Use of peptides containing PCNA interaction motifs in the treatment of solid tumors

Abstract

The present invention relates to pharmaceutical compositions and methods for treating carcinomas and sarcomas. In particular, the present invention relates to a pharmaceutical composition comprising a peptide or a pharmaceutically acceptable salt thereof for use in treating carcinoma or sarcoma in a human subject, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 1 and a cell-penetrating peptide, and the pharmaceutical composition is administered systemically to the subject weekly to achieve a weekly dose of about 15 to 65 mg / m2 per body surface area (BSA), calculated as the free form of the peptide. 2 The present invention provides a pharmaceutical composition that provides a dosage of the peptide of

Description

[Technical Field]

[0001] The present invention relates to pharmaceutical compositions and methods for the treatment of carcinomas and sarcomas. More specifically, the present invention relates to a dosage regimen for the use of a pharmaceutical composition comprising a peptide having the amino acid sequence described in SEQ ID NO: 1 in the treatment of carcinoma or sarcoma. [Background technology]

[0002] Cancers are classified into more than 100 types based on the type of cells they originate from. The National Cancer Institute (NCI) lists the major types of cancer (https: / / www.cancer.gov / types). Each type of cancer can be further grouped and classified based on the organ or tissue it originates in, the expression of molecular markers, its gene expression profile, its mutational burden, the oncogenic mutations it transforms, and its stage of development.

[0003] While treatment protocols often differ depending on the morphology and stage of cancer, some therapeutic molecules have been found to be useful in treating various cancers due to their overall activity against rapidly proliferating cells.

[0004] APIM peptides are a group of peptides that interact with PCNA (proliferating cell nuclear antigen) via a novel PCNA interaction motif (Gilljam et al., 2009. Identification of a novel, widespread, and functionally important PCNA-binding motif, J. Cell Biol. 186(5), pp. 645-654). This motif was initially identified as mediating the interaction between hABH2 and PCNA, hence the name APIM (AlkB homologue 2 (hABH2) PCNA-interacting motif), but APIM sequences have now been identified in various proteins. The PCNA-binding motif found in APIM peptides is typically defined using the consensus sequence [R / K]-[F / W / Y]-[L / I / V / A]-[L / I / V / A]-[K / R] (SEQ ID NO: 2), and a more diverse motif, [R / K / H]-[W / F / Y]-[L / I / V / A / M / S / T / N / Q / C]-[L / I / V / A / M / G / S / T / N / Q / R / H / K / C]-[K / R / H / P] (SEQ ID NO: 3), has been determined to be present in various proteins that act on PCNA (see WO2015 / 067713, referenced herein). Furthermore, "extended" motifs consisting of additional amino acids have also been identified, including [R / K / H]-[W / F / Y]-[W / F / Y / L / I / V / / M]-[L / I / V / A / M / S / T / N / Q / C]-[L / I / V / A / M / G / S / T / N / Q / R / H / K / C / P]-[K / R / H / P / L / I / V / A / M / G / S / T / N / Q / C] (see Sequence ID No. 4) (see WO2016 / 177899 as incorporated herein by reference).

[0005] PCNA is a member of the sliding clamp family of proteins known to be involved in both DNA replication and DNA repair. A key function of PCNA is to provide replication polymerases with the high processing capacity necessary for genome replication.

[0006] APIM peptides have been shown to be therapeutically useful. Specifically, APIM peptides have been shown to be effective in sensitizing cells to cytotoxic and cell division arresting agents, particularly DNA damaging agents (WO 2009 / 104001), microtubule-targeting agents (Soegaard et al, 2018, Oncogene, Vol.9(14), pp.11752-11766), and kinase inhibitors (Soegaard et al, 2019, Oncogene, Vol.10(68), pp.7185-7197). Thus, APIM peptides have been shown to be useful in combination with other therapeutic agents such as cytotoxic and / or cell division arresting agents in the treatment of diseases and conditions in which suppressing cell proliferation is desirable, and in intravenous therapies, i.e., therapies that prevent or suppress unwanted cell proliferation, such as in the treatment of cancer.

[0007] While some studies suggest that APIM peptides themselves act as apoptosis-inducing cytotoxic agents, these peptides do not induce apoptosis in healthy cells. For example, APIM peptides regulate monocyte cytokine production without inducing apoptosis (e.g., Mueller et al., 2013, PLOS One, 8(7), e70430, pp.1-12 and Olaisen et al., 2015, Cell Signal., Vol. 27(7), pp. 1478-1487). Several animal studies have shown that APIM peptides are not useful as standalone activators for cancer treatment. For example, Soegaard et al. (Oncotarget, 2018, Vol. 9(65), pp. 32448-32465) demonstrated that APIM peptide monotherapy was ineffective in a muscle-invasive bladder cancer model. [Overview of the project]

[0008] In the process leading to the present invention, the inventors surprisingly discovered that APIM-peptide alone is particularly effective in treating carcinomas and sarcomas in human patients. Furthermore, the inventors unexpectedly determined that APIM-peptide is effective at significantly lower concentrations than those used in animal studies to enhance the effects of cytotoxic agents.

[0009] The therapeutic effect in humans was particularly surprising because APIM peptides are typically not detectable in the blood within 10 to 960 minutes after administration. While not theoretically constrained, it is hypothesized that the peptides enter cells, remain within them for several days, and exert long-term effects. [Modes for carrying out the invention]

[0010] According to one aspect of the present invention, the present invention relates to a pharmaceutical composition comprising a peptide or a pharmaceutically acceptable salt thereof for use in the treatment of carcinoma or sarcoma in a human subject, wherein the peptide comprises the amino acid sequence and transcellular peptide described in SEQ ID NO: 1, and the pharmaceutical composition is administered systemically to the subject weekly, with the free form of the peptide yielding approximately 15-65 mg / m² of body surface area (BSA) per week. 2 (about 15~50mg / m 2 The present invention provides a pharmaceutical composition that provides the dosage of the peptide.

[0011] Alternatively, the present invention relates to a method for treating carcinoma or sarcoma in a human subject requiring treatment, comprising administering a pharmaceutical composition comprising a peptide or a pharmaceutically acceptable salt thereof to the subject, wherein the peptide comprises the amino acid sequence and transcellular peptide described in SEQ ID NO: 1, and the pharmaceutical composition is administered systemically to the subject weekly, with a dose of approximately 15-65 mg / m² of free peptide per week per body surface area (BSA). 2 (about 15~50mg / m 2 The present invention provides a method for providing the dosage of the peptide.

[0012] In yet another aspect, the present invention is the use of a peptide or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical composition for the treatment of carcinoma or sarcoma in a human subject, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 1 and a cell-penetrating peptide, and the pharmaceutical composition is administered systemically to the subject weekly, providing a dosage of the peptide calculated as the free form of the peptide of about 15 - 65 mg / m 2 (about 15 - 50 mg / m 2 ) per body surface area (BSA) per week.

Brief Description of the Drawings

[0013] [Figure 1] Figure 1 shows a Swimmer plot of the treatment duration of subjects treated in the long-term follow-up study (ATX-101-02) shown in Tables 2 - 4. The numbers on each bar graph indicate the total number of months until disease stabilization, disease progression, or final study termination due to study discontinuation by treatment. The dotted line indicates the end of the first treatment part (ATX-101-01) 6 weeks after the time point of the first tumor evaluation based on RECIST V1.1. At this time point, all patients achieved disease stabilization and transitioned to long-term follow-up treatment (ATX101-02 study).

[0014] (Detailed Description) The term "carcinoma" refers to cancers arising from epithelial cells and includes cancers derived from the endothelium of organs. Carcinomas can be further classified histologically.

[0015] For example, carcinomas that contain histiocytes, tissue structures related to glands, and / or molecular products related to glands (e.g., mucin) are classified as adenocarcinomas.

[0016] Squamous cell carcinoma includes cancers having characteristics indicating squamous epithelial differentiation (intercellular bridges, keratinization, squamous pearls).

[0017] Adenosquamous carcinoma is a mixed tumor that includes both adenocarcinoma and squamous cell carcinoma, and usually each of these cell types occupies at least 10% of the tumor volume.

[0018] Anaplastic or undifferentiated carcinomas are a heterogeneous group of cancers characterized by cells that lack clear histological or cytological evidence of being more specifically differentiated neoplasms.

[0019] Large cell carcinoma consists of large, monotonous, rounded, or overtly polygonal cells with abundant cytoplasm.

[0020] Small cell carcinoma typically consists of round cells, approximately three times or less in diameter than a resting lymphocyte, with little apparent cytoplasm. Occasionally, the small cell malignancy itself may contain significant components of slightly polygonal and / or spindle-shaped cells.

[0021] Therefore, in some embodiments, the carcinomas treated according to the present invention are adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, or small cell carcinoma.

[0022] In some embodiments, the carcinoma is a carcinoma of the lung, pancreas, cervix, urethra, or ovary, such as adenocarcinoma or squamous cell carcinoma.

[0023] Therefore, in some embodiments, the adenocarcinoma is pancreatic adenocarcinoma or lung adenocarcinoma.

[0024] In some embodiments, squamous cell carcinoma is squamous cell carcinoma of the cervix or urethra.

[0025] In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the non-small cell lung cancer is large cell carcinoma or adenocarcinoma.

[0026] In some embodiments, the ovarian carcinoma is an ovarian granulosa cell tumor. In some embodiments, the ovarian carcinoma includes epithelial carcinoma of the ovary, fallopian tube, or primary peritoneal carcinoma. Thus, in some embodiments, the carcinoma is an epithelial carcinoma of the ovary, an epithelial carcinoma of the fallopian tube, or a primary peritoneal carcinoma.

[0027] Sarcoma is a type of cancer that originates from mesenchymal cells that form connective tissue (such as bone, cartilage, fat, blood vessels, and hematopoietic tissue). Sarcomas are generally classified based on the specific tissue and cell type from which the tumor originates, and are often broadly categorized into osteosarcoma and soft tissue sarcoma.

[0028] In some embodiments, the sarcoma treated according to the present invention is a soft tissue sarcoma. In some embodiments, the sarcoma is a myosarcoma (e.g., rhabdomyosarcoma or leiomyosarcoma), liposarcoma, undifferentiated pleomorphic sarcoma, or synovial sarcoma.

[0029] In some embodiments, the myosarcoma is a leiomyosarcoma such as a uterine leiomyosarcoma.

[0030] In some embodiments, undifferentiated pleomorphic sarcoma is metastatic undifferentiated pleomorphic sarcoma.

[0031] As used herein, the terms “to treat” or “treatment” broadly refer to any effect or step (or intervention) that is beneficial in the management of a clinical condition or disorder. Therefore, treatment may refer to reducing, alleviating, improving, slowing the progression of, or eliminating one or more symptoms of the carcinoma or sarcoma being treated, compared to the symptoms before treatment, or improving the clinical condition in question in any way. Treatment may include clinical steps or interventions that contribute to or are part of a treatment program or regimen. In particular, such treatment may include reducing the size or volume of the carcinoma or sarcoma being treated.

[0032] Treatment may include, for example, delaying, limiting, reducing, or preventing the onset of one or more symptoms of a carcinoma or sarcoma compared to the carcinoma or sarcoma or symptoms before treatment. Thus, treatment explicitly includes both absolute prevention of the development or onset of symptoms of a carcinoma or sarcoma, and delaying the onset of symptoms of a carcinoma or sarcoma or its symptoms, or reducing or limiting the onset or progression of symptoms of a carcinoma or sarcoma or its symptoms.

