Cyclic peptides targeting the epidermal growth factor receptor and its variants for drug delivery

Abstract

Provided in the present application are a family of cyclic peptides that exhibit specific affinity for EGFR-expressing cells and can be used as a drug delivery vehicle for labeling agents and cytotoxic drugs for the above cells in the form of a peptide-drug conjugate, a pharmaceutical composition containing the same, and their use for cancer treatment.

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 344,657, filed May 23, 2022, the entire contents of which are incorporated herein by reference.

[0002] Statement Regarding the Sequence Listing A 20,480-byte file named 96504.xml, created on May 23, 2023, and filed concurrently with this application, is hereby incorporated by reference.

[0003] In some embodiments, the present invention relates to cancer treatment, and more specifically but not limited to, a family of cyclic peptides having affinity for epidermal growth factor receptor and its variants for drug delivery.

Background Art

[0004] The genetic and cellular heterogeneity of almost all cancers continuously hinders the development of effective cancer chemotherapy. To address this problem, several studies have highlighted the potential of combination therapies using multiple drugs with different mechanisms of action. The rationale for this strategy is that such combinations significantly reduce the probability of proliferation of clones resistant to multiple drugs. However, such resistance occurs. One example is the use of small molecules and antibodies against cancers overexpressing epidermal growth factor receptor (EGFR).

[0005] EGFR plays a critical role in the control of cell proliferation, differentiation, and migration. Its overexpression is frequently detected in various human tumors of epithelial origin, including non-small cell lung cancer (NSCLC), breast cancer, head and neck cancer, gastric cancer, colorectal cancer, prostate cancer, esophageal cancer, bladder cancer, renal cancer, pancreatic cancer, and ovarian cancer. EGRF undergoes several mutations, particularly mutations within exons 18 - 21 that encode a part of the EGFR kinase domain, which are associated with several forms of lung cancer. The most common variant is EGFR variant III (EGFRvIII), which requires deletions in exons 2 - 7. The EGFRvIII mutation has been suggested as a marker for cancer stem cells or tumor-initiating populations, and its presence has been associated with more progressive disease and poor prognosis. EGFR overexpression in NSCLC has been identified in 40% - 89% of cases.

[0006] NSCLC is the most common form of lung cancer, with a high incidence rate and a 5-year survival rate of only 11% - 15%. Drug resistance is the main cause of treatment failure in this disease. Two classes of EGFR inhibitors have been clinically approved, which are monoclonal antibodies (cetuximab, panitumumab) that target the extracellular domain of EGFR and small molecule kinase inhibitors (gefitinib, erlotinib) that block intracellular phosphorylation of the receptor. Despite the good response rates of these drugs, most patients eventually acquire drug resistance, which becomes a major limitation in reducing the long-term efficacy of treatment. Therefore, new treatment methods are still needed.

[0007] Peptide-drug conjugates (PDCs) are targeted therapeutic approaches for cancer therapy that combine the specificity of peptides with the efficacy of small molecule drugs. In PDCs, a peptide that specifically targets cancer cells is chemically conjugated to a cytotoxic drug. The peptide guides the drug to the tumor site where it is released to exert the therapeutic effect of the drug. This approach increases the selectivity and effectiveness of cancer treatment while reducing side effects on healthy tissues. In clinical trials of PDCs, two therapeutic PDCs have been approved on the market: 177 Lu-dotatate (Lutathera) and melphufen. 177 Lu-dotatate, the first PDC approved by the US Food and Drug Administration (FDA), is used for the treatment of tumors of the gastrointestinal tract, pancreas, and neuroendocrine system. Others are in various stages of research and development.

[0008] WO2015 / 187540A1 provides peptide-drug conjugates containing a self-immolative linker of p-aminobenzylcarbamoyl or p-aminobenzoyl carbonate. The peptide-drug conjugate contains a peptide moiety cleavable by cellular proteases conjugated to the self-immolative linker, which is conjugated to the cytotoxic drug moiety. Upon cleavage of the peptide moiety, the linker self-destructs and releases the active form of the cytotoxic drug. A dimer structure of the peptide-drug conjugate containing two molecules of the cytotoxic drug per conjugate is also disclosed.

[0009] WO2022 / 155172A1 provides conjugates of therapeutic molecules (e.g., cytotoxic agents) and targeting moieties (e.g., peptides) useful for the treatment of diseases such as cancer. SUMMARY OF THE INVENTION

[0010] The present disclosure provides a methodology for identifying and validating a novel cyclic peptide that not only has a high specific affinity for EGFR and / or EGFR variants expressed in cancer cells but also exhibits the ability to be internalized within cells and thus can be used as a delivery vehicle in the form of a conjugate carrying a payload (drug molecule, detectable label / reporter agent, etc.).

[0011] Thus, according to one aspect of some embodiments of the present invention, there is provided a cyclic peptide having two terminal cysteine residues and looped by a disulfide bond between the terminal cysteine residues, comprising 7 to 11 amino acid residues, which is characterized by at least one of the following properties. Affinity for EGFR and / or EGFR variants expressed by cancer cell lines selected from the group consisting of H1299, H1975 and DKMG, and the ability to be internalized by cancer cell lines determined by flow cytometry analysis using the cyclic peptide conjugated to a labeling moiety, and Specificity for EGFR and / or EGFR variants determined by competitive binding and / or internalization assays against the native ligand of EGFR and / or EGFR variants in cancer cell lines.

[0012] In some embodiments, the cyclic peptide is a nonameric peptide having a heptameric sequence sandwiched between two terminal cysteine residues at both ends.

[0013] In some embodiments, the cyclic peptide has a sequence individually selected from the group consisting of the following. CLRWRFGRC (SEQ ID NO: 1), CSAETVESC (SEQ ID NO: 2), CVRWRFGRC (SEQ ID NO: 3), CLAVEVRPC (SEQ ID NO: 4), CPNDSYHQC (SEQ ID NO: 5), CHVPGSYIC (SEQ ID NO: 6), CWHSLSLAC (SEQ ID NO: 7), CSALWASHC (SEQ ID NO: 8), CVNAMQSYC (SEQ ID NO: 9), CNWLSRTEC (SEQ ID NO: 10), and CAQYTPGRC (SEQ ID NO: 11), and any C-terminal amidated products, salts, hydrates or solvates thereof.

[0014] In some embodiments, the cyclic peptide is CLRWRFGRC (SEQ ID NO: 1), and any C-terminal amidated product, salt, hydrate or solvate thereof.

[0015] In some embodiments, the cyclic peptide is CSAETVESC (SEQ ID NO: 2), and any C-terminal amidated product, salt, hydrate or solvate thereof.

[0016] In some embodiments, the cyclic peptide is CVRWRFGRC (SEQ ID NO: 3), and any C-terminal amidated product, salt, hydrate or solvate thereof.

[0017] In some embodiments, the cyclic peptide is CLAVEVRPC (SEQ ID NO: 4), and any C-terminal amidated product, salt, hydrate or solvate thereof.

[0018] In some embodiments, the cyclic peptide is CPNDSYHQC (SEQ ID NO: 5), and any C-terminal amidated product, salt, hydrate or solvate thereof.

[0019] In some embodiments, the cyclic peptide is CHVPGSYIC (SEQ ID NO: 6), and any C-terminal amidated product, salt, hydrate or solvate thereof.

