the cell(s) (see Figure 1). Next, the cells are washed and fixed to a glass slide. Other parts of the cell (e.g., cell membrane, organelles, or nucleus) are stained. Fluorescence microscopy is performed to evaluate the dyes in the cells. A series of stratified images that can be used to assemble a 3D image (like MRI) are acquired using confocal microscopy.

[0074] Example 2: Internal transfer of HCC15.2 is mediated by the receptor. Experiments have shown that HCC15.2 can internalize cells, and that this internalization is receptor-mediated (see Figure 3). More specifically, HCC15.2 binds to specific subsets of NSCLC and other tumor cells (see Figure 12). Phage blocking was used as a control. HCC15.2 internalized in a specific panel of cell lines, but not in others. The results also indicate that the receptor-mediated internalization was sequence-specific. Treatment with trypsin for 2 minutes before adding HCC15.2 prevented internalization. Furthermore, HCC15.2 did not internalize at 4°C.

[0075] Figure 4 shows that tetramerization of HCC15.2 does not significantly improve receptor-mediated internal transport. This result is surprising, considering that most peptides studied and identified through the FOX3 molecular induction system showed a non-additive increase in binding / internal transport.

[0076] Example 3: Cutting of HCC15.2 Figures 5 and 15 show that the minimal binding sequence was revealed by cleavage at the N-terminus and C-terminus of the HCC15.2 peptide.

[0077] Figure 6 shows that acetylation improves the binding of cleaved peptides.

[0078] Example 4: The peptide coexists with lysosomes. Figure 7 shows that the MGS peptide coexists with lysosomes and accumulates in the lysosomes over time (see Figures 8 and 16).

[0079] Example 5: HCC15.2 complexed with saporin. Further experiments were conducted to test HCC15.2 complexed with saporin in cell viability assays (see Figure 9 and Table 2). For this purpose, tumor cell lines were seeded in black-walled, transparent, flat-bottomed 96-well plates. The following day, the culture medium was changed with Hcc15.2-saporin, saporin alone, or untreated, with a concentration gradient. After 1 hour of incubation, the treatment was removed and the medium was replaced with fresh culture medium. After 72 hours, cell viability was measured using Cell Titer Glo®, and fluorescence was measured using Analyst HT from LjL Biosystems. IC50 was calculated using Graphpad Prizm.

[0080] A cancer cell killing assay was performed (see Figure 22). Cancer cells were placed in a dish. After 24 hours, different doses of peptide saporin were added to the cells and left for 1 hour. The peptide saporin was washed off, and the cells were left for a further 72 hours. An assay was performed to measure the viability of cells. Figure 21 shows that HCC15.2 directs saporin towards cancer cells in vitro. These results demonstrate that saporin did not enter cells on its own, but entered cells when it formed a complex with the required MGS peptide (e.g., HCC15.2). These results also show that the HCC15.2 saporin reduces off-target effects and evades intracellular vesicles. Table 2. Cell viability in multiple cell lines. [Table 2]

[0081] Figure 10 shows that HCC15.2, when complexed with saporin, reduces tumor growth compared to free saporin in a human NSCLC xenograft model.

[0082] Example 6: Tetramerization does not significantly improve the maximum bonded half. Flow cytometry is performed to measure KD. Cells are treated for 1 hour while increasing the peptide-dye concentration. MGS peptides (or multiple peptides) entering or entering the cells carry the dye with them. Cells are washed, placed in test tubes, and flow cytometry is performed. The flow cytometer can measure the brightness of more than 10,000 individual cells. Each concentration is quantified and plotted (see Figures 13 and 14). When the curve is mathematically fitted to the data, the midpoint is the KD (e.g., a measure of how well the binding is).

[0083] Example 7: HCC15.2-targeted NIR dyes accumulate in tumors in vivo. In vivo imaging was performed. H2009 tumor cells were suspended in sterile PBS at a ratio of 10⁶ cells / 100 μl and subcutaneously injected into the flank of female thymus-deficient nude mice (Jackson Labs.). Cysteine-labeled peptide was complexed with maleimide Alexafluor-750 C5 (1:1.1) in sterile PBS (pH 7.4) for 1 hour. The peptide dye complex was diluted in sterile PBS to 15 μg of dye per 100 μl, and 100 μl was intravenously injected into the lateral tail vein of 4 mice / group. At 12, 24, 48, and 72 hours, the mice were anesthetized with isothesia, and whole animal images were taken with IVIS. Then, at 72 hours, tumors and organs were weighed and imaged ex vivo.