[0033] Therefore, therapies according to the present invention include killing, suppressing or slowing the proliferation of carcinoma or sarcoma cells, or increasing the size of the carcinoma or sarcoma cell body or population (e.g., in tissue, tumor or proliferation), decreasing the number of carcinoma or sarcoma cells, or preventing the spread of carcinoma or sarcoma cells (e.g., to another anatomical site), or reducing the size of cell proliferation, etc. The term “therapy” does not necessarily mean a cure or complete disappearance or elimination of the proliferation of carcinoma or sarcoma cells.

[0034] In some embodiments, treatment is measured using the RECIST (Response Evaluation Criteria In Solid Tumors) criteria. The RECIST criteria are based on measurements such as X-ray, CT scan, and MRI scan, indicating whether the tumor shrinks, remains unchanged, or grows. The types of responses a patient may have include complete response (CR), partial response (PR), disease progression (PD), and stable disease (SD). Therefore, treatment refers to a patient demonstrating one of the following: complete response (CR), partial response (PR), or stable disease (SD). In some embodiments, treatment refers to a patient demonstrating SD. In some embodiments, treatment refers to a patient demonstrating SD for at least three months from the start of treatment, for example, four, five, or six months or longer.

[0035] "Complete response" refers to a state in which all target lesions have disappeared.

[0036] "Partial response" refers to a reduction of 30% or more in the total lesion diameter (LD) of target lesions, relative to the baseline total LD.

[0037] "Stable disease activity" refers to a state where, based on the lowest LD since the start of treatment, there is neither a reduction that corresponds to a partial response (PR) nor an increase that corresponds to a progressive disease (PD).

[0038] "Disease progression" refers to a situation where the total LD ​​of target lesions increases by 20% or more, based on the lowest total LD ​​recorded since the start of treatment, or when one or more new lesions appear.

[0039] In this specification, the terms “subject” and “patient” refer to a human being, i.e., a human being having a carcinoma or sarcoma as defined herein that requires treatment.

[0040] The peptide used in this invention includes the amino acid sequence described in Sequence ID No. 1, which can be considered a "PCNA interaction motif."

[0041] A “PCNA interaction motif” refers to a sequence of amino acids within a peptide that has the function of promoting the interaction between the peptide and PCNA. Therefore, the peptides of the present invention can be characterized insofar as they need to be able to interact with PCNA proteins. In other words, the peptides used in the present invention need to be competent and / or proficient molecules that interact with PCNA. The terms “PCNA interaction motif” and “APIM sequence” can be used interchangeably herein.

[0042] The PCNA protein used to determine the ability and / or affinity of peptide:PCNA interactions may be PCNA from any suitable source, e.g., any animal, particularly mammals such as humans, rodents (e.g., mice, rats), or any other non-human animal. In a preferred embodiment, peptide:PCNA interactions are determined, characterized, or evaluated using human PCNA proteins. These interactions include direct binding between the peptide and the PCNA protein.

[0043] A peptide is an isolated peptide, most preferably a synthetic peptide. In other words, a peptide is a non-native molecule, meaning it does not exist in nature.

[0044] For a peptide capable of interacting with PCNA to function in the methods and uses of the present invention, the peptide must be able to enter the target cell, that is, to cross the cell membrane into the cytosol (cytoplasm), and possibly into one or more other cellular locations, such as the nucleus. As mentioned above, since this peptide can remain in the cell for several days, it is expected to have a long-lasting effect.

[0045] Therefore, peptides contain domains that assist in the passage of peptides across the cell membrane; that is, peptides are provided as fusion peptides or chimeric peptides (peptides formed from two or more domains not typically found together in nature). In particular, the peptides used in this invention include transcellular peptides (CPPs), which may alternatively be called internalized peptides or imported peptides, or peptide transduction domains.

[0046] The final size of a peptide depends on the size and number of domains that make up the peptide. That is, PCNA interaction motifs and CPPs can be considered as domains of the peptide. Therefore, a domain can be considered a distinct part of a peptide (i.e., a sequence within the full-length amino acid sequence) to which a specific function or property is assigned or attributed.

[0047] This peptide comprises at least two domains, namely a PCNA interaction motif domain (SEQ ID NO: 1) and a CPP. However, the peptide may also contain additional domains that can enhance its function and / or stability, for example, its ability to interact with its target PCNA. Thus, the peptide may contain two, three, four, or five domains, e.g., six, seven, eight, nine, ten, twelve, fifteen, or more domains. For example, in some embodiments, the peptide may contain one or more linker domains, i.e., domains that interpose between two other domains, i.e., domains that occupy and connect the space between two domains of the peptide.

[0048] In some embodiments, the linker domain may be inactive, meaning it has no physiological function in the target cell where the peptide is activated, and merely functions to physically separate other domains in the peptide. However, in some embodiments, the linker domain may have additional functions. For example, the linker domain may also function as a cleavage domain. That is, the linker domain may contain a peptide bond that is easily cleaved under physiological conditions, for example, inside the target cell, so that the peptide is cleaved after its uptake.

[0049] In some embodiments, the peptide may include a domain that directs the peptide to an intracellular or extracellular location, such as a signal peptide (also known as a target or transit peptide), such as a nuclear localization signal (NLS) sequence. Thus, in some embodiments, one or more linker domains function as a signal peptide, e.g., an NLS, i.e., the linker may be a signal peptide, preferably such as an NLS. Alternatively, a signal peptide domain may function as a linker domain in some embodiments. In some embodiments, the peptide may include one or more linker domains, e.g., an inactive linker domain, in addition to a signal peptide (e.g., an NLS).

[0050] In exemplary embodiments, the peptide comprises the PCNA interaction motif domain, CPP, and linker domain described in SEQ ID NO: 1. In further exemplary embodiments, the peptide may also comprise a nuclear localization signal sequence domain. In yet another embodiment, the nuclear localization signal sequence domain may function as a linker domain.

[0051] Therefore, in such embodiments, it will be found that the peptide of the present invention may take the form of a construct comprising the PCNA interaction motif described in Sequence ID No. 1, a CPP domain that promotes its uptake into cells, and optionally additional domains. From this viewpoint, the present invention can be considered to provide a construct comprising a peptide capable of interacting with PCNA.

[0052] Accordingly, the present invention may provide a pharmaceutical composition comprising (i) a peptide comprising the PCNA interaction motif described in SEQ ID NO: 1, and (ii) a construct for use in the methods and uses of the present invention comprising a transcellular peptide.

[0053] Cell-penetrating peptide (CPP) technology has developed significantly in recent years, and a wide variety of transcellular peptides are known and described in this field. In fact, such peptides are commercially available. Although transcellular peptides differ greatly in size, sequence, charge, and functional mechanisms (currently, the mechanisms of some peptides are unknown, and those of others are not fully understood), they share the common ability to cross the cell membrane and deliver an attachment or associated site (the so-called "cargo") into the cell's cytoplasm. Thus, CPPs are peptide-based delivery vectors.

[0054] Although CPPs are not characterized by a single structural or functional motif, tools for identifying CPPs are available, and those skilled in the art can easily determine whether a peptide sequence has a function that promotes the uptake of peptides forming a domain, i.e., whether a peptide sequence can function as a CPP. For example, Hansen et al. (Predicting cell-penetrating peptides, Advanced Drug Delivery Reviews, 2008, 60, pp. 572-579) provide a review of a CPP prediction method using principal component analysis ("z-prediction") and a corresponding algorithm based on the original paper by Haellbrink et al. (Prediction of Cell-Penetrating Peptides, International Journal of Peptide Research and Therapeutics, 2005, 11(4), pp. 249-259). Specifically, a Z-score is calculated from the numerical value and its range for candidate peptides. If the z-score falls within the range of known CPP z-scores, the peptide examined is classified as a CPP. This method has been shown to have high accuracy (predicting known CPPs with approximately 95% accuracy).

[0055] Additional methods for predicting CPPs have since been developed (e.g., Sanders et al., Prediction of Cell Penetrating Peptides by Support Vector Machines, PLOS Computational Biology, 2011, 7(7), pp. 1-12, incorporated herein by reference), and CPP databases are available (Gautam et al., CPPSite: a curated database of cell penetrating peptides, Database, 2012, Article ID bas015 and http: / / crdd.osdd.net / raghava / cppsite / index.php, both incorporated herein by reference). Therefore, any suitable CPP may find usefulness in the present invention, and as will be discussed later, various CPPs have already been identified and tested, forming a basis for determining and identifying novel CPPs.

[0056] CPPs may be derived from naturally occurring proteins capable of traversing the cell membrane, such as the Drosophila homeobox protein Antennapedia (transcription factor), from viral proteins such as the HIV-1 transcription factor TAT or the HSV-1 capsid protein VP22, and / or synthetically from synthetic polypeptides such as chimeric proteins or polyarginines. As mentioned above, there is not a single mechanism responsible for the transduction effect, so various structures and sequences are possible for CPP design. Jarver et al. 2006 (Biochimica et Biophysica Acta 1758, pages 260-263) also reviews transcellular peptides. US 6,645,501, WO2015 / 067713 and WO2016 / 177898 (all incorporated herein by reference) further describe various transcellular peptides that may be used.

[0057] The Antennapedia-derived CPP (Antp class) is a class of CPPs centered around a 16-amino acid penetratin sequence corresponding to the third loop of the Antennapedia protein, and has been shown to be involved in protein translocation. Penetratin has been widely developed as a delivery vehicle, particularly for pharmaceutical applications, and various penetratin derivatives and modified sequences have been proposed and described. See, in particular, WO 91 / 1891, WO 00 / 1417, WO 00 / 29427, WO 2004 / 069279 and US 6,080,724 (incorporated herein by reference). Thus, the 16-amino acid sequence of penetratin may be modified and / or cleaved, or the peptide may be chemically modified, or retro, inverso, or retroinverso analogs may be created while retaining cell entry activity.

[0058] Another group of transcellular peptides that can be used are based on HIV-TAT sequences and HIV-TAT and its fragments. Various TAT-based CPPs are described in US 5,656,122 (incorporated herein by reference). An exemplary HIV-TAT peptide, such as the one used in the following examples, is RKKRRQRRR (SEQ ID NO: 38), but it will be readily apparent that longer or shorter TAT fragments may be used.

[0059] As mentioned above, there are no specific structural features or sequence motifs common to all CPPs. However, different classes of CPPs may be distinguished by specific features, such as amphiphilic and positively charged peptides. Other groups of CPPs may have structures exhibiting high α-helical content. There is also another group of peptides characterized by a high basic amino acid content. CPPs may therefore be oligomers of basic amino acids such as arginine, for example, R residues 5-20, 6-15 or 6-12, e.g., R7 (SEQ ID NO: 37), R8 (SEQ ID NO: 39) or R 11This may include (SEQ ID NO: 40) or QSR8 (SEQ ID NO: 41). These CPPs represent a preferred group of CPPs for use in the present invention.