[0020] In some embodiments, the cyclic peptide is CWHSLSLAC (SEQ ID NO: 7), and any C-terminal amidated product, salt, hydrate or solvate thereof.

[0021] In some embodiments, the cyclic peptide is CSALWASHC (SEQ ID NO: 8), and any C-terminal amidated product, salt, hydrate or solvate thereof.

[0022] In some embodiments, the cyclic peptide is CVNAMQSYC (SEQ ID NO: 9), and any C-terminal amidated product, salt, hydrate or solvate thereof.

[0023] In some embodiments, the cyclic peptide is CNWLSRTEC (SEQ ID NO: 10), and any C-terminal amidated product, salt, hydrate or solvate thereof.

[0024] In some embodiments, the cyclic peptide is CAQYTPGRC (SEQ ID NO: 11), and any C-terminal amidated product, salt, hydrate or solvate thereof.

[0025] In some embodiments, the cyclic peptide is bound to a moiety (conjugate) consisting of a bioactive agent.

[0026] In some embodiments, the bioactive agent is selected from the group consisting of drugs, cytotoxic agents, imaging agents, diagnostic agents and labeling agents.

[0027] In some embodiments, the cyclic peptide provided in the present application is for use in targeted drug delivery to cells overexpressing EGFR and EGFRvIII variants.

[0028] According to another aspect of some embodiments of the present invention, a conjugate is provided that includes a moiety consisting of the cyclic peptide provided in the present application and a moiety consisting of a bioactive agent. In some embodiments, the two moieties are linked by a covalent bond.

[0029] In some embodiments, the moiety consisting of the cyclic peptide and the moiety consisting of the bioactive agent are bound via a linking moiety.

[0030] In some embodiments, the linking moiety is labile, bio-cleavable, or biodegradable.

[0031] In some embodiments, the linking moiety includes a spacer moiety.

[0032] In some embodiments, the linking moiety is selected from the group consisting of a gamma-aminobutyric acid (GABA) moiety, a glutathione moiety, a lysine moiety, a succinic acid moiety, a 2-amino-5-(carbamoylamino) pentanoic acid (PABA) moiety, a citrulline moiety, a valine-citrulline-PABA moiety, and any combination thereof.

[0033] In some embodiments, the bioactive agent is a cytotoxic agent.

[0034] In some embodiments, the cytotoxic agent is selected from the group consisting of camptothecin (CTP), doxorubicin (DOX), monomethyl auristatin F (MMFA), and 7-ethyl-10-hydroxycamptothecin (SN38).

[0035] In some embodiments, the peptide-drug conjugate exhibits higher cytotoxicity against cells that overexpress EGFR and the EGFRvIII variant compared to the free cytotoxic agent.

[0036] According to another aspect of some embodiments of the present invention, there is provided a pharmaceutical composition comprising the conjugate provided herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0037] In some embodiments, the pharmaceutical composition is packaged by a packaging material, and it is specified by printing on the inside or surface of the packaging material that it is for the treatment of a medical condition. In some embodiments, the medical condition is treatable by a bioactive agent (drug). In some embodiments, the medical condition is cancer.

[0038] According to another aspect of some embodiments of the present invention, there is provided the peptide-drug conjugate provided herein for the treatment of a medical condition. In some embodiments, the medical condition is treatable by a bioactive agent (drug). In some embodiments, the medical condition is cancer.

[0039] According to another aspect of some embodiments of the present invention, there is provided a peptide-drug conjugate provided herein for use in the manufacture of a medicament.

[0040] According to another aspect of some embodiments of the present invention, there is provided the use of a peptide-drug conjugate provided herein in the manufacture of a medicament.

[0041] In some embodiments, the medicament is for the treatment of a medical condition. In some embodiments, the medical condition is treatable with a bioactive agent. In some embodiments, the medical condition is cancer.

[0042] According to another aspect of some embodiments of the present invention, there is provided the use of a conjugate provided herein for treating a medical condition. In some embodiments, the medical condition is treatable with a bioactive agent. In some embodiments, the medical condition is cancer.

[0043] According to another aspect of some embodiments of the present invention, there is provided a method for treating a medical condition associated with cells that overexpress EGFR and EGFRvIII variants, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a conjugate or pharmaceutical composition or medicament provided herein.

[0044] In some embodiments, the medical condition is treatable with a bioactive agent.

[0045] In some embodiments, the medical condition is cancer.

[0046] In some embodiments, the medical condition is associated with cells that overexpress EGFR and EGFRvIII variants.

[0047] In some embodiments, the bioactive agent is a cytotoxic agent or an anti-cancer agent.

[0048] According to another aspect of some embodiments of the present invention, there is provided a method for diagnosing a disease associated with cells that overexpress EGFR and the EGFRvIII variant, the method comprising using the conjugate provided herein as a diagnostic agent in an imaging technique or a detection technique. In some embodiments, the bioactive agent is a detectable label moiety.

[0049] As used herein, the term "about" refers to ±10%. For example, the term "about 100 μm" includes, together with the value of 100 μm, the values of 90 μm, 91 μm, 92 μm, 93 μm, 94 μm, 95 μm, 96 μm, 97 μm, 98 μm, 98 μm, 99 μm, 100 μm, 101 μm, 102 μm, 103 μm, 104 μm, 105 μm, 106 μm, 107 μm, 108 μm, 109 μm, and 110 μm.

[0050] The terms "comprises", "comprising", "includes", "including", "having" and their conjugations mean "including but not limited to".

[0051] The term "consisting of" means "including and limited to".

[0052] The term "consisting essentially of" means that a composition, method or structure may include additional components, steps, and / or parts, but only to the extent that the additional components, steps, and / or parts do not materially change the basic and novel features of the composition, method, or structure recited in the claims.

[0053] As used herein, the phrase "selected from the group consisting of" includes all members of the recited group, each member of the recited group, and all possible combinations. For example, being selected from the group consisting of A, B, and C includes only A, as well as only B, further only C, further A and B, further A and C, further B and C, and further A, B, and C.

[0054] As used herein, the phrases "substantially devoid of" and / or "essentially devoid of" in relation to a particular substance refer to a composition that either contains none of this substance or contains less than about 5%, 1%, 0.5%, or 0.1% based on the total weight or volume of the composition. Alternatively, the phrases "substantially without" and / or "essentially without" in relation to a process, method, property, or feature refer to a process, composition, structure, or article that either has none of a particular process / method step, or a particular property or a particular feature, or a particular process / method step is carried out at less than about 5%, 1%, 0.5%, or 0.1% compared to a given standard process / method, or a property or feature characterized by being less than about 5%, 1%, 0.5%, or 0.1% compared to a given standard.

[0055] When applied to the original properties, or desired properties, or given properties of an object or composition, the term "substantially maintained" as used herein means that in the processed object or composition, the property does not change by more than 20%, 10%, or 5%.

[0056] The term "exemplary" as used herein is used to mean "serving as an example, instance, or illustration". Any embodiment described as "exemplary" should not necessarily be construed as being more preferred or advantageous than other embodiments, and / or does not exclude incorporating features from other embodiments.

[0057] The terms "optionally" or "alternatively" are used herein to mean that "there are embodiments that are provided and embodiments that are not provided." Any particular embodiment of the invention can include a plurality of "optional" features as long as such features are not inconsistent.