[0084] In vivo animal imaging studies were conducted. For these experiments, human cancer cells were inoculated subcutaneously into mice, and tumors were allowed to grow to a specific size. When the tumors reached the predetermined size, a dye-containing peptide was intravenously injected. Animals were anesthetized and imaged at 12, 24, 48, and 72 hours. Organs and tumors were imaged at 72 hours. Yellow indicates a high proportion of peptide, reddish-brown / red indicates a low proportion of peptide, and colorless indicates a very low / absent proportion of peptide. The HCC15.2-targeted NIR dye accumulated in the tumor (see Figure 17) and remained in the tumor over time (see Figure 18). Figures 19 and 20 show the results of ex vivo imaging.

[0085] Subcutaneous tumor growth assay. H2009 tumors were established subcutaneously in the flank of thymus-deficient nude mice (Jackson Labs). Saporin complexed with streptavidin (SAZAP) was purchased from Advanced Targeting Systems and complexed in a 1:1 ratio with optimized biotinylated Hcc15.2 and a control peptide. When the tumor reached approximately 1003 mM in size, the mice were injected IV via the lateral tail vein with either untargeted saporin (without MGS peptide) or the 15.2-saporin complex, or received no injection. Targeted and untargeted saporin toxins were administered twice weekly at approximately 7 ug / dose for 2.5 weeks. Tumors were blinded and measured every other day with calipers, and tumor volume was calculated using the following formula. π / 6 * (length * width)^3 / 2.

[0086] For the following experimental setup, human cancer cells were inoculated subcutaneously into mice, and tumors were allowed to grow to a specific predetermined size. Once the tumors reached the predetermined size, peptides complexed or linked to saporin were administered intravenously. The animals received five doses of 7 or 7.5 μg / dose over 2.5 weeks. Tumors were measured every other day. Figure 23 shows that targeting of saporin by HCC15.2 significantly slowed tumor growth.

[0087] Example 8: Further characterization of H1299.3 peptide. We evaluated a modified MGS peptide (SEQ ID NO: 31): LQWRRNFGVWARYRL (SEQ ID NO: 31), which was created by fusing amino acids 1-5 and 11-20 of the original MGS peptide. This MGS peptide maintains its oncological properties and has an increased ability to deliver to autophagosomes. Acetylation protects it from degradation in vivo and improves its solubility. This MGS peptide is enhanced in animals in its ability against NSCLC tumors (see Figure 27). The data show that no tumor targeting was observed with the non-acetylated form. This MGS peptide (e.g., modified 15-mer) shows a >2-fold reduction in cardiac, lung, and renal tumors (see Figure 27). The binding results of the 1299.3Ac-15-mer to cells are shown in Figure 28.

[0088] Cell culture. Human NSCLC cell line was provided. The cells were treated with L-glutamine and 5% FB. The cells were cultured in RPMI 1640 containing S at 37°C and 5% CO2.

[0089] Example 9: In vitro delivery of saporin combined with other peptides. Figure 29 shows the IC50 values ​​in various cell lines administered with saporins that were complexed or linked to selected MGS peptides.

[0090] As will be apparent to those skilled in the art, various modifications and alterations can be made to the present invention without departing from the scope or spirit of the invention. Other aspects of the invention will become apparent to those skilled in the art by examining the specifications and practices of the invention disclosed herein. The specification and examples are for illustrative purposes only, and the true scope and spirit of the invention are intended to be shown by the following claims.

[0091] Many equivalents to specific embodiments of the methods and compositions described herein will be apparent to those skilled in the art, or can be confirmed by mere standard experiments. Such equivalents shall be included in the following claims.

Claims

1. A composition comprising one or more molecular induction system (MGS) peptides and a cytotoxic agent.

2. The composition according to claim 1, wherein the one or more MGS peptides include SEQ ID NOs: 1, 2, 3, 34, 35, 36, 37, 38, 39, 40, 41, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or a combination thereof.

3. The composition according to claim 1 or 2, wherein the one or more MGS peptides include SEQ ID NOs: 1, 2, 3, 34, 35, 36, 37, 38, 39, 40, 41, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84.

4. The composition according to any one of claims 1 to 3, wherein the cytotoxic agent is a saporin or a biologically active variant thereof.

5. The composition according to claim 1, wherein the one or more MGS peptides are Sequence ID No. 3, and the cytotoxic agent is a saporin.