[0060] Therefore, in some embodiments, the domain that promotes the uptake of oligopeptide compounds (e.g., CPP) may be defined as a peptide of 4 to 30 amino acids (e.g., 5 to 29, 6 to 28, 7 to 27, 8 to 26, 9 to 25, etc.). At least four amino acids, optionally at least four consecutive amino acids (e.g., at least 5, 6, 7, 8, 9, 10, or 11 amino acids, e.g., 4 to 20, 5 to 19, 6 to 18, 7 to 17, 8 to 16, 9 to 15, 10 to 14, 11 to 13 amino acids) are positively charged amino acids, preferably selected from K, R, or H.

[0061] Proline-rich amphiphilic peptides are another class of CPPs, characterized by the presence of a pyrrolidine ring from proline. Such peptides are described in Pujalset al. 2008 Advanced Drug Delivery Reviews 60, pages 473-484 (incorporated herein by reference).

[0062] Other CPPs that have been successfully developed include pVEC (Elmquistet al. 2003 Biol. Chem 384, pages 387-393; Holmet al. 2005 Febs Lett. 579, pages 5217-5222, all incorporated herein by reference) and calcitonin-derived peptide (Krausset al. 2004 Bioorg. Med. Chem. Lett., 14, pages 51-54, incorporated herein by reference).

[0063] Commercially available CPPs include Chariot (Active Motif, France), which is based on the Pep-1 peptide; Syn-B vector (Syntem, France), which is based on the protegrin peptide PG-1; and Express-Si Delivery (Genospectra, USA), which is based on the MPG peptide.

[0064] Other CPPs include R41, R8, M918, and YTA-4 peptides (sequence numbers 866-869, respectively) disclosed in Eriksson et al. 2013, Antimicrobial Agents and Chemotherapy, vol. 57(8), pp. 3704-3712 (referenced herein).

[0065] In some embodiments, the CPP may be a cyclic peptide, e.g., Ohet al, 2014, Mol. Pharmaceuticals, Vol. 11, pp. 3528-3536 (as incorporated herein by reference). In particular, the CPP may be an amphiphilic cyclic CPP, particularly containing tryptophan and arginine residues. In some embodiments, the CPP may be a cyclic polyarginine peptide, which may be modified by the addition of a fatty acyl moiety, e.g., octanoyl, dodecanoyl, hexadecanoyl, N-acetyl-L-tryptophanyl-12-aminododecanoyl. Suitable cyclic CPPs for use in the present invention are presented in SEQ ID NOs: 870-876.

[0066] In addition to known and reported CPPs, novel or derivative CPP peptides can be designed and synthesized based on known or reported criteria (e.g., known CPP sequences or characteristics such as the basic amino acid content and α-helical content mentioned above). Furthermore, randomly designed or other peptides can be screened for CPP activity by, for example, attaching such peptides containing a detectable label or tag, such as a reporter molecule, e.g., a fluorescent tag, to a desired cargo (e.g., a peptide containing SEQ ID NO: 1), and then testing whether the constructs move across the cell membrane by, for example, examining cell importation using a confocal microscope after adding these peptides to living cells.

[0067] In some cases, successful or efficient delivery may depend on, or vary accordingly, the exact properties of the cargo (e.g., cargo peptide sequence) and / or the CPP used. Determining the optimal peptide sequence and combination, and testing and / or modifying the cargo and / or CPP sequence or structure, would be within the scope of the routine art of those skilled in the art.

[0068] Therefore, in some embodiments, the CPP is selected from one of the following: (i) Antennapedia class peptides, (ii) Protegrin claspeptide, (iii) HIV-TAT class peptides, (iv) Amphiphilic peptides selected from amphiphilic and positively charged peptides, proline-rich amphiphilic peptides, peptides based on Pep-1 peptides and peptides based on MPG peptides, (v) Peptides exhibiting a high α-helical content, (vi) Peptides containing oligomers of basic amino acids, (vii) pVEC, (viii) Calcitonin-derived peptides, and (ix) Amphiphilic cyclic CPP.

[0069] In some embodiments, the CPP is selected from a sequence selected from any one of SEQ ID NOs. 5-876, or from a fragment and / or derivative thereof. Details and properties of the CPPs identified in SEQ ID NOs. 43-865 are available on the CPPSite database at http: / / crdd.osdd.net / raghava / cppsite / index.php (referenced herein).

[0070] In a preferred embodiment, the CPP comprises the amino acid sequence described in SEQ ID NO: 37, 39, or 40.

[0071] In some embodiments, the peptide also includes one or more domains that provide a signal (target or pass-through) sequence. In some embodiments, the signal sequence can target the peptide to a specific cell type. In addition, or alternatively, in some embodiments, the peptide may include a signal peptide that localizes the peptide to a specific intracellular compartment, such as the nucleus. In some embodiments, the peptide is targeted to the cytoplasm, which can be achieved without an additional signal peptide; i.e., the CPP is sufficient to guide or localize the peptide to the cytoplasm of the cell.

[0072] Therefore, a signal sequence or signal sequence domain can be considered any sequence that acts to localize a peptide to any desired location, e.g., any cell type or intracellular location (e.g., the nucleus), or, conversely, to induce, move, or transport it.

[0073] As described above, the peptides used in the present invention may include one or more signal sequences (i.e., one or more domains that function as signal sequences), for example, a signal peptide that induces the peptide to a specific intracellular compartment such as the nucleus.

[0074] Nuclear localization signals (NLS) are once again well-known in the art and are widely described in the literature. For example, searchable databases of known and predicted NLS are available; see, for instance, Cokol et al. (Finding nuclear localization signals, EMBO Reports, 2000, 1(5), pp.411-415, incorporated herein by reference). For predicting the nuclear localization of proteins based on NLS, the PSORT II database, http: / / psort.hgc.jp / (incorporated herein by reference) can be used. Therefore, any known or functional NLS can be useful in the present invention.

[0075] NLSs can vary in length and / or sequence, and a wide variety of NLS sequences have been described. However, peptides containing positively charged amino acids (particularly lysine (K), arginine (R), and / or histidine (H)) have generally been found to function as NLSs. Thus, exemplary NLSs may be peptides of, for example, 4-20, more specifically 4-15, 4-12, 4-10, or 4-8 amino acids, where at least four amino acids (more specifically at least 60, 70, 75, 80, 85, or 90% of the amino acid residues of the NLS peptide) are selected from positively charged amino acids, preferably K, R, or H. Such exemplary NLSs may have, for example, the sequence RKRH (SEQ ID NO: 877) or contain them.

[0076] The nuclear localization signals include both those that have actually been experimentally determined and those predicted or proposed NLS sequences, and strategies for identifying NLSs are also described in Lange et al., J. Biol.Chem. 2007, 282(8), 5101-5105; Makkerh et al., Current Biology 1996, 6(8), 1025-1027; Leslie et al., Methods 2006, 39, 291-308; and Lusk et al., Nature Reviews MCB 2007, 8, 414-420 (all of which are incorporated herein by reference).

[0077] Classical NLSs consist of one (monopartite) or two (bipartite) stretches of basic amino acids. The monopartite NLS can be exemplified by the SV40 large T antigen NLS ( 126 PKKKRKV 132 [SEQ ID NO: 878]) and the bipartite NLS by nucleoplasmin NLS ( 155 KRPAATKKAGQA KKKK 170 [SEQ ID NO: 879]). A monopartite NLS consensus sequence K-[K / R]-X-[K / R] (SEQ ID NO: 880) has been proposed, and accordingly, the NLSs according to the present invention can, in one embodiment, include or consist of such a consensus sequence (where X is any amino acid).

[0078] A representative bipartite NLS according to the present invention can have the sequence KR-[X] 5-20 -KKKK (SEQ ID NO: 881), for example KR-X 10 -KKKK (SEQ ID NO: 882) (where X is any amino acid).

[0079] An alternative exemplary bipartite NLS can take the form of RKRH-[X] 2-10 -KK (SEQ ID NO: 883), for example RKRH-X2-KK (SEQ ID NO: 884), for example RKRH-II-KK (SEQ ID NO: 885).

[0080] Oncoprotein c-myc NLS differs from classical NLS in that only three of its nine amino acid residues are basic (PAAKRVKLD [SEQ ID NO: 886]), demonstrating that NLS does not necessarily need to conform to the consensus or classical sequences mentioned above. Makkerh et al. (see above) describe an NLS sequence in which a cluster of basic amino acids (e.g., KKKK [SEQ ID NO: 887]) is flanked by neutral and acidic residues, e.g., PAAKKKKLD (SEQ ID NO: 888).

[0081] Other possible NLS sequences that can be cited as examples include: PKKKRKVL (SEQ ID NO: 889), KKKRK (SEQ ID NO: 890), KKKRVK (SEQ ID NO: 891), KKKRKVL (SEQ ID NO: 892), and RKKRKVL (SEQ ID NO: 893). Any NLS can be used as a derivative of known NLS such as SV40, nucleoplasmin, UNG2, and c-myc NLS.

[0082] Estimated, proposed, or predicted NLS sequences can be tested for NLS activity using principles and assays known and described in the art. For example, a candidate NLS sequence can be attached to a desired cargo (in this case, a peptide as defined herein), a detectable reporter molecule (e.g., a tag or label that can be visualized, such as a fluorescent label) can be provided to the construct, and it can be brought into contact with test cells. The distribution of the construct within the cells can then be determined.

[0083] Thus, in summary, those skilled in the art will recognize the appropriate signal sequence. In a particularly preferred embodiment, the peptide contains an NLS signal sequence from the SV40 protein, which includes the amino acid sequence KKKRK (SEQ ID NO: 890).

[0084] Therefore, in some embodiments, the peptide may include a signal sequence (i.e., a domain containing the signal peptide) that localizes or induces the peptide to an intracellular location, such as an NLS, and may be selected from any one of the following: (i) A peptide comprising 4 to 20 amino acids, wherein at least 4 amino acids are positively charged, preferably selected from K, R, or H; and / or (ii) A sequence, fragment thereof, and / or derivative selected from any one of sequence numbers 877-893.

[0085] In some embodiments, the nuclear localization signal sequence comprises a sequence selected from any one of sequence numbers 877 to 893, or a fragment and / or derivative thereof, preferably the fragment and / or derivative comprising at least four positively charged amino acids selected from K, R, or H.

[0086] In some embodiments, the peptide or construct according to the present invention may include (i) the PCNA interaction motif described in SEQ ID NO: 1 (APIM sequence), (ii) a linker domain which may in some embodiments include a nuclear localization signal sequence, and (iii) at least three domains including CPP.

[0087] The distinct elements or components (domains) of the peptide according to the present invention may be included or presented in any order, but preferably in the order shown above (for example, APIM sequence-CPP or APIM sequence-linker-CPP).

[0088] In some embodiments, the APIM motif is located at or toward the N-terminus of the peptide. For example, the APIM motif may be described as being at the N-terminus of the CPP, and optionally at the N-terminus of the linker sequence, if one exists.

[0089] The domains (which can be considered as components, elements, or separate parts) of the peptides of the present invention described herein can be attached to or linked to one another in any desired or convenient way according to art well known. Thus, the domains may be chemically linked or bonded using, for example, known chemical coupling techniques, or the compound or construct may be formed as a single whole using, for example, genetic engineering techniques such as techniques for forming fusion proteins, or it may simply be synthesized as a whole using, for example, peptide synthesis techniques. In preferred embodiments, the domains are linked by peptide bonds.