[0058] As used herein, the singular forms "a", "an", and "the" are intended to include the plural as well, unless the context clearly indicates otherwise. For example, "a compound" or "at least one compound" includes a plurality of compounds and can also include mixtures thereof.

[0059] Throughout this application, various embodiments of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as imposing a limitation that lacks flexibility in the scope of the invention. Thus, a description of a range should be considered to specifically disclose all the sub-ranges within the possible range, as well as the individual numerical values within that range. For example, a description of a range such as 1 - 6 should be considered to specifically disclose not only the sub-ranges such as 1 - 3, 1 - 4, 1 - 5, 2 - 4, 2 - 6, 3 - 6, etc., but also the individual numerical values within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the size of the range.

[0060] Whenever a numerical range is indicated herein, it is intended to include any recited number (fractional or integral) within the indicated range. The phrases "a range between" a first recited number and a second recited number, and "a range from" a first recited number "to" a second recited number are used interchangeably herein and are intended to include the first and second recited numbers, and all the fractions and integers between the first and second recited numbers.

[0061] As used herein, the terms "process" and "method" mean a manner, means, technique, and procedure for achieving a given objective, including, but not limited to, those known to, or readily developed by, persons of ordinary skill in the fields of chemistry, materials, mechanical, computer, and digital arts.

[0062] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the present patent specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and not intended to be limiting.

Best Mode for Carrying Out the Invention

[0063] In some embodiments thereof, the present invention relates to cancer treatment, and more specifically, but not limited thereto, to a family of cyclic peptides having affinity for epidermal growth factor receptor and its variants for drug delivery.

[0064] Before describing in detail at least one embodiment of the present invention, it is to be understood that the use of the present invention is not necessarily limited to the details shown in the following detailed description or exemplified embodiments for carrying out the following invention. The present disclosure is intended to cover other embodiments or to be practiced or realized in various ways.

[0065] While considering the present invention, the inventors conceived that a strategy capable of effectively overcoming the hurdle of drug resistance is to use a drug delivery system targeting overexpressed survival and proliferation-related receptors for cell-specific delivery of cytotoxic drugs to cancer cells. In this scenario, the use of S-S bridged cyclic peptides incorporated into peptide-drug conjugates (PDCs) has several key advantages. PDCs have a more constrained structure with enhanced metabolic stability and binding affinity / specificity for target molecules compared to their corresponding linear counterparts, which enhances the pharmacokinetics and efficacy of drug delivery to cancer cells.

[0066] In practicing the present invention, the inventors discovered and elucidated a family of cyclic peptide ligands that selectively bind to both EGFR and EGFRvIII, and evaluated the targeted cytotoxicity of those PDCs containing camptothecin (CPT), a Topo I inhibitor of NSCLC cell lines.

[0067] Cyclic peptide: As described above, the EGF-EGFR pathway has become a major focus in selective chemotherapeutic intervention. As a result, two classes of EGFR inhibitors have been clinically approved, which are monoclonal antibodies (cetuximab, panitumumab) targeting the extracellular domain of EGFR and small molecule kinase inhibitors (gefitinib, erlotinib) that block intracellular phosphorylation of the receptor. Despite the good response rates of these drugs, most patients eventually acquire drug resistance. Therefore, new treatment methods are still needed.

[0068] In the process of commercializing the present invention, phage display technology was used to discover the EGFR-specific cyclic peptides provided in the present application, which can function as drug carriers in a targeted drug delivery system. For the selection of peptides, the inventors utilized live cells instead of purified proteins. Selecting for isolated biomarkers is more accurate because unrelated molecules do not obscure the target receptor, and screening against whole cells brings the experiment closer to the natural environment. To mimic a more natural environment, phage display biopanning procedures were used on H1299 cells, H1975 cells, and DKMG cells that overexpress EGFR or variants of EGFR on the cell surface.

[0069] Furthermore, the combination of phage display biopanning and next-generation sequencing (NGS) technology enabled the inventors to perform high-throughput screening and selection of EGFR-specific peptides. NGS is a high-throughput parallel sequencing technology that offers ultra-high throughput, scalability, and speed. This technology is used to determine the nucleotide sequence in the entire genome or in targeted regions of DNA or RNA.

[0070] General structure: In the context of the present invention, a cyclic peptide is a type of peptide whose amino acid sequence forms a loop structure rather than a linear structure. The cyclic peptides in some embodiments of the present invention contain 7 to 11 amino acid residues in their sequences, and each peptide sequence has terminal cysteine residues at its N-terminus and C-terminus, respectively.

[0071] The termini of the peptide can each be an unbonded amino group and / or carboxyl group, or a protected amino group and / or carboxyl group. In some embodiments, the C-terminus is an amide group (C-amide).

[0072] The peptide or its conjugate can be in the form of a salt, hydrate, or solvate.

[0073] Affinity Criteria for Peptide Selection: The cyclic peptides provided in the present application are characterized by their affinity for EGFR variants such as epidermal growth factor receptor (EGFR) or EGFRvIII variants. This means that the peptides can specifically bind to these receptors, which are proteins found on the cell surface. Cells with EGFR or EGFR variants expressed on their surface are defined in the present application as cells overexpressing EGFR and / or EGFRvIII variants, and are abbreviated as "EGFR-positive cells" in the present application. In the context of the present invention, cell lines that do not express the target receptor are referred to as "EGFR-negative cells".

[0074] The affinity of the cyclic peptides provided in the present application for EGFR or EGFR variants is determined by a process called biopanning, where a phage display library of random cyclic peptides is used against cell lines expressing EGFR or EGFR variants for positive (specific) binding and cell lines that do not express the target receptor as a negative (nonspecific) binding control.

[0075] As is known in the art and understood by those of ordinary skill in the art, biopanning is an affinity selection technique for selecting peptides that bind to a given target. This includes multiple steps or cycles, including the provision of a phage display library, the incubation of the phage library with the desired target, the washing away of unbound phages, and the elution of the bound phages. A peptide "passing" through a biopanning cycle means that it has successfully bound to the target protein and was not washed away in the washing step. Next, the bound phages are eluted to enrich for phages presenting peptides with high affinity for the target protein and amplified in additional biopanning steps.

[0076] More specifically, the biopanning process for determining that a certain cyclic peptide exhibits specific affinity for a target receptor involves a large number (about 1 billion, 10 9Incubate a library of bacteriophages (viruses that infect bacteria) presenting a random and unique cyclic peptide on their surface with at least one EGFR-positive cell line (specific binding) and at least one EGFR-negative cell line (non-specific binding). Phages with non-specific or weak binding are then removed, and the remaining phages are amplified and subjected to additional biopanning steps to enrich for phages presenting peptides with high affinity for the target protein.

[0077] In one example of some embodiments of the present invention, the phage display peptide library that can be used for biopanning is the Ph.D.-C7C™ phage display peptide library from New England Biolabs Inc., which includes the Ph.D.-C7C™ phage display peptide library and the -96gIII sequencing primer for >50 sequencing reactions (7-mer cyclic peptide). In this embodiment, the Ph.D.-C7C phage display peptide library is based on a combinatorial library of random disulfide-looped peptides fused to the N-terminus of the minor coat protein (pIII) of M13 phage. This library consists of approximately 10 9 electroporated (i.e., unique) sequences.