6. The composition according to claim 1, wherein the one or more of the above-mentioned types are four MGS peptides.

7. The composition according to claim 6, wherein the one or more MGS peptides form a tetrameric scaffold protein.

8. The composition according to claim 1, wherein one or more MGS peptides are acetylated at their N-terminus.

9. The composition according to any one of claims 1 to 8, wherein one or more MGS peptides are chemically bonded to the cytotoxic agent.

10. The composition according to claim 9, wherein the chemical bond is polyethylene glycol (PEG).

11. The composition according to claim 10, wherein the PEG has a length of 11 units.

12. The composition according to claim 1, wherein the one or more MGS proteins include SEQ ID NO: 3, the N-terminus of SEQ ID NO: 3 is acetylated and chemically bonded to PEG; and the cytotoxic agent is a saporin, the saporin is covalently bonded to PEG.

13. A membrane-permeable complex for transport across a lipid membrane, comprising one or more molecular induction system (MGS) peptides and a cytotoxic agent.

14. The one or more MGS peptides mentioned above are sequence numbers 1, 2, 3, 34, 35, 36, 37, 38, 39, 40, 41, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 The composite according to claim 13, comprising 78, 79, 80, 81, 82, 83, 84, or a combination thereof.

15. The complex according to claim 13 or 14, wherein the one or more MGS peptides include SEQ ID NOs: 1, 2, 3, 34, 35, 36, 37, 38, 39, 40, 41, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84.

16. The composition according to any one of claims 13 to 15, wherein the cytotoxic agent is a saporin or a biologically active variant thereof.

17. The composition according to claim 13, wherein the one or more MGS peptides are Sequence ID No. 3, and the cytotoxic agent is a saporin.

18. The composition according to claim 13, wherein the one or more MGS peptides are four MGS peptides.

19. The composition according to claim 13, wherein the one or more MGS peptides form a tetrameric scaffold protein.

20. The composition according to claim 13, wherein one or more MGS peptides are acetylated at their N-terminus.

21. The composition according to any one of claims 13 to 20, wherein one or more MGS peptides are chemically bonded to the cytotoxic agent.

22. The composition according to claim 21, wherein the chemical bond is polyethylene glycol (PEG).

23. The composition according to claim 22, wherein the PEG has a length of 11 units.

24. The composition according to claim 13, wherein the one or more MGS proteins include SEQ ID NO: 3, the N-terminus of SEQ ID NO: 3 is acetylated and chemically bonded to PEG; and the cytotoxic agent is a saporin, the saporin is covalently bonded to PEG.

25. A pharmaceutical composition comprising the composition described in claim 1 and a pharmaceutically acceptable carrier.

26. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is formulated for intravenous administration.

27. A method for treating cancer, comprising: (a) identifying a patient in need of treatment; (b) administering to the patient a therapeutically effective amount of the composition according to any one of claims 1 to 27; and (c) a pharmaceutically acceptable carrier.

28. The method according to claim 27, wherein the patient is a human patient.

29. The method according to claim 27, wherein the cancer is a primary, secondary, refractory, or recurrent tumor.

30. The cancer is lung cancer, breast cancer, colorectal cancer, ovarian cancer, or pancreatic cancer, as described in claim 27. The method.

31. The method according to claim 27, further comprising administering to the patient a therapeutically effective dose of radiotherapy, immunotherapy, or chemotherapy, or a combination thereof.

32. A method for targeting an intracellular target, comprising administering one or more MGS peptides conjugated with a cytotoxic agent, wherein the cytotoxic agent targets an intracellular target.

33. The method according to claim 32, wherein the intracellular target is a lysosome.

34. A composition comprising one or more molecular induction system (MGS) peptides and a cytotoxic agent, wherein the one or more MGS peptides are selected from the group listed in Table 1.

35. MGS peptide as disclosed herein.

36. A nucleic acid sequence capable of encoding the MGS peptide disclosed herein.

37. A vector comprising the nucleic acid sequence described in claim 36.

38. A cell line comprising the MGS peptide described in claim 34, the nucleic acid sequence described in claim 36, or the vector described in claim 37.

39. MGS peptides containing the sequence of SEQ ID NOs: 1, 2, 3, 34, 35, 36, 37, 38, 39, 40, 41, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84.

40. A nucleic acid sequence capable of encoding the MGS peptide described in claim 39.

41. A vector comprising the nucleic acid sequence described in claim 40.

42. A cell line comprising the MGS peptide described in claim 39, the nucleic acid sequence described in claim 40, or the vector described in claim 41.

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