[0090] Domains may be directly linked to each other or indirectly linked by one or more linker (or spacer) sequences. Thus, a linker sequence can separate or link two or more individual domains (i.e., parts, e.g., or separate motif elements) in a peptide. The exact nature of the linker sequence is not important; it may be of variable length and / or sequence, for example, it may have 0-40, more particularly 0-20, 0-15, 0-12, 0-10, 0-8, 0-7, 0-6, 0-5, 0-4, or 0-3 residues, e.g., 1, 2, or 3 or more. Typical examples include having 1-15, 1-12, 1-10, 1-8, 1-7, 1-6, 1-5, or 1-4 residues, etc. The nature of the residues is not important; for example, they may be any amino acid such as a neutral amino acid or an aliphatic amino acid, or they may instead be hydrophobic, polar, charged, or structure-forming, e.g., proline. Various different linker sequences have been shown to be useful, including short (e.g., 1-7) sequences of neutral and / or aliphatic amino acids.

[0091] Therefore, exemplary linker sequences include any single amino acid residue, e.g., A, I, L, V, G, R, Q, T, or W, or a di, tri, tetra, penta, or hexapeptide consisting of such residues.

[0092] Representative linkers include I, II, IL, R, W, WW, WWW, RIL, RIW, GAQ, GAW, VAT, IILVI (sequence number 894), IILVIII (sequence number 895), GILQ (sequence number 896), and GILQWRK (sequence number 897).

[0093] As described above, in some embodiments, the linker includes an NLS sequence. Therefore, in a particularly preferred embodiment, the linker includes WKKKRKI (sequence number 898).

[0094] In preferred embodiments, the peptide comprises the PCNA interaction motif (APIM sequence) described in SEQ ID NO: 1 and the transcellular signal sequence described in SEQ ID NO: 37, 39, or 40. For example, in some embodiments, the peptide comprises the PCNA interaction motif described in SEQ ID NO: 1, the linker domain described in SEQ ID NO: 890 or 898, and the transcellular signal sequence described in SEQ ID NO: 37, 39, or 40, preferably SEQ ID NO: 40. In some specific embodiments, the peptide comprises the PCNA interaction motif described in SEQ ID NO: 1, the linker domain described in SEQ ID NO: 898, and the transcellular signal sequence described in SEQ ID NO: 40, for example, the sequence described in any one of SEQ ID NOs. 914-916 or 918-920, preferably SEQ ID NO: 914 or 918, most preferably SEQ ID NO: 914.

[0095] Furthermore, in some embodiments, the peptide according to the present invention may contain one or more PCNA-interaction motifs. The peptide may contain, for example, 1 to 10 motifs, for example, 1 to 6 motifs, or 1 to 4 motifs, or 1 to 3 motifs, or 1 or 2 motifs. In some embodiments, the motifs may be identical, i.e., the peptide may contain two or more sequences described in SEQ ID NO: 1. In some embodiments, the motifs may be different, i.e., SEQ ID NO: 1 and one or more other motifs. Suitable alternative motifs have been described in the Art as described above. Within a peptide containing a signal sequence, such motifs may be spaced apart according to selection, for example, they may be grouped together, or they may be separated by other domains, e.g., motif-motif-CPP, motif-linker-motif-CPP; or motif-linker-motif-CPP; or motif-motif-linker-CPP, etc.

[0096] As used herein, a “fragment” may contain at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99% of the amino acids of the sequence from which it originates. The fragment may be obtained from the central, N-terminal, or C-terminal portion of the sequence. The size of the fragment depends on the size of the original sequence, but in some embodiments, the fragment may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more shorter amino acid residues than the sequence from which it originates, for example, 1 to 10, 2 to 9, 3 to 8, or 4 to 7 amino acid residues shorter than the sequence from which it originates.

[0097] As used herein, a “derivative” of a sequence means that it is at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence being compared.

[0098] Sequence identity can be determined, for example, using the SWISS-PROT protein sequence databank, with FASTA pep-cmp set to the variable pamfactor, a gap creation penalty of 12.0, a gap expansion penalty of 4.0, and a two-amino acid window. Preferably, the comparison is performed over the entire length of the sequence, but it may also be performed over smaller comparison windows, e.g., over fewer than 200, 100, 50, 20, or 10 consecutive amino acids.

[0099] Preferably, such sequence identity-related peptides, i.e., derivatives, are functionally equivalent to the peptide described in the cited sequence number. Similarly, peptides having the sequence described in the sequence number can be modified without affecting the polypeptide sequence, as described later.

[0100] Furthermore, the “fragments” described herein may also be functional equivalents. Preferably, these fragments satisfy the identity (relative to comparable regions) conditions referred to herein.

[0101] As referred to herein, in order to achieve “functional equivalence,” the peptide may exhibit some reduced potency in performing its function compared to the parent molecule (i.e., the molecule derived, for example, by amino acid substitution), but preferably it is of the same or higher potency. Thus, functional equivalence may relate to peptides that are effective in localizing or inducing the peptide into the cell, for example, to promote the uptake of the peptide as described above. This can be tested by qualitatively or quantitatively comparing the effect of the derivative peptide on the peptide from which it is derived, for example, by performing the in vitro analysis described above. If quantitative results are possible, the derivative is at least 30, 50, 70, or 90% more effective than the parent peptide.

[0102] Functionally equivalent peptides related to or derived from a parent peptide can be obtained by modifying the parent amino acid sequence by one or more amino acid substitutions, additions, and / or deletions (while satisfying the sequence identity requirement described above), without disrupting the molecular function. Preferably, the parent sequence has fewer than 20 substitutions, additions, or deletions, for example, 10, 5, 4, 3, or fewer than 2 such modifications. Such peptides may be encoded by “functionally equivalent nucleic acid molecules” that can be produced by appropriate substitutions, additions, and / or deletions of one or more bases. The following are representative peptides containing the PCNA interaction motif described in Sequence ID No. 1. MDRWLVKRILVATK (Sequence ID 899), MDRWLVKRILKKKRKVATKG (Sequence ID 900), MDRWLVKGAQPKKKRKVLRQIKIWFQNRRMKWKK(Sequence ID 901), MDRWLVKGAWKKKRVKIIRKKRRQRRRK(Sequence ID 902), MDRWLVKGAWKKKRKIIRKKRRQRRRG (Sequence ID 903), MDRWLVKGAWKKKRKIIRKKRRQRRRK(Sequence ID 904), MDRWLVKRIWKKKRKIIRKKRRQRRRK(Sequence ID 905), MDRWLVKWWWKKKRKIIRKKRRQRRRK(Sequence ID 906), MDRWLVKWWRKRHIIKKRKKRRQRRRK (Sequence ID 907), MDRWLVKRIWKKKRKIIRRRRRRRRRRRK(Sequence ID 908), MDRWLVKRIWKKKRKIIRQIKIWFQNRRMKWKK (Sequence ID 909), MDRWLVKWKKKRKIRRRRRRRRRRRK(Sequence ID 910), MDRWLVKWKKKRKIRKKRRQRRRK(Sequence ID 911), MDRWLVKWRKRHIRKKRRQRRRK (Sequence ID 912), MDRWLVKGAWRKRHIRKKRRQRRRK(Sequence ID 913), MDRWLVKWKKKRKIRRRRRRRRRRR(Sequence ID 914), MDRWLVKKKKRKRRRRRRRRRRRK(Sequence ID 915), MDRWLVKKKKRKRRRRRRRRRRR(Sequence ID 916), MDRWLVKRIWKKKRKIIRWLVKWWWRKKRRQRRRK (Sequence ID 917).

[0103] The peptides described above contain an N-terminal amino acid sequence, i.e., an "MD" sequence, that does not form part of the domain essential for the peptide to be active in the methods and uses of the present invention. Some peptides may contain N-terminal modifications, such as an acetyl group. These additional amino acids and modifications help to facilitate the production of the peptide, for example, in vitro or in vivo, and / or protect the peptide from degradation in vivo. It will be apparent that the peptide does not require these additional amino acids or modifications for its activity. Therefore, further representative sequences according to the present invention include any of SEQ ID NOs. 899-917 with the N-terminal "MD" omitted, for example, the peptide may contain the following amino acid sequences: RWLVKWKKKRKIRRRRRRRRRRR,RWLVKKKKRKRRRRRRRRRRRK or RWLVKKKKRKRRRRRRRRRRRR (SEQ ID NOs. 918-920). Furthermore, the presence of additional amino acids or modifications at any of the terminals does not appear to disrupt or inhibit the function of the peptides described herein. Therefore, in some embodiments, the peptide may include an N-terminal sequence, for example, an N-terminal sequence that does not include the domain defined above, for example, a so-called N-terminal flanking sequence. In some embodiments, the peptide may include a C-terminal sequence, for example, a C-terminal sequence that does not include the domain defined above, for example, a so-called C-terminal flanking sequence. In some embodiments, the peptide may include N-terminal and C-terminal flanking sequences. The peptide may also include a C-terminal modification, for example, an amide group. Therefore, in some embodiments, the C-terminal residue may be amidated. In some preferred embodiments, the peptide includes an amidated C-terminal arginine residue.

[0104] A flanking sequence may contain approximately 1 to 50 amino acids, for example, approximately 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, etc. Therefore, a flanking sequence may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids, for example, 1 to 40, 2 to 39, 3 to 38, 4 to 37, 5 to 36, 6 to 35, 7 to 34, 8 to 33, 9 to 32, 10 to 31, 11 to 30, 12 to 29, 13 to 28, 14 to 27, 15 to 26 amino acids or any combination thereof.

[0105] In some embodiments, the peptide of the present invention may be in the form of a salt, i.e., a pharmaceutically acceptable salt. For example, the peptide may be in the form of an acidic salt or a basic salt, and is preferably an acidic salt. In some embodiments, the peptide is in the form of a neutral salt.

[0106] Pharmaceutically acceptable salts include pharmaceutically acceptable base addition salts and acid addition salts, such as metal salts like alkali metal salts and alkaline earth metal salts, ammonium salts, organic amine addition salts and amino acid addition salts, and sulfonates. Examples of acid addition salts include inorganic acid addition salts such as hydrochloride, sulfate, and phosphate, and organic acid addition salts such as alkyl sulfonates, aryl sulfonates, acetates, maleates, fumarates, tartrates, citrates, and lactates. Examples of metal salts include alkali metal salts such as lithium salts, sodium salts, and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, aluminum salts, and zinc salts. Examples of ammonium salts include ammonium salt and tetramethylammonium salt. Examples of organic amine addition salts include salts with morpholine and piperidine. Examples of amino acid addition salts include salts with glycine, phenylalanine, glutamic acid, and lysine. Examples of sulfonates include mesylates, tosylates, and benzenesulfonates.

[0107] Preferred salts include acidic salts such as hydrochloride salts, and organic acid addition salts such as acetate salts, alkyl sulfonates, aryl sulfonates, maleates, fumarates, tartrates, citrates, and lactates. In some embodiments, the peptide may be in the form of an acetate salt or its derivatives, such as trichloroacetate (TCA) or trifluoroacetate (TFA). In some embodiments, the peptide may be stabilized by being prepared in the form of a salt, such as an acetate salt. Hydrochloride salts are particularly preferred.

[0108] As used herein, "pharmaceutically acceptable" means a component that is compatible with other components used in the method or use of the present invention and is physiologically acceptable to the recipient.