[0078] According to some embodiments of the present invention, the cyclic peptide is characterized by having "passed through" the three bio-panning cycles described above. Instead, each of the cyclic peptides provided in the present application is determined by binding to EGFR and / or EGFR variants in at least three bio-pannings using a phage display library of random cyclic peptides against at least one EGFR-positive cell line and at least one EGFR-negative cell line, and is also characterized by having an affinity for EGFR and / or EGFR variants. This process is typically repeated at least three times to ensure that the selected peptide has a high affinity for the target protein. This method enables the identification and synthesis of a family of exemplary cyclic peptide candidates and the study of their EGFR specificity and selectivity.

[0079] Internalization (uptake) by EGFR-positive cell line: The cyclic peptides provided in the present application are characterized by their ability to internalize in EGFR-positive cells when bound to EFGR or its variants. For example, the cyclic peptides exhibit binding and internalization to cancer cells such as the H1299 cell line, the H1975 cell line, and the DKMG cell line. This ability can be determined by methods known in the art. For example, the internalization of the cyclic peptide can be determined by flow cytometry using a cyclic peptide labeled with a labeled moiety such as a detectable moiety or a fluorescent moiety. An exemplary labeled moiety in some embodiments of the present invention can be fluorescein-5-isothiocyanate (FITC).

[0080] An exemplary internalization assay is provided in the Examples section described below.

[0081] Specificity for EGFR: The cyclic peptides provided herein are also characterized by their specificity for EGFR, as determined by competitive binding and internalization assays against ligands of EGFR, preferably the natural ligands of receptors that are overexpressed in cancer cell lines. An example of a natural ligand is human epidermal growth factor (hEGF).

[0082] The binding specificity of the cyclic peptide for EGFR can be determined by comparing the level of binding and / or internalization of the peptide to the receptor in the absence or presence of a known ligand for the receptor, and if the binding and / or internalization of the peptide is decreased, it can be determined in this way.

[0083] Those of ordinary skill in the art are familiar with and recognize tools and methodologies for verifying specificity, such as competitive flow cytometry using labeled cyclic peptides, EGF, and at least one EGFR-positive cell line.

[0084] An exemplary competitive binding assay is provided in the Examples section below.

[0085] Further properties of the cyclic peptide: As shown in the Examples section below, confocal microscopy and peptide docking were used to confirm the results of fluorescence-activated cell sorting (FACS). Cytotoxicity assays were performed to study the activity of the peptides and peptide-drug conjugates.

[0086] Specific exemplary cyclic peptides: Following identification using biopanning binding, internalization assays, and specificity by competitive assays, an exemplary group of 11 peptides was selected for further exploration.

[0087] Thus, according to some embodiments of the present invention, there is provided a family of cyclic peptides presenting sequences as cyclic (“c”) peptides closed by disulfide bonds between the sulfur atoms of cysteine residues, listed in Table 1 below.

[0088]

Table 1

[0089] Table 1 shows the structure of the cyclic peptide in the form of a C-terminal amidate, although the C-terminus of each peptide can be in the form of a carboxyl group or an amide group.

[0090] In some embodiments, the cyclic peptide is cyclized via the side-chain thiol of a terminal cysteine, and thus can be in the form of an unbound N-terminal amine, an unbound C-terminal carboxyl, a terminal amide, a terminal ester, a salt, preferably a pharmaceutically acceptable salt, its hydrate, or its solvate.

[0091] The term "solvate" refers to a complex formed by a solute (the compounds described herein) and a solvent that does not interfere with the biological activity of the solute, and the stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, and so on) varies. Suitable solvents include, for example, ethanol, acetic acid, etc. The term "hydrate" refers to a solvate where the solvent is water, as defined above.

[0092] In some embodiments, the cyclic peptide contains only residues of naturally occurring amino acids. In some embodiments, the cyclic peptide contains at least one residue that is a D-amino acid other than glycine.

[0093] The cyclic peptides showed specific and selective binding and internalization to EGFR-positive cells. Studies on off-target cells showed that two cyclic peptides named P6 and P9 exhibited high specific activity against EGFR. Confocal microscopy showed the high specificity of P6 for NSCLC while being more specific for glioblastoma cells. The XTT assay on EGFR-overexpressing cell lines showed that the conjugates of P6 and P9 with camptothecin (CPT) had higher cytotoxicity compared to free CPT. The results led to the discovery that the cyclic peptides disclosed herein specifically target the EGF receptor and its specific variant EGFRvIII.

[0094] Peptide-drug conjugate: According to one aspect of some embodiments of the present invention, a family of EGFR-specific short-chain cyclic peptides that can be used for targeted drug delivery in the form of peptide-drug conjugates (PDCs) is provided.

[0095] The ability of the peptide-drug conjugate to maintain stability and further specifically penetrate target cells is an important factor in the delivery of highly toxic drugs. Indeed, the cyclic peptides selected as candidates for targeted cell binding have the ability to be taken up by cells and showed high internalization in DKMG cells that have the EGFRvIII mutation, which is a genetic marker for multiple cancers. The EGFRvIII mutation has also been suggested as a marker for cancer stem cells or tumor-initiating populations because this mutation is associated with more aggressive disease and poor prognosis.

[0096] As described below, a competitive binding assay was used to determine the ability of the labeled peptide to compete with the natural ligand for the EGF receptor. FACS analysis showed that the competition between the cyclic peptide provided in the present application and EGF was successful, and thus the number of FITC-positive cells having the bound peptide was significantly reduced after incubation. It was found that the binding activity was significantly reduced after co-incubation with EGF, the natural ligand. This result demonstrated the interaction with the EGFR receptor and suggested the expected role in the transport of the cyclic peptide delivery system. Studies on the target specificity of the cyclic peptides provided in the present application have revealed that some show high specificity for EGFR-expressing cells.

[0097] The section of the examples described below also provides peptide docking performed on the cyclic peptides provided in the present application, which demonstrated target specificity and showed binding at the active site of the receptor. The cyclic peptides showed good binding energies in the range of -13.0 to -11.3 kcal mol -1 within the range.

[0098] Since EGF promotes cell proliferation, the cyclic peptides provided in the present application had to be tested under conditions where there was a risk of promoting cell proliferation. The results described below, by themselves, demonstrated that the peptides did not promote cell proliferation and thus could be safely used as a ligand targeting and drug carrier in the form of a peptide-drug conjugate. This was further confirmed by the lower cytotoxic effect observed for the free peptide compared to the more pronounced cytotoxicity when the peptide was conjugated to CPT.

[0099] Therefore, according to one aspect of some embodiments of the present invention, there is provided a peptide-drug conjugate comprising a cyclic peptide according to an embodiment of the present invention and at least one bioactive agent, for example, a drug, wherein the cyclic peptide and the bioactive agent are covalently bonded to each other via a linking moiety represented as "L" in the scheme shown below.

[0100] [Chemistry]

[0101] According to aspects of some embodiments of the present invention, a bioactive agent or a portion thereof means a compound (molecule) that is carried to a target and bound to a cyclic peptide with the intention of exerting an effect under physiological conditions. In the context of this embodiment, the terms "bioactive agent", "pharmaceutically active agent" and "drug" are used interchangeably, although a bioactive agent may be a molecule used as a labeling agent in diagnostic purposes and / or imaging, radiotherapy, and research.