[0109] This specification uses standard single-letter codes for amino acids, so K represents lysine (Lys), I represents isoleucine (Ile), and so on.

[0110] In some embodiments, the peptide may contain unconventional or non-standard amino acids in domains other than the APIM sequence (SEQ ID NO: 1). In some embodiments, the peptide may contain one or more unconventional amino acids, for example, 1, 2, 3, 4, 5 or more, i.e., amino acids having side chains not encoded by the standard genetic code, referred to here as “non-coding amino acids,” which are well known in the art. For example, amino acids produced in metabolic processes such as ornithine and taurine, and / or artificially modified amino acids such as 9H-fluoren-9-ylmethoxycarbonyl (Fmoc), (tert)-(B)utyl(o)xy(c)arbonyl (Boc), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) protected amino acids, and amino acids having a benzyloxycarbonyl group (Z). In some embodiments, the non-coding amino acids are present in two or more domains of the peptide.

[0111] The in vitro and / or in vivo stability of peptides can be improved or enhanced by stabilization or protection means known in the art, such as the addition of protecting or stabilizing groups, the incorporation of amino acid derivatives or analogs, or the use of chemical modifications of amino acids. Such protecting or stabilizing groups may be added, for example, to the N-terminus and / or C-terminus. An example of such a group is the acetyl group, and other protecting or peptide-stabilizing groups are known in the art.

[0112] The peptides of the present invention typically contain only L-coordinated amino acids, but may also contain one or more D-coordinated amino acids. In some embodiments, the peptide contains 1, 2, 3, 4, 5 or more D-amino acids. In some embodiments, the D-amino acids are located within a motif, while in other embodiments, they are located only outside the motif. In even further embodiments, the D-amino acids may be found in two or more domains of the peptide. The peptides may be linear or cyclic, and are preferably linear.

[0113] In a preferred embodiment, the peptide consists of L-amino acids. In a more preferred embodiment, the peptide consists of standard amino acids or encoded L-amino acids.

[0114] As described above, peptides may contain non-standard amino acids. Therefore, in some embodiments, peptides may incorporate di-amino acids and / or β-amino acids. However, in preferred embodiments, at least the APIM motif domain consists of α-amino acids. Most preferably, the peptide, i.e., all domains and possibly all flanking sequences, consists of α-amino acids.

[0115] Peptides as defined herein consist of five or more amino acids, but the length of a peptide depends on the size of the CPP sequence and, if other domains are present, their number and size. Therefore, the term peptide refers to a molecule containing a relatively small number of amino acids, i.e., 100 or fewer, preferably 90, 80, 70, 60, or 50 or fewer amino acids. The peptides of the present invention contain at least 10, 11, or 12 amino acids, for example, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. Alternatively, they are defined as containing 50, 45, 40, 35, 34, 33, 32, 31, or 30 or fewer amino acids. Therefore, typical subunit ranges include 12-50, 12-45, 12-40, 12-35, 12-30, 12-25, 12-23, 12-20, 12-18, etc., with 12-30 and 12-40 being preferred. Further typical subunit ranges include 20-50, 21-45, 22-40, 23-35, 24-30, for example, 25, 26, 27, 28, 29, or 30.

[0116] In some embodiments, the peptide may form part of a larger unit, for example, by fusing with a polypeptide to form a recombinant fusion protein, or by attaching to a scaffold to form a peptide aptamer. Thus, fusion proteins or aptamers incorporating peptides may also find useful in the uses and methods of the present invention, i.e., in some embodiments, the pharmaceutical composition may contain a fusion protein or aptamer comprising the peptide defined above.

[0117] Pharmaceutical compositions comprising peptides, fusion proteins, or aptamers, or pharmaceutically acceptable salts thereof, can be formulated with at least one pharmaceutically acceptable carrier or excipient.

[0118] The excipients may include any excipients known in the art, such as any vehicle or diluent, or any other components or agents such as solvents (e.g., water), buffers (e.g., physiological saline), antioxidants, chelators, solubilizers, emulsifiers and / or preservatives.

[0119] The pharmaceutical compositions described herein can be administered systemically to a subject by any suitable means, and the route of administration will depend on the formulation of the pharmaceutical composition.

[0120] "Systemic administration" includes non-local administration, where the drug is administered to a site other than the site directly adjacent to or near the carcinoma or sarcoma, resulting in the systemic reception of the administered peptide. Conveniently, systemic administration is carried out by parenteral administration (e.g., intravenous, intraperitoneal, intramuscular, or subcutaneous).

[0121] The pharmaceutical composition may be provided in any suitable form known in the art, such as a liquid, suspension, emulsion, lyophilized product, or any mixture thereof.

[0122] In preferred embodiments, the peptide is provided in the form of a liquid pharmaceutical composition, and methods for preparing such formulations are well known in the art. Any such formulation can be used in the methods and applications of the present invention.

[0123] In some embodiments, the pharmaceutical composition is a “ready to use” formulation containing a soluble or solubilized peptide, intended for use as is or further diluted with an intravenous diluent. However, in some embodiments, the pharmaceutical composition may be provided in solid form, for example, as a lyophilized product, for dissolution in a suitable solvent to provide a liquid formulation.

[0124] A typical example involves storing peptides as lyophilized products and preparing pharmaceutical compositions from them. For instance, the peptides are dissolved in a small amount of sterile water (e.g., 0.5-10 mL, or approximately 1-5 mL), and if necessary, further diluted (e.g., with physiological saline) to provide a suitable amount for injection.

[0125] In some preferred embodiments, the pharmaceutical composition is formulated for parenteral infusion or injection, preferably intravenous or intraperitoneal infusion or injection.

[0126] In a preferred embodiment, the pharmaceutical composition is formulated as an infusion (e.g., intravenous infusion). The volume and duration of the infusion are determined by those skilled in the art and may depend on the characteristics of the person being treated, such as age, weight, sex, etc. In a typical embodiment, the infusion may have a volume of about 100 to 750 mL, for example, about 200 to 500 mL. In a further typical embodiment, the infusion may be administered over a period of about 30 minutes to about 8 hours, for example, at least about 1 hour, for example, 1 to 6 hours or 1 to 4 hours, for example, about 1 hour, 1.5 hours, 2 hours, 2.5 hours or 3 hours. In a further typical embodiment, the infusion may be administered over a period of about 30 minutes to about 24 hours, for example, about 23 hours, 22 hours, 21 hours or 20 hours, for example, 1 to 24 hours, 1 to 23 hours, 1 to 20 hours or 1 to 16 hours.

[0127] The infusion flow rate can be easily determined by those skilled in the art. In some embodiments, the infusion flow rate is started at approximately 5 mg / hr and increased approximately every 30 minutes until the required dose is administered. Typical embodiments of the infusion flow rate are outlined in the examples. In some embodiments, it is desirable that the maximum infusion rate does not exceed approximately 120 mg / hour and / or approximately 500 mL / hour. In some embodiments, approximately 45 mg / m 2 (For example, about 60 mg / m²) 2 The dosage is approximately 400-600 mL, for example, administered in a volume of about 500 mL.

[0128] The pharmaceutical composition, calculated as the free form of the peptide, is approximately 15-65 mg / m² per week per body surface area (BSA). 2 (For example, 15-50 mg / m²) 2 To provide a dosage of ) it is administered weekly. In some embodiments, the pharmaceutical composition is calculated as the free form of the peptide and is approximately 20-60 or 20-45 mg / m² per week per body surface area (BSA). 2 For example, approximately 20, 30, 45, or 60 mg / m² per week, per body surface area (BSA). 2 It is administered weekly to provide the dosage of the peptide.

[0129] BSA (body surface area) can be calculated, for example, using Mosteller's formula (√([height (cm) × weight (kg)] / 3600). If necessary, use a conversion factor of 0.025 mg / kg = 1 mg / m² for the average adult. 2 It can be converted to mg / kg using this method.

[0130] In some embodiments of the present invention, the pharmaceutical composition is administered weekly for at least three weeks, for example, three, four, five, six, seven, eight, nine, ten weeks or more (for example, two, three, four, five, fifty weeks or more). This administration may be in a single cycle or as a sum of multiple cycles.

[0131] Weekly administration typically refers to regular administration, such as on day 1, day 8, and day 15 of a three-week cycle. However, it is clear that some flexibility is necessary to achieve patient compliance; that is, "weekly" does not strictly mean a 7-day interval. Therefore, for example, a weekly administration could be on day 1, day 8±1, day 15±1 of a three-week cycle, such as day 1, day 7, day 15, day 1, day 9, day 14, or day 1, day 7, day 16, etc.

[0132] In this book, a "cycle" refers to a period of time during which a particular treatment regimen is applied, and is generally repeated to perform periodic treatment. The treatment in each cycle may be the same or different (for example, different dosages, timings, etc. may be used). In some embodiments, one cycle may be 3 to 6 weeks or 3 to 12 weeks long, for example, a 3, 4, 6, 9, or 12-week cycle. In some embodiments, one cycle may be about 1 to 6 months long, i.e., about 1 to 6 months, for example, about 1 to 4 months or 1 to 3 months, for example, about 1 to 2 months (for example, 3 to 26 weeks, for example, about 3 to 16 weeks or 3 to 12 weeks, for example, about 4 to 8 weeks), and may be administered weekly. In preferred embodiments, this cycle is repeated at least once. Therefore, multiple cycles may be used, for example, at least 2, 3, 4, or 5 cycles, for example, 6, 7, 8, 9, or 10 (for example, 10, 20, 30, or more) cycles may be used. In some embodiments, the treatment cycle may be continued until disease regression or progression occurs. In some embodiments, the treatment cycle may be continued as long as the patient exhibits stable disease according to RECIST criteria. In some embodiments, treatment may be resumed after a regression period.

[0133] In some embodiments, treatment cycles may be delimited by interruptions in treatment, i.e., periods during which the pharmaceutical composition is not administered weekly. In some embodiments, the period between cycles is at least one week, e.g., two, three, four weeks or longer. In some embodiments, the period between cycles is at least one month, e.g., two, three, four months or longer.

[0134] However, in some embodiments, a second or subsequent treatment cycle may immediately follow a first or previous cycle. For example, if the third week dose of the first cycle is administered on day 15 ± 1, the first week dose of the second cycle may be administered on day 22 ± 1.

[0135] In some embodiments of the present invention, the patient may receive other treatments before, concurrently with, or after the treatment of the present invention. For example, in some embodiments, the patient may be treated with radiotherapy and / or surgery according to procedures known in the art.

[0136] Therefore, in some embodiments, the patient being treated has previously received or is scheduled to receive other treatments, such as radiotherapy and / or surgery according to procedures known in the Art. In some embodiments, the other treatments may be immunotherapy, targeted therapy, hormone therapy, stem cell transplantation, or a combination thereof.

[0137] Therefore, in some embodiments, the method of the present invention may include a further step of treating the subject with radiotherapy and / or surgery (before, simultaneously with, or after treatment with the pharmaceutical composition of the present invention). The surgery may involve excision of carcinoma or sarcoma tumors.

[0138] In some embodiments, the pharmaceutical composition may contain one or more additional therapeutic agents, or may be intended to be administered together with one or more additional therapeutic agents.

[0139] For example, the inventors have found that administration of a pharmaceutical composition (especially the first dose) may cause an allergic reaction (typically grade 1 or 2) characterized by one or more symptoms selected from rash or itching (especially at the injection site), sweating, tachycardia, hives, and fever, which can be treated with antihistamines.

[0140] Therefore, in some embodiments, the pharmaceutical composition may include, or be administered together with (sequentially or simultaneously with) agents for preventing or treating allergic reactions, such as antihistamines, corticosteroids, and / or anti-inflammatory agents.

[0141] In some embodiments, the subject may be administered an agent for preventing or treating an allergic reaction prior to administration of the pharmaceutical composition of the present invention. In some embodiments, the subject may be administered a corticosteroid (e.g., dexamethasone or an equivalent dose of a pharmaceutically alternative corticosteroid), one or more histamine receptor antagonists (e.g., promethazine and / or ranitidine), an analgesic (e.g., acetaminophen) and / or an anti-leukotriene (e.g., monterukas) prior to administration of the pharmaceutical composition of the present invention. In some embodiments, the subject may be administered a corticosteroid (e.g., dexamethasone or an equivalent dose of a pharmaceutically alternative corticosteroid), one or more histamine receptor antagonists (e.g., promethazine and / or ranitidine), an analgesic (e.g., acetaminophen) and an anti-leukotriene (e.g., monterukas) prior to administration of the pharmaceutical composition of the present invention.

[0142] The inventors have advantageously found that the peptide of the present invention is effective in treating carcinomas and sarcomas when administered alone (e.g., in first-line therapy), but in some embodiments, the pharmaceutical composition may include or be administered with one or more further therapeutic agents useful for treating carcinomas or sarcomas. Examples include chemotherapeutic agents (e.g., cytotoxic agents or small molecule targeted drugs), immunotherapeutic agents (e.g., immune checkpoint inhibitors, monoclonal antibodies), hormones or their agonists or antagonists (e.g., gonadotropin-releasing hormone (GnRH) agonists, aromatase inhibitors, luteinizing hormone-releasing hormone (LHRH) agonists), stem cells or combinations thereof. For example, in some embodiments, the peptide of the present invention may be used as a second-line therapy, i.e., in combination with one or more further therapies or therapeutic agents (e.g., cytotoxic agents, immunotherapeutic agents, etc.) for subjects refractory to initial therapy, e.g., treatment with the peptide of the present invention or other therapies or therapeutic agents alone. Therefore, in some embodiments, the target being treated is refractory to one or more other therapies or therapeutic agents, such as chemotherapy or immunotherapy. In some embodiments, the peptides of the present invention can be used as a first-line treatment, either alone or in combination with one or more further therapies or therapeutic agents (e.g., cytotoxic agents, immunotherapies, etc.).

[0143] In some preferred embodiments, the peptide of the present invention is not administered as part of a combination therapy with another therapy or therapeutic agent, such as another chemotherapeutic agent or immunotherapeutic agent.

[0144] In some embodiments, further or other therapies or therapeutic agents are immunotherapies, such as immunotherapy selected from immune checkpoint inhibitors, T-cell transfer therapy, antibody therapy, therapeutic vaccines, or combinations thereof.

[0145] In some embodiments, further or other therapies or therapeutic agents are monoclonal antibodies, such as antibody-toxin conjugates, or targeted therapies such as CAR-T cell therapy. Thus, in some embodiments, further or other therapies or therapeutic agents are targeted immunotherapy or immunotherapies.

[0146] In some embodiments, further or other therapies or therapeutic agents are chemotherapy or chemotherapeutic agents, such as cytotoxic agents.

[0147] In some embodiments, the cytotoxic agents described herein (e.g., anticancer agents) may be used to provide a sensitizing effect, in other words, to enhance (or alternatively, increase, reinforce, or enhance) the effects of the peptides of the present invention (e.g., in the treatment of carcinoma or sarcoma), or to make a subject (or more specifically, carcinoma or sarcoma cells or tumors(s) present in the subject) more sensitive to the effects of the peptides of the present invention.

[0148] Accordingly, according to one embodiment, the present invention relates to a pharmaceutical composition for use in the treatment of carcinoma or sarcoma in human subjects, comprising a peptide or a pharmaceutically acceptable salt thereof, for use in combination with other therapeutic agents (e.g., cytotoxic agents), for isolated, co-administered, or sequential administration, wherein the peptide comprises the amino acid sequence and transcellular peptide described in SEQ ID NO: 1, and the pharmaceutical composition is administered systemically to a subject weekly, with the free form of the peptide yielding approximately 15-65 mg / m² of body surface area (BSA) per week. 2 (about 15~50mg / m 2 The present invention provides a pharmaceutical composition that provides the dosage of the peptide.

[0149] Alternatively, the method of the present invention further comprises administering another therapeutic agent (e.g., a cytotoxic agent) to the subject, wherein the therapeutic agent (e.g., a cytotoxic agent) is administered separately, simultaneously, or sequentially to a pharmaceutical composition comprising the peptide as defined herein.

[0150] In some embodiments, further therapeutic agents (e.g., cytotoxic agents) are agents capable of inhibiting, suppressing (e.g., killing) the proliferation, viability, and / or growth (replication / growth) of animal cells. In some embodiments, further therapeutic agents (e.g., cytotoxic agents) are capable of inhibiting, suppressing (e.g., killing) the proliferation, viability, and / or growth (replication / growth) of human carcinoma and / or sarcoma cells.

[0151] Cytotoxic agents include antitumor agents and all drugs indicated for oncological use. Therefore, this includes drugs used in chemotherapy treatment protocols ("chemotherapeutic agents" or "anticancer agents").

[0152] Cytotoxic agents are typically classified into different classes according to their mechanism of action, and all of these classes are intended herein. Therefore, cytotoxic agents may, for example, alkylating agents, crosslinking agents, intercalation agents, nucleotide analogs, spindle formation inhibitors, and / or topoisomerase I and / or II inhibitors. Other types or classes of agents include antimetabolites, plant alkaloids and terpenoids, or antitumor antibiotics.

[0153] Alkylating agents modify DNA by alkylating nucleosides, inhibiting correct DNA replication. Nucleotide analogs are incorporated into DNA during replication, inhibiting DNA synthesis. Spindle formation inhibitors inhibit spindle formation, causing it to arrest at metaphase. Intercalation agents interpose between DNA bases, inhibiting DNA synthesis. Topoisomerase I or II inhibitors affect DNA twisting, thereby inhibiting DNA replication.

[0154] Appropriate cytotoxic agents are known in the art, including, for example: actinomycin D, bortezomib, BCNU (carmustine), BI 2536, buparlisib, carboplatin, CCNU, camptothecin (CPT), cantharidin, cisplatin, combretastatin A4, CUDC-907, cyclophosphamide, cytarabine, dasatanib, dacarbazine, dactosilib, daporinad, daunorubicin, docetaxel, doxorubicin, dubelisib, DTIC, elethcromol, epirubicin, etoposide, gefitib, gemcitabine, idelalisib Ifosamide, ispinesib, irinotecan, ionomisin, luminsip, melphalan, methotrexate, mitomycin C (MMC), mitozantron mercaptopurine, molyblesib, oxaliplatin, ovatoclax, paclitaxel (Taxol), PARP-1 inhibitors, pelitinib, perifosine, PX-866, sepantronium bromide, SB-743921, tasericib, taxotere, temozolomide (TZM), teniposide, topotecan, trametinib, treosulfan tryptolide, umbralisib, vinorelbine, vincristine, vinblastine, borasertib, boxtalisib, 5-azacitidine, 5,6-dihydro-5-azacitidine, and 5-fluorouracil. Any of the above-mentioned cytotoxic agents may be used in the combination therapy of the present invention.

[0155] Cytotoxic agents for use in combination with pharmaceutical compositions comprising the peptides defined herein may be provided in the pharmaceutical compositions as defined above and may be administered as defined above. In some embodiments, the pharmaceutical compositions comprising the cytotoxic agents may be formulated for parenteral administration. Accordingly, the compositions may comprise pharmaceutically acceptable excipients, solvents, and diluents suitable for such formulations, e.g., intravenous bolus or injection.

[0156] Those skilled in the art will know the appropriate dosage range for any cytotoxic agent. In preferred embodiments, the cytotoxic agent is present in the pharmaceutical composition or administered to the subject within its typical dose range.

[0157] A preferred embodiment of the present invention is one in which one or more parameters or components used in the examples may be used as preferred features of the methods described herein.

[0158] Next, the present invention will be further described with reference to the following non-limiting embodiments and figures. [Examples]

[0159] Example 1 - Clinical trial of ATX-101 in patients with various carcinomas and sarcomas (SEQ ID NO: 914) Details of the clinical trial A Phase I, open-label, single-arm safety and tolerability study (ATX101-01 study) was conducted to evaluate an escalation cohort of ATX-101 (SEQ ID NO: 914) in patients with advanced solid tumors. The study aimed to systematically evaluate safety and tolerability and determine the maximum tolerated dose (MTD) and Phase II recommended dose of ATX-101. Pharmacokinetics and preliminary efficacy (antitum activity) were also evaluated.

[0160] If no signs of tumor progression are observed in the patient at the end of the 6-week Phase I trial (ATX101-01 trial), treatment can be continued in the long-term follow-up trial (ATX101-02 trial) until a reason for discontinuing treatment arises, such as disease progression or withdrawal of consent. Eligibility to participate in clinical trials Participation criteria 1. Women or men aged 18 or older 2. Signing of informed consent 3. Progressive disease for which conventional antitumor drug therapy is ineffective or refused. 4. There are lesions that are measurable or unmeasurable by CT / MRI scans (but radiologically evaluable), and there is at least one lesion in an area other than the previously irradiated area.

[0161] 5. The ECOG performance status is 0-2. 6. Life expectancy of more than 3 months 7. The following laboratory requirements must be met. • Absolute neutrophil count (ANC) ≥ 1.5 × 10⁻⁶ 9 / L • Platelet count ≥ 75 × 10 9 / L aPTT / PT ≤ 1.5 × ULN • Total bilirubin level ≤ 1.5 × ULN • AST and ALT ≤ 2.5 × ULN (≤ 5 × ULN if liver metastases are present) • Creatinine ≤ 1.5 × ULN • Albumin ≥ 30 g / L

[0162] 8. Women of childbearing potential (WOCBP) must use a highly effective method of contraception (with an annual failure rate of less than 1% when used consistently and correctly) and be willing to continue contraception for at least one month after the last infusion. Highly effective methods of contraception include combination hormonal contraception (containing estrogen and progesterone) with ovulation suppression, progesterone-only hormonal contraception with ovulation suppression, intrauterine devices, intrauterine hormone-releasing systems, bilateral fallopian tube obstruction, vasectomy partner, and sexual abstinence.

[0163] 9. Men who are not surgically azoospermic must use condoms until the end of the trial and for 30 days after the last treatment dose, unless they have a surgically azoospermic or postmenopausal female partner. During this time, they must refrain from trying to conceive.