[0102] As used herein, the terms "bioactive agent" and "drug" mean small molecules or biomolecules that modify, inhibit, activate, or affect biological mechanisms or events. Bioactive agents that can be linked to the cyclic peptides according to embodiments of the present invention include anti-cancer substances, anti-proliferative agents, chemosensitizing agents, anti-inflammatory agents (including steroid and non-steroid anti-inflammatory agents and antipyretics), antibacterial agents (including antibiotics, antiviral agents, antifungal agents, anti-parasitic agents, anti-protozoal agents, etc.), antioxidants, hormones, antihypertensive agents, anti-AIDS substances, anti-diabetic substances, immunosuppressive agents, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, lubricants, sedatives, anti-convulsants, muscle relaxants and anti-Parkinson substances, anti-itch agents, anti-spasmodics and muscle contractants (including channel blockers, miotics and anti-cholinergic agents), anti-glaucoma compounds, regulators of cell-cell extracellular matrix interactions (including cell growth inhibitors and anti-adhesion molecules), vitamins, vasodilators, DNA, RNA or protein synthesis inhibitors, analgesics, anti-angiogenic factors, anti-secretory factors, anticoagulants and / or anti-thrombotics, anesthetics, ophthalmic agents, prostaglandins, anti-depressants, anti-psychotics, anti-emetics, radioactive substances and imaging agents, but are not limited thereto. A more comprehensive list of exemplary drugs suitable for use in the present invention can be found in "Pharmaceutical Substances: Syntheses, Patents, Applications" by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999, "Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals", edited by Susan Budavari et al., CRC Press, 1996, and The United States Pharmacopeia-25 / National Formulary-20, published by the United States Pharmacopeial Convention, Rockville, Maryland, 2001.

[0103] As used herein, the term "small molecule" refers to a molecule, whether natural or artificial (e.g., chemically synthesized), that has a relatively small molecular weight. Typically, small molecules are monomers and have a molecular weight of less than about 1500 Da. Preferred small molecules are biologically active and produce a local or systemic effect in animals, preferably mammals, and more preferably humans. In certain preferred embodiments, the small molecule is a drug. Preferably, but not necessarily, the drug is one that has already been approved as safe and effective by an appropriate government agency or for living organisms. For example, drugs that can be used in humans are listed by the FDA in 21 CFR §§ 330.5, 331 to 361, and 440 to 460, and drugs for veterinary use are listed by the FDA in 21 CFR §§ 500 to 589, all of which are considered acceptable for use according to the present invention.

[0104] Anticancer drugs: Particularly interesting as a partner for conjugation with the cyclic peptides provided in the present application is an anticancer agent due to its affinity for EGFR-expressing cells. Anticancer agents that can serve as bioactive agents in the form of peptide-drug conjugates include, but are not limited to, the following according to embodiments of the present invention. Acivicin, Aclarubicin, Acodazole Hydrochloride, Acronine, Adriamycin, Adozelesin, Aldesleukin, Altretamine, Ambomycin, Ametantrone Acetate, Aminoglutethimide, Amsacrine, Anastrozole, Anthramycin, Asparaginase, Asperlin, Azacitidine, Azetepa, Azotomycin, Batimastat, Benzodepa, Bicalutamide, Bisantrene Hydrochloride, Bisfanidide Dimethyl Sulfate, Bizelesin, Bleomycin Sulfate, Brequinar Sodium, Broxuridine, Busulfan, Cactinomycin, Camptothecin (CPT), Caracemide, Carbetimer, Carboplatin, Carmustine, Carvicine Hydrochloride, Carzelesin, Cerebroside Sulfate, Chlorambucil, Cirolemycin, Cisplatin, Cladribine, Crisnatol Mesylate, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Daunorubicin Hydrochloride, Decitabine, Dexormaplatin, Dezaguanine, Dezaguanine Mesylate, Diaziquone, Docetaxel, Doxorubicin Hydrochloride, Droloxifene, Droloxifene Citrate, Drostanolone Propionate, Duazomycin, Edatrexate, Efloxatin Hydrochloride, Elsamicolcin, Enoplatin, Epmate, Epipropidine, Epirubicin Hydrochloride, Elbuzole, Esorubicin Hydrochloride, Estramustine, Estramustine Sodium Phosphate, Ethanidazole, Etoposide, Etoposide Phosphate, Etoprine, Fadrozole Hydrochloride, Fazarabine, Fenretinide, Floxuridine, Fludarabine Phosphate, Fluorouracil, Flurocitabine, Fosquidone, Fostriecin Sodium, Gemcitabine, Gemcitabine Hydrochloride, Hydroxyurea, Idarubicin Hydrochloride, Ifosfamide, Ilmofosine, Interferon Alpha-2a, Interferon Alpha-2b, Interferon Alpha-n1, Interferon Alpha-n3, Interferon Beta-Ia, InterferonGamma-Ib, Iproplatin, Irinotecan Hydrochloride, Lanreotide Acetate, Letrozole, Leuprolide Acetate, Rialoxazole Hydrochloride, Sodium Romidexol, Lomustine, Losoxantrone Hydrochloride, Masoprocol, Mithramycin, Mechlorethamine Hydrochloride, Megestrol Acetate, Melenegestrol Acetate, Melphalan, Menogaril, Mercaptopurine, Methotrexate, Methotrexate Sodium, Methopterin, Methyluredepa, Mitindomide, Mitocarcin, Mitochromin, Mitogillin, Mitomalcin, Mitomycin, Mitosper, Mitotan, Mitoxantrone Hydrochloride, Mycophenolic Acid, Nocodazole, Nogalamycin, Ormaplatin, Oxisuran, Paclitaxel, Pegaspargase, Periomycin, Pentamustine, Pepromycin Sulfate, Perfosfamide, Pipobroman, Piposulfan, Pyroxantrone Hydrochloride, Plicamycin, Promestane, Porfimer Sodium, Porfiromycin, Prednimustine, Procarbazine Hydrochloride, Puromycin, Puromycin Hydrochloride, Pyrazofurin, Riboprine, Logretimide, Safingol, Safingol Hydrochloride, Semustine, Simtrazene, Sparfosate Sodium, Sparsomycin, Spirogermanium Hydrochloride, Spiro-mustine, Spiroplatin, Streptozocin, Streptozocin, Slofenur, Talisomycin, Taxol, Tegafur Sodium, Tegafur, Teroxantrone Hydrochloride, Temoporfin, Teniposide, Teroxirone, Testolactone, Thiamiprine, Thioguanine, Thiotepa, Thiazofurin, Tirapazamine, Topotecan Hydrochloride, Toremifene Citrate, Trestolone Acetate, Trisciribine Phosphate, Trimethoprim, Trimethoprim Glucuronide, Triptorelin, Tubroluzole Hydrochloride, Uracil Mustard, Uredepa, Bapreotide, Verteporfin, Vinblastine Sulfate, Vincristine Sulfate, Vindesine, Vindesine Sulfate, Vinetepidine Sulfate, Vincoglycinate Sulfate, Vinleurosine Sulfate, Vinorelbine Tartrate, Vinrosidine Sulfate, Vinzolidine Sulfate, Borozole, Zeniplatin, Dinostatin, Zorubicin Hydrochloride. Additional antineoplastic agents include Goodman and Gilman's "The Pharmacological Basis ofThose disclosed in "Therapeutics", 8th Edition, 1990, McGraw-Hill, Inc. (Health Professions Division), Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner), and its preface, pages 1202-1263 are included.