[0164] Exclusion criteria 1. Individuals who have received the investigational drug within four weeks prior to administration of the investigational drug (within six weeks for investigational immunotherapy agents), or who are scheduled to receive such treatment during the prescribed treatment period or post-treatment period of this study. 2. The patient has received concomitant anticancer drug treatment (such as cytoreductive therapy, radiotherapy other than palliative bone-targeted radiotherapy, immunotherapy, cytokine therapy other than erythropoietin, etc.) within 21 days prior to the first dose of the investigational drug or within 5 times (5 times) the half-life of the investigational drug. 3. Use of hormone therapy within 7 days prior to the start of the clinical trial treatment. However, in patients with castration-resistant prostate cancer (CRPC), treatment with luteinizing hormone-releasing hormone agonists or antagonists may be continued.

[0165] a. For patients receiving bisphosphonate or denosumab, treatment must have been initiated at least 14 days prior to the date of the first dose. 4. Patients who are expected to require surgery or initiate chemotherapy during the trial period. 5. Side effects from medication administered more than four weeks prior (excluding hair loss, CTCAE Grade 2 or higher) have not resolved.

[0166] 6. Heart failure (according to the New York Heart Association [NYHA] functional classification) is grade 2 or higher. 7. Within the past six months, there is evidence or a history of clinically significant cardiac disease such as congestive heart failure, unstable angina, acute myocardial infarction, or cerebrovascular disease, or symptomatic arrhythmias requiring treatment (excluding off-systolic or mild conduction abnormalities, or well-controlled and well-treated chronic atrial fibrillation). 8. QTcF > 460 ms

[0167] 9. Active central nervous system (CNS) metastases. Patients with confirmed CNS metastases must have received radiation therapy or surgery at least two weeks prior to ATX-101 administration. If neuropathy persists, corticosteroid administration should be discontinued and the patient should be stable. 10. Lymphatic carcinomatosis 11. Piatric Lesions 12. Major surgery within 3 weeks of screening 13. If the principal investigator determines that exposure to the investigational drug would increase the expected risk or interfere with the planned evaluation of an acute or chronic disease other than the indication for the clinical trial.

[0168] 14. The patient is either breastfeeding or pregnant, with a positive serum β-human chorionic gonadotropin (β-HCG) pregnancy test confirmed during a screening test or subsequent consultation. 15. Unwilling to or unable to comply with the protocol requirements. 16. Known positive status for human immunodeficiency virus (HIV) and active hepatitis B or C. In patients with a history of hepatitis B or C infection, clearance of infection must be demonstrated by negative serological testing for hepatitis B surface antigen (HBsAg) and negative ribonucleic acid (RNA) for hepatitis C virus (HCV) within at least 6 weeks after antiviral therapy.

[0169] 17. You have a history of severe allergies (requiring hospital treatment), severe reactions to any medication, or known or suspected allergies or hypersensitivity to any component of the test drug. 18. Insufficient venous access for blood collection.

[0170] ATX-101 active pharmaceutical ingredient The peptide active pharmaceutical ingredient is amorphous; its crystalline and polymorphic forms are unknown, and it is freely soluble in water and aqueous media. It was available as a hydrochloride salt (the chloride counterion is ionically bonded to the basic side chain of the peptide; 18 mol / mol peptide, theoretical value; molecular formula). C 158 H 285 N 71 O 29 S,18HCl; relative molecular weight: 4320.9. Free base: 3673.3).

[0171] ATX-101 Pharmaceutical Product This peptide was provided as a sterile lyophilized product for reconstitution and dilution before injection. The lyophilized product was placed in 5 mg or 80 mg single-use colorless vials, sealed with rubber stoppers, aluminum flip-off seals, and plastic discs, and stored in a light-shielded place at -20°C ± 5°C. Prior to administration, the test drug was removed from the freezer and reconstituted with 1 ml (5 mg) or 4 mL (80 mg) of sterile water for injection. To aid in reconstitution before intravenous infusion, the vials were gently swirled without shaking before dilution in an infusion bag with an appropriate volume (e.g., 100 mL, 250 mL, and 500 mL) of normal saline.

[0172] Treatment and Dosage In the Phase I trial (ATX101-01 trial), intravenous administration of ATX-101 (net peptide with salt content subtracted) was conducted at four dose levels (20, 35, 45, and 60 mg / m², calculated based on the patient's height and weight). 2 The following was tested: ATX-101 was administered by intravenous drip infusion over at least one hour using a constant infusion rate or an adaptive infusion method starting at 5 mg / hr and increasing the infusion rate every 30 minutes, as shown in the infusion rate table below (Table 1). The maximum infusion rate was not to exceed 120 mg / hr and 500 mL / hr. 45 and 60 mg / m³ 2 It was administered in 500 mL doses.

[0173] [Table 1]

[0174] The treatment consisted of 21-day cycles, with a single intravenous infusion of ATX-101 administered on days 1, 8, and 15 of each cycle. Patients received the treatment weekly for a maximum of two cycles (2 x 21 days = 6 weeks).

[0175] After the initial 6-week treatment in the Phase I trial (ATX101-01 trial), patients could continue treatment in the long-term follow-up trial (ATX101-02 trial). These patients received the same dose regimen as in the 01 trial and were treated for up to 15.6 months.

[0176] Assessments conducted Safety: The incidence, severity, duration, and treatment-related adverse events (TEAEs) were evaluated according to the Common Terminology Criteria for Adverse Events (CTCAE) v4.03. Adverse events (AEs) not covered by CTCAE v4.03 were graded as mild, moderate, or severe.

[0177] Efficacy: Tumor evaluation was performed according to RECIST V1.1 (Response Evaluation Criteria In Solid Tumors, https: / / ctep.cancer.gov / protocolDevelopment / docs / recist_guideline.pdf).

[0178] Tumor imaging was performed using CT or MRI of the chest, abdomen, or pelvis (and other areas as needed depending on the type of tumor). Based on RECIST V1.1, evaluations were performed at baseline and after up to six weekly treatments with ATX-101 (at the end of the ATX101-01 trial). Patients who received longer-term treatment (i.e., the long-term follow-up trial ATX101-02) were further evaluated every three months (±14 days). In general, lesions detected at baseline were to be followed up at subsequent tumor evaluation visits using the same imaging methods and imaging equipment.

[0179] Clinical signs of tumor progression were observed throughout the entire treatment period. This includes evaluations of specific symptoms, physical examinations, and laboratory values.

[0180] For each patient, the principal investigator used the most appropriate method from the above to assess the patient's tumor status. Patients with clinical signs of progression were recommended to undergo unscheduled tumor scans and evaluations. In general, the metrics selected for individual patient tumor assessment were consistent throughout the study and corresponded to the metrics used for patient enrollment.

[0181] Based on the aforementioned evaluation, the following parameters were assessed.

[0182] DCR (Disease Control Rate): Defined as the percentage of patients who did not show signs of disease progression based on RECIST V1.1 during the trial period.

[0183] ORR (Objective Response Rate): Defined as the percentage of patients who achieved a complete response (CR) or partial response (PR) based on the RECIST 1.1 criteria.

[0184] Number of patients In the Phase I trial, 22 patients received treatment. Of these 22 patients, 10 (45%) showed no signs of disease progression 6 weeks after the start of administration (at the end of the ATX101-01 trial). Nine of these 10 patients were enrolled in a long-term follow-up study, which is part of the efficacy evaluation described below. Table 2 summarizes the disease, tumor status at the start of the trial, and the dose of ATX-101 administered to these nine patients. At the start of the trial, all patients, except for patient #1 whose disease was stable, had progressive tumors.

[0185] [Table 2]

[0186] Table 3 shows the total number of chemotherapy treatments (including surgery, chemotherapy, and radiotherapy) received by patients who underwent long-term follow-up before participating in the trial. Based on the progression of their disease, most patients had received numerous prior treatments. [Table 3]

[0187] Safety data ATX-101 demonstrated a favorable safety profile in all 22 patients treated in Phase 1 trials (ATX101-01 and -02 trials). No treatment-related deaths or dose-limiting toxicities (DLTs) were reported. There were no serious or severe treatment-related adverse events. Furthermore, there were no treatment discontinuations due to treatment-related toxicity. Treatment-related adverse events were mild to moderate.

[0188] The most common treatment-related event was a grade 1 or 2 infusion-related reaction (IRR), observed in 73% of patients. These are a type of allergic reaction presenting with symptoms such as itching, redness, urticaria, fever, rash, swelling, flushing, and hives. Symptoms resolved rapidly upon discontinuation of the infusion, regardless of whether symptomatic treatment with antihistamines or corticosteroids was administered. In the vast majority of patients, ATX-101 infusions could be safely resumed, and treatment was completed. IRRs were reported not only during the initial infusion but also during subsequent infusions. Repeated administration of ATX-101 did not worsen the IRRs. To manage IRRs, two measures were implemented: gradually increasing the infusion rate with each infusion (see Table 1) and mandating premedication consisting of dexamethasone, paracetamol (acetaminophen), montelukast, and histamine receptor antagonists.

[0189] Safety Overview ATX-101 can be safely administered as a monotherapy at all doses investigated. Treatment-related adverse events were mild to moderate in severity. IRRs were observed in most patients but were manageable without causing serious or life-threatening complications.

[0190] Effectiveness data Table 4 and Figure 1 show the total treatment duration of ATX-101 for patients treated for more than 6 weeks. At the end of treatment, 3 patients had stable disease, 1 patient had no tumor assessment, and the remaining 5 patients had disease progression. The median treatment duration was 4.2 [2.1-15.6] months.

[0191] [Table 4]

[0192] Below are brief case reports of nine patients who received treatment during a long-term follow-up study. Case 1: Pancreatic Cancer This 67-year-old male patient was diagnosed with metastatic pancreatic cancer in May 2018. He was treated with gemcitabine and nab-paclitaxel until August 2018. He was enrolled in a clinical trial in October 2018. At that time, his condition was stable, and metastases were observed in the liver (segment 4 / 5) and abdomen (coeliac axis). He was treated with ATX-101 at a dose of 20 mg / m². 2 During the first six weeks of weekly intravenous infusions, the tumor remained stable. As a result, treatment was continued. After a total treatment period of 7.2 months, treatment was discontinued due to a deterioration in performance status despite no disease progression.

[0193] Case 2: Uterine leiomyosarcoma This woman was diagnosed with uterine leiomyosarcoma in August 2015, at the age of 45. Prior to ATX-101 treatment, she had received four types of anticancer drug therapy, including hormone therapy (tamoxifen, letrozole, medroxyprogesterone) and chemotherapy (gemcitabine + docetaxel).

[0194] At the time of participation in the trial in October 2018, the sarcoma had progressed, with lesions found in the lungs (right middle lobe, left lower lobe) and lymph nodes (left external iliac crest), indicating metastasis. 20 mg / m² 2After administering ATX-101 six times a week, tumor growth stopped, and the disease stabilized according to standard RECIST tumor evaluation criteria. Weekly infusions were continued for a total of 15.6 months, with regular tumor assessments. The disease remained stable, and there were no signs of tumor growth. The patient decided to discontinue treatment, but tumor control continued. In November 2020, two years after the start of ATX-101 treatment, no signs of tumor growth were observed.

[0195] Case 3: Urethral squamous cell carcinoma This male patient is 61 years old and was diagnosed with urethral cancer in December 2017. Prior to joining the trial, he had received combination chemotherapy consisting of paclitaxel, ifosfamide, and cisplatin. This combination therapy was discontinued in June 2018 because the tumor progressed during treatment.