[0105] Approved chemotherapeutic drugs that can be bioactive agents in the form of peptide-drug conjugates include, but are not limited to, according to embodiments of the present invention. Abarelix, Aldesleukin, Alemtuzumab, Alitretinoin, Allopurinol, Altretamine, Amifostine, Anastrozole, Arsenic Trioxide, Asparaginase, Azacitidine, Bevacizumab, Bexarotene, Bleomycin, Bortezomib, Busulfan, Calusterone, Capecitabine, Carboplatin, Carmustine, Celecoxib, Cetuximab, Cisplatin, Cladribine, Clofarabine, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Actinomycin D, Darbepoetin Alfa, Darbepoetin Alfa, Liposomal Daunorubicin, Daunorubicin, Decitabine, Denileukin Diftitox, Dexrazoxane, Dexrazoxane, Docetaxel Doxorubicin, Drostanolone Propionate, Elliot B Solution, Epirubicin, Epoetin Alfa, Erlotinib, Estramustine, Etoposide, Exemestane, Filgrastim, Floxuridine, Fludarabine, Fluorouracil 5-FU, Fulvestrant, Gefitinib, Gemcitabine, Gemtuzumab Ozogamicin, Goserelin Acetate, Histrelin Acetate, Hydroxyurea, Ibritumomab Tiuxetan, Idarubicin, Ifosfamide, Imatinib Mesylate, Interferon Alfa2a, Interferon Alfa-2b, Irinotecan, Lenalidomide, Letrozole, Leucovorin, Leuprolide Acetate, Levamisole, Lomustine, CCNU, Mechlorethamine, Nitrogen Mustard, Megestrol Acetate, Melphalan, L-PAM, Mercaptopurine6-MP, Mesna, Methotrexate, Mitomycin C, Mitotane, Mitoxantrone, Nandrolone phenylpropionate, Nelarabine, Ofatumumab, Oprelvekin, Oxaliplatin, Paclitaxel, Palifermin, Pamidronic acid, Pegademase, Pegaspargase, Pegfilgrastim, Pemetrexed disodium, Pentostatin, Pipobroman, Plicamycin, Mitramycin, Porfimer sodium, Procarbazine, Quinacrine, Rasburicase, Rituximab, Sargramostim, Sorafenib, Streptozocin, Sunitinib maleate, Tamoxifen, Temozolomide, Teniposide VM-26, Testolactone, Thioguanine 6-TG, Thiotepa, Topotecan, Toremifene, Tositumomab, Trastuzumab, Tretinoin ATRA, Uracil mustard, Valrubicin, Vinblastine, Vinorelbine, Zoledronic acid ester and Zoledronic acid.

[0106] Linking moiety: Generally, a linking moiety can be formed during a chemical reaction and is formed as a new chemical substance that can contain a bond (between two atoms) or one or more bonding atoms by reacting two or more reactive groups. Alternatively, a linking moiety can be an independent compound moiety having two or more reactive groups that can be added either directly or indirectly to the reactive groups of other compounds, as detailed below.

[0107] In the above exemplary scheme, the variable group "L" represents a linking moiety connecting a moiety consisting of a cyclic peptide and a moiety consisting of a bioactive agent (drug, detection / diagnostic drug). The linking moiety is the result of a conjugation reaction between the two parts of the conjugate and can be a bond (a pair of electrons forming a covalent bond), an atom, typically a heteroatom (N, O, S, etc.), or a group of atoms.

[0108] As used herein, the terms "link", "linked", "linking", "linker", "attached" or "attached" are used interchangeably herein and, unless otherwise expressly indicated, refer to the presence of at least one covalent bond between molecular species. As used herein, the term "moiety" refers to a part of a molecule, typically its principal part, or a group of atoms related to a particular function.

[0109] As used herein, the term "linking moiety" refers to a chemical moiety (group of atoms, or covalent bond) that links two chemical moieties via one or more covalent bonds. The linking moiety may include atoms that form part of one or both of the chemical moieties it links, and / or may include atoms that do not form part of one or both of the chemical moieties it links. For example, a peptide bond (amide) linking moiety that links two amino acids includes at least one nitrogen atom and one hydrogen atom of one amino acid and at least one carboxyl of the other amino acid.

[0110] The position at which the bioactive agent is linked to the cyclic peptide moiety is generally selected such that any residue extending from the linking moiety on the bioactive agent, if any, after cleavage once, does not substantially interfere with the bioactivity (mechanism of bioactivity). The appropriate position depends on the type of bioactive agent. According to some embodiments of the present invention, the form of the linking moiety is such that the bioactivity of the bioactive agent once released from the cyclic peptide is practically the same as the bioactivity of a similar original bioactive agent without loss of bioactivity.

[0111] As used herein, the term "reactive group" typically refers to a chemical group capable of undergoing a chemical reaction that results in the formation of a covalent bond. Chemical reactions that result in the formation of a bond include, for example, cycloaddition reactions (e.g., Diels-Alder reactions, 1,3-dipolar cycloaddition Huisgen reactions, and similar "click reactions"), condensations, nucleophilic and electrophilic addition reactions, nucleophilic and electrophilic substitutions, addition and elimination reactions, alkylation reactions, rearrangement reactions, and any other known organic reactions in which the reactive group participates.

[0112] Representative examples of reactive groups include, but are not limited to, acyl halide, aldehyde, alkoxy, alkyne, amide, amine, aryloxy, azide, aziridine, azo, carbamate, carbonyl, carboxyl, carboxylate, cyano, diene, dienophile, epoxy, guanidine, guanyl, halide, hydrazide, hydrazine, hydroxy, hydroxylamine, imino, isocyanate, nitro, phosphate, phosphonate, sulfinyl, sulfonamide, sulfonate, thioalkoxy, thioaryloxy, thiocarbamate, thiocarbonyl, thiol, thiourea, and urea, and these terms are as defined hereinafter.

[0113] According to some embodiments of the present invention, various elements of the cyclic peptide moiety provided herein are attached to one or more linking moieties via a spacer moiety. As used herein, the phrase "spacer moiety" typically means a chemical moiety that extends between two chemical moieties and is attached to each chemical moiety via a covalent bond. The spacer moiety may be linear or cyclic, branched or unbranched, rigid or flexible.

[0114] The nature of the spacer moiety can be seen to affect from the following two aspects. The synthetic aspect, i.e., the influence of the spacer moiety on the production of the cyclic peptide moiety, and the influence of the spacer moiety on the biological activity, bioavailability, and other ADME-Tox considerations of the cyclic peptide moiety or the bioactive agent moiety (i.e., the drug).

[0115] According to some embodiments of the present invention, the spacer portion is selected to enable and / or facilitate the conjugation reaction between the cyclic peptide portion and the bioactive agent portion and to reduce the formation rate of by-products due to unwanted reactions. Such properties can be selected from the perspective of the length, flexibility, structure, and specific chemical reactivity or lack thereof of the spacer. A spacer portion with fewer reactive groups presents simpler synthetic challenges, requires fewer protection / deprotection steps, and results in higher chemical yields. For example, a saturated straight-chain alkyl having 1 to 10 or 1 to 5 carbon atoms and having one reactive group at the terminal atom for conjugation with the corresponding reactive group results in substantially higher yields and fewer by-products. Similarly, a spacer portion based on one or two benzyl rings will lead to an efficient conjugation reaction.

[0116] According to some embodiments of the present invention, the spacer portion is selected to provide favorable cleavage conditions for releasing the bioactive agent from the cyclic peptide as contemplated herein. For example, by the spacer changing the proximity to the ligation portion of the enzyme, it becomes possible to cleave the linkage between the bioactive agent portion and the cyclic peptide portion by the enzyme.

[0117] According to some embodiments of the present invention, the spacer portion includes, but is not limited to, the following: -CH2-, -CH2-O-, -(CH2)2-, -(CH2)2-O-, -(CH2)3-, -(CH2)3-O-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH(CH3))-CH2-, -CH=CH-CH=CH-, -C≡C-C≡C-, -CH2CH(OH)CH2-, -CH2-O-CH2-, -CH2-O-CH2-O-, -(CH2)2-O-(CH2)2-, -(CH2)2-O-(CH2)2-O-, -CH2-mC6H4-CH2-, -CH2-mC6H4-CH2-O-, -CH2-pC6H4-CH2-, -CH2-pC6H4-CH2-O-, -CH2-NHCO-, -C6H4-NHCO-, -CH2-O-CH2- and -CH=CH-CH2-NH-(CH2)2-.

[0118] In some embodiments, the spacer portion can be considered part of the linking portion.

[0119] According to some embodiments of the present invention, the linking portion is stable under physiological conditions. In particular, the linking portion does not disintegrate during the period of exposure to the physiological environment in the body. Such a linking portion is referred to in the present application as "biostable". The biostable linking portion has the advantage of extending the time during which its molecular structure exerts its biological activity (release of the bioactive agent in the target body part) until the time it is secreted or removed from the body part. Biostability is used as a relative term in the sense that the biostable linking portion requires a longer time to disintegrate or requires specific cleavage conditions that the molecular structure encounters less frequently under physiological conditions. Note that this is also the case.

[0120] In the context of some embodiments of the present invention, the biodegradable linking portion is selected to break under specific conditions referred to in the present application as "drug release conditions" or "cleavage conditions" and release the bioactive agent bound thereto.

[0121] According to some embodiments of the present invention, further non-limiting examples of the linking portion include amines (N, secondary and tertiary), ethers (O), thioethers (S), amides, carbonates, lactones, lactams, carboxylates, carbamates, esters, boroalkyls, boronate, sulfones, sulfates, phosphonates, phosphines, phosphites, cycloalkenes, cyclohexenes, heteroalicyclics, heteroaryls, triazines, triazoles, disulfides, imines, imides, oximes, aldimines, ketimines, hydrazones, semicarbazones, acetals, ketals, aminals, aminoacetals, thioacetals, thioketals, phosphate esters, and the like. Other linking portions are defined below, and still other linking portions are contemplated as being within the scope of the terms used herein.

[0122] According to some embodiments, the linking moiety is selected from the group consisting of the following.

[0123]

Chemical formula

[0124] According to some embodiments of the present invention, some of the linking moieties are biodegradable linking moieties. As used herein, the terms "biocleavable" and "biodegradable" are used interchangeably to refer to moieties that break down (i.e., separate and / or lose at least a portion of their covalent structure) under physiological or endosomal conditions. Biodegradable moieties need not necessarily be hydrolyzable and may require enzymatic action for breakdown.

[0125] As used herein, the term "biocleavable moiety" or "biodegradable moiety" represents a compound moiety that is cleaved in a biological system, such as the digestive system of a living body or the metabolic system of living cells.

[0126] In some embodiments, the binding site is selected and the cleavage conditions are defined based on the sensitivity to a specific enzyme that is likely to be present in the target body site or any other body site where biodegradation of the biodegradable linking moiety is planned.

[0127] Representative examples of biodegradable moieties include, but are not limited to, amides, esters, carboxylates, carbamates, phosphates, hydrazides, thiohydrazides, disulfides, epoxides, peroxides, and methyleneamines. Such moieties are typically cleaved in biological systems by enzymes such as hydrolases, amidases, kinases, peptidases, phospholipases, lipases, proteases, esterases, epoxide hydrolases, nitrilases, glycosidases, and the like.

[0128] For example, hydrolases (EC numbers starting with 3) catalyze the hydrolysis of chemical bonds by the general reaction scheme A-B + H2O → A-OH + B-H. A subgroup of hydrolases, known as esterases (EC numbers starting with 3.1) and including nucleases, phosphodiesterases, lipases, and phosphatases, cleave ester bonds. Hydrolases with EC numbers starting with 3.4 are peptidases that act on peptide bonds.

[0129] Additional information on enzymes, enzyme reactions, and enzyme-linked moiety correlations can be found in various publicly accessible information sources such as Bairoch A., “The ENZYME database in 2000”, Nucleic Acids Res, 2000, 28, pp. 304-305.

[0130] The definitions of specific functional groups, chemical terms, and general terms used throughout this specification are described in more detail below. For the purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements, CAS version, as described on the inside front cover of the Handbook of Chemistry and Physics, 75th Edition, and specific functional groups are generally defined as described in that publication. Further, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999, Smith and March March’s Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001, Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989, Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.

[0131] Exemplary conjugates: In some embodiments of the present invention, the cyclic peptide may be used for delivery of one or more bioactive agent moieties, which may be the same, similar but different in linkage (different positions of the peptide or drug molecule and / or different linking moieties), or different bioactive agent moieties.

[0132] In some embodiments of the present invention, the linking moiety is part of one amino acid residue of the cyclic peptide or is attached to its side chain. In some embodiments of the present invention, the linking moiety is part of one (main chain) end of the cyclic peptide or is attached to its side chain. In some embodiments of the present invention, the linking moiety is part of the amino group at the N-terminus of the peptide or is attached thereto as an amide. In some embodiments of the present invention, the linking moiety is part of the carboxy group at the C-terminus of the peptide or is attached thereto as an ester or an amide. In some embodiments of the present invention, the linking moiety is part of the amino group of a residue of the side chain of the peptide or is attached thereto as an amide. In some embodiments of the present invention, the linking moiety is part of the carboxy group of a residue of the side chain of the peptide or is attached thereto as an ester or an amide. In some embodiments of the present invention, the linking moiety is part of the hydroxy group of a residue of the side chain of the peptide or is attached thereto.

[0133] Table 2a below shows some exemplary conjugates according to some embodiments of the present invention, which include a cyclic peptide moiety, a GABA (γ-aminobutyric acid) linking moiety, and a CPT drug moiety.

[0134] [Table 2a] TIFF2025522286000007.tif234163TIFF2025522286000008.tif52157

[0135] Table 2b below shows some exemplary conjugates according to some embodiments of the present invention, which include a cyclic peptide moiety, a GABA (γ-aminobutyric acid) linking moiety, and a labeling moiety derived from fluorescein-5-isothiocyanate (FITC).