[0196] In January 2019, the patient was enrolled in a clinical trial. At that time, the advanced cancer had metastasized to the lymph nodes (both inguinal regions and the right external iliac crest). The initial dosage was 20 mg / m² for the first six weeks. 2 The patient was administered ATX-101 weekly, and there were no signs of disease progression; the disease remained stable. After a total of 6.9 months of treatment, the tumor progressed, and treatment was discontinued.

[0197] Case 4: Cervical squamous cell carcinoma This woman was diagnosed with cervical cancer in January 1985 at the age of 32. Prior to receiving ATX-101, she underwent chemotherapy including surgery, chemotherapy, and radiation therapy. The chemotherapy regimens included carboplatin + paclitaxel, cisplatin + radiation therapy (pelvis), combination therapy of carboplatin + paclitaxel + bevacizumab, cisplatin monotherapy, and finally, an experimental anti-PD-1 agent. The latter treatment was her most recent treatment before joining the trial and was discontinued in August 2018 due to disease progression.

[0198] This patient was enrolled in a clinical trial in January 2019. At that time, the tumor had metastasized to the soft tissue of the right iliac crest and the left supraclavicular lymph node. For the first six weeks, the dosage was 20 mg / m² per week. 2After administering ATX-101 intravenously, the tumor stabilized, and treatment was continued. Overall, the disease progressed during the 4.2-month treatment period, and treatment was discontinued.

[0199] Case 5: Undifferentiated Pleomorphic Sarcoma This male patient was diagnosed with metastatic sarcoma in December 2010 at the age of 63. Prior to ATX-101 treatment, he underwent left lower limb amputation, gemcitabine + docetaxel, pazopanib, radiation therapy (left hip and right arm), doxorubicin, and right upper limb amputation. Chemotherapy with pazopanib was his most recent systemic therapy, but it was discontinued in December 2017 due to disease progression.

[0200] The patient was enrolled in a clinical trial in April 2019. At that time, the sarcoma showed lesions in the lungs and thighs. ATX-101 was administered at 30 mg / m². 2 After intravenous administration six times a week, the patient's tumor showed no signs of disease progression, and treatment was continued for a total of 4.1 months before disease progression was diagnosed.

[0201] Case 6: Lung adenocarcinoma (non-small cell lung cancer) ) This male patient was diagnosed with metastatic lung adenocarcinoma in October 2013 at the age of 56. He underwent a right upper lobectomy followed by radiation therapy in 2013, and a left upper lobectomy in 2015. He also received five different lines of chemotherapy: two cycles of cisplatin + vinorelbine, nivolumab, pemetrexed, and carboplatin + gemcitabine. The final treatment, carboplatin + gemcitabine, was discontinued in April 2019 due to disease progression.

[0202] This patient was enrolled in a clinical trial in September 2019. At that time, multiple lesions appeared in the lungs and adrenal glands (left and right). The patient was given ATX-101 at a dose of 45 mg / m². 2 The patient received weekly intravenous infusions. After 6 weeks of treatment, the patient's condition stabilized, and ATX-101 treatment was continued. Overall, the patient received infusions for 4.1 months. Although no signs of disease progression were reported, treatment was discontinued.

[0203] Case 7: Non-small cell lung cancer (NSCLC) This female patient was diagnosed with NSCLC in September 2017 at the age of 64. She underwent local therapies including right upper / middle lobectomy, stereotactic radiosurgery, and radiation therapy to multiple sites. She received three lines of systemic therapy: carboplatin + pemetrexed, atezolizumab, and an experimental drug (PD-1 / CTLA-4 bispecific antibody). Treatment with the experimental drug was discontinued in August 2019 due to disease progression. Between the discontinuation and the start of the trial, the patient received radiation therapy to the skull and brain.

[0204] This patient was enrolled in a clinical trial in September 2019. At that time, different lesions were observed in the kidneys, left adrenal gland, thyroid gland, and lungs. The patient received ATX-101 at a dose of 45 mg / m². 2 The medication was administered intravenously once a week. After 6 weeks of treatment, the patient's condition stabilized. Treatment continued for a total of 3.5 months, but was discontinued because the patient's condition worsened.

[0205] Case 8: Cervical Cancer This woman was diagnosed with cervical cancer in June 2018 at the age of 47. The tumor was surgically removed, and the area was treated with radiation. In 2019, she began three types of systemic anticancer drug therapy: carboplatin + paclitaxel + bevacizumab chemotherapy, followed by bevacizumab maintenance therapy, and finally treatment with an investigational drug (anit-PD-1 antibody). The latter treatment was discontinued in March 2020 due to disease progression.

[0206] The patient was enrolled in a clinical trial in May 2020. At that time, the tumor had metastasized to the soft tissues within the pelvis. The dosage was ATX-101 60 mg / m². 2 The patient received an intravenous infusion once a week. After 6 weeks of treatment, the patient's condition stabilized. Treatment was continued for another 2.1 months, but due to urinary tract obstruction and obstruction of the urethral stent by the tumor, the clinical progression of the disease was judged, and treatment was terminated prematurely. Imaging studies of the tumor were not performed.

[0207] Case 9: Ovarian granulosa cell tumor This woman was diagnosed with ovarian cancer in December 2009 at the age of 48. Prior to participating in this study, she had received various chemotherapy treatments: five debulking surgeries, two rounds of hormone therapy, and two lines of combination chemotherapy (etoposide + ifosfamide + cisplatin, and carboplatin + gemcitabine). The most recent systemic therapy prior to ATX-101, carboplatin + gemcitabine, was discontinued in November 2017 after approximately two years of treatment due to disease progression.

[0208] This patient underwent their fifth debulking surgery in February 2020 and was subsequently enrolled in this trial in August 2020. This patient received ATX-101 at a rate of 60 mg / m² weekly. 2 The patient received intravenous treatment for five months, but no signs of disease progression were observed. Treatment was discontinued because another debulking surgery was scheduled, even though no disease progression was observed.

[0209] Summary of effectiveness In the Phase I trial, 95% (n=21) of patients had advanced disease at the start of the trial. 45% (n=10) of all patients experienced disease stabilization after the first six weeks of ATX-101 treatment. Nine patients (42% of all patients) continued treatment, with a median total treatment duration of 4.2 months. Of these nine patients, only five discontinued treatment due to disease progression. The median progression-free survival for these patients was concluded to be 4.2 months or longer. Considering the tumor status at the start of treatment, this stabilization of disease is thought to be attributable to the activity of ATX-101.

[0210] Clinical overall conclusion ATX-101 is a first-in-class compound that is well-tolerated with once-weekly intravenous administration. The only mild to moderate adverse events associated with ATX-101 are fluid-related reactions, which are easily manageable.

[0211] ATX-101 was able to stabilize the disease for a clinically meaningful period in cancer patients with a long history of prior treatment and no standard treatment options. This effect was not anticipated at all in the Phase I trial aimed at determining the safety and tolerability of the compound. Prior to this trial, the maximum tolerated dose was 60 mg / m². 2 Doses exceeding this level were tested, and it was expected that only these high doses would be an effective treatment. Notably, the stabilizing effect was observed at the lowest dose tested, 20 mg / m². 2 This has already been observed at all other investigated dose levels (30, 45, 60 mg / m²). 2 This was also evident in the study. Surprisingly, no dose-dependent effect was observed, with approximately 15-65 mg / m² being used. 2 The dosage range indicates that it is effective in treating various types of tumors.

[0212] Data from the Phase I trial showed that the patient risk-benefit ratio outweighed the benefits. In particular, the effectiveness of the tested dosing regimen was surprising, given that the patients enrolled in the Phase I trial were often resistant to other treatments. Furthermore, the efficacy observed across various disease environments supports the efficacy of ATX-101 in the present invention's dose range and patient population, and the characteristics of ATX-101 enable further clinical development as a monotherapy and combination therapy.

Claims

1. A pharmaceutical composition for the treatment of soft tissue sarcoma in human subjects, wherein the pharmaceutical composition comprises a peptide or a pharmaceutically acceptable salt thereof, the amino acid sequence of the peptide being the sequence described in Sequence ID No. 914, and the free form of the peptide is calculated to be 15 to 65 mg / m² per week. ２ A pharmaceutical composition in which the pharmaceutical composition is administered systemically to a subject weekly in order to provide a dosage of peptides based on body surface area (BSA).

2. The peptide dosage, calculated as the free form of the peptide, is 15-50 mg / m² per week. ２ The pharmaceutical composition according to claim 1, wherein body surface area (BSA) is the body surface area.

3. The peptide dosage, calculated as the free form of the peptide, is 20-60 mg / m² per week. ２ The pharmaceutical composition according to claim 1, wherein body surface area (BSA) is the body surface area.

4. The peptide dosage, calculated as the free form of the peptide, is 20–45 mg / m² per week. ２ The pharmaceutical composition according to claim 1 or 3, wherein body surface area (BSA) is the body surface area.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the soft tissue sarcoma is a myosarcoma, liposarcoma, or undifferentiated pleomorphic sarcoma.

6. The pharmaceutical composition according to claim 5, wherein the sarcoma is a leiomyosarcoma.

7. The pharmaceutical composition according to claim 6, wherein the leiomyosarcoma is uterine leiomyosarcoma.

8. The pharmaceutical composition according to claim 5, wherein the undifferentiated pleomorphic sarcoma is metastatic undifferentiated pleomorphic sarcoma.

9. A pharmaceutical composition according to any one of claims 1 to 8, wherein the pharmaceutical composition is administered weekly to a subject for a treatment cycle of at least three weeks.

10. The pharmaceutical composition according to claim 9, wherein the treatment cycle is repeated at least once.

11. A pharmaceutical composition according to any one of claims 1 to 10, wherein the pharmaceutical composition is administered to a subject parenterally.

12. A pharmaceutical composition according to any one of claims 1 to 11, wherein the pharmaceutical composition is administered to a subject by intravenous injection.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the pharmaceutical composition is administered to a subject by intravenous infusion over at least one hour.

14. The pharmaceutical composition according to any one of claims 1 to 8 and 11 to 13, wherein the pharmaceutical composition is administered to a subject weekly in multiple treatment cycles.

15. The pharmaceutical composition according to claim 14, wherein there is an interruption during the treatment cycle.

16. The pharmaceutical composition according to claim 15, wherein the interruption period between treatment cycles is at least one week.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the peptide is provided as a hydrochloride salt.

18. The pharmaceutical composition according to any one of claims 1 to 16, wherein the peptide comprises an acetyl group at its N-terminus.

19. The use of a peptide or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical composition for the treatment of soft tissue sarcoma in humans, wherein the amino acid sequence of the peptide is the sequence described in SEQ ID NO: 914, and the free form of the peptide is calculated to be 15 to 65 mg / m². ２ The pharmaceutical composition is administered systemically to the subject weekly to provide a dose of BSA / week of peptide.

20. The use according to claim 19, wherein the dose of the peptide is as defined in any one of claims 2 to 4.

21. The use according to claim 19 or 20, wherein the soft tissue sarcoma is as defined in any one of claims 5 to 8.

22. The use according to any one of claims 19 to 21, wherein the pharmaceutical composition is administered to a subject as defined in any one of claims 9 to 16.

23. The use according to any one of claims 19 to 22, wherein the pharmaceutically acceptable salt is a hydrochloride salt.

24. The use according to any one of claims 19 to 23, wherein the peptide comprises an acetyl group at its N-terminus.

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