[0136] [Table 2b] TIFF2025522286000010.tif154130

[0137] As additional PDCs according to embodiments of the present invention, as illustrated in the schemes described later, cyclic peptides are conjugated via their N-terminal amines with one or more linking moieties [GABA, glutathione, lysine, succinic acid, 2-amino-5-(carbamoylamino) pentanoic acid (para-aminobenzoic acid, PABA), valine-citrulline-PABA, citrulline, and / or 3-(2-aminoethyl) disulfanylpropanoic acid, etc.] to a drug moiety [doxorubicin (DOX), monomethyl auristatin F (MMFA), 7-ethyl-10-hydroxycamptothecin (SN38, an analogue of CPT), etc.] and / or a labeling moiety [cyanine (Cy5), sulfocyanine 5 or iodinated xanthene-cyanine NIR dye, etc.], but are not limited thereto.

[0138]

Chemical formula

[0139]

Chemical formula

[0140]

Chemical formula

[0141]

Chemical formula

[0142] Practicality and uses: Specific to the EGFR-overexpressing H1299 cell line (also known as NCI-H1299 or CRL-5803) and its mutant EGRFvIII-expressing DKMG cell line, the family of cyclic heptamers + two terminal cysteine peptides (a total of nine-mer cyclic peptides) discovered by a combination of phage display cell-based positive biopanning selection method and subsequent NGS sequencing of modified DNA phages has been shown to be active in vivo, as shown in the examples section described below.

[0143] For P6 and P9, which are at least two exemplary candidates of these peptide families, when testing the important therapeutic properties for peptide-drug conjugates, it was confirmed by the use of FACS and fluorescence microscopy that they exhibit effective binding and internalization to EGFR cell lines and EGFRvIII cell lines. In addition, P6 and P9 conjugated to CPT enabled the nuclear accumulation of cytotoxic CPT. The intracellular accumulation of CPT leads to DNA damage and induction of cell death. Thus, the coupling of CPT to the cyclic peptides provided in the present application provides a specific targeting tool to EGFR-overexpressing cell lines and EGFRvIII-overexpressing cell lines, especially cancer cells, without interfering with the cytotoxic effect of CPT.

[0144] Docking simulations revealed that the peptides interact with the EGFR receptor while showing substantial overlap with EGF in the vicinity of the EGF binding site. Overall, the cyclic peptides provided here have been shown to be excellent carriers in drug delivery to EGFR-overexpressing cancers and their EGFRvIII variants.

[0145] Furthermore, the cytotoxicity of the cyclic peptides provided in the present application is minimal, and moreover, it has been shown not only to be non-cytotoxic but also to bind efficiently to EGFR. Therefore, not only the cyclic peptides provided in the present application but also one or more of their salts or esters are useful in the form of a single agent or in combination with one or more additional cyclic peptides as an active ingredient of a medicament or as a medicament for cancer treatment.

[0146] The cyclic peptides provided in the present application have been shown to be capable of delivering diagnostic and / or therapeutic payloads to EGFR-positive cells (such as cancer cells).

[0147] Pharmaceutical composition: Thus, according to one aspect of some embodiments of the present invention, there is provided a pharmaceutical composition comprising a peptide-drug conjugate provided and described in the present application as an active material. Similarly, there is provided the use of the conjugate according to embodiments of the present invention in the manufacture of a medicament. According to some embodiments of the present invention, a pharmaceutical composition or medicament is used to treat a medical condition treatable by at least one drug linked to a peptide-drug conjugate and released under control.

[0148] In some of any corresponding embodiments of the present invention, the pharmaceutical composition or medicament is packaged by a packaging material, and printed on the inside or surface of the packaging material, for the treatment of a medical condition or symptom associated with EGFR-positive cells, and / or for the treatment of a medical condition treatable by a drug linked to a peptide-drug conjugate and released under control, and / or for the treatment of cancer. The conjugate according to some embodiments of the present invention may be incorporated into any suitable pharmaceutically acceptable carrier before its use.

[0149] The conjugate may be administered by any conventional approach known and / or used in the art. In any use described in the present application, the PDC provided in the present application can be administered as part of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier described hereinafter in the present application. The carrier is selected to be suitable for the selected route of administration.

[0150] The PDC provided in the present application can be administered by any route of administration including, but not limited to, oral, inhalation, systemic (e.g., intravenous drip, or injection into the peritoneal cavity, subcutaneous, intramuscular or intravenous), or topical (including intraocular, intravaginal, rectal, intranasal).

[0151] The formulations for the pharmaceutical use of the conjugates according to this embodiment typically include the agent in association with a pharmaceutically acceptable carrier and any other therapeutic material. The carrier should be "acceptable" in the sense of being compatible with the other materials in the formulation and not harmful to the recipient. From this perspective, pharmaceutically acceptable carriers are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are compatible with pharmaceutical administration. Such media and agents for such pharmaceutically active substances are known in the art. The use of any conventional media and agents in their compositions is contemplated as long as they are compatible with the active ligand. Additional active agents identified or designed by the present invention and / or known in the art can also be incorporated into the composition. The formulations may be conveniently provided in unit dosage form and prepared by methods known in the pharmaceutical / microbiological arts. Usually, some formulations are prepared by combining the active ligand with a liquid carrier or a finely divided solid carrier or both, and then shaping the product into the desired formulation if necessary.

[0152] The pharmaceutical compositions in some embodiments of the present invention are formulated to be compatible with the intended route of administration. Solutions or suspensions intended for use herein may contain the following components: sterile diluents such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetate buffer, citrate buffer, or phosphate buffer, and reagents for adjusting isotonicity such as sodium chloride and dextrose. The pH can be adjusted with acids and bases such as hydrochloric acid and sodium hydroxide.

[0153] According to some embodiments of the present invention, a pharmaceutical composition suitable for injection use includes a sterile aqueous solution (when water-soluble) or dispersion for immediate preparation of a sterile injectable solution or dispersion, and a sterile powder. Carriers suitable for intravenous administration include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ), or phosphate buffered saline (PBS).

[0154] In addition to the conjugate and a pharmaceutically acceptable carrier, the pharmaceutical composition may include other agents having an effect on nerve cells, for example, agents that cause peripheral neuropathy.

[0155] Treatment method: The present application further provides a method for treating a medical condition, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a cyclic peptide-drug conjugate provided herein according to embodiments of the present invention.

[0156] As used in the present application, the phrase "therapeutically effective amount" refers to the amount of the active agent or cyclic peptide-drug conjugate administered that improves to some extent one or more symptoms of the medical condition being treated. In the context of this embodiment, the phrase "therapeutically effective amount" refers to the amount of the cyclic peptide-drug conjugate administered and / or readministered that, by being at a level that causes disruption of the life cycle of the target cell or microorganism, improves to some extent one or more symptoms of the condition being treated.

[0157] In the context of embodiments of the present invention, the therapeutically effective amount may be referred to in terms of the amount of the entire cyclic peptide-drug conjugate or one or more bioactive agents releasably bound thereto. The effectiveness of any bioactive agent comprising the cyclic peptide-drug conjugate provided herein can be determined by multiple methodologies known in the art.

[0158] According to another aspect of an embodiment of the present invention, any one of the cyclic peptide-drug conjugates described in the present application is identified for the treatment of a subject diagnosed as having a medical condition treatable by at least one drug that binds to the peptide-drug conjugate and is releasable under control.

[0159] According to another aspect of an embodiment of the present invention, there is provided the use of any of the peptide-drug conjugates described in the present application as a medicament or a material for a medicament. In some embodiments, the medicament is for the treatment of a subject diagnosed as having a medical condition treatable by at least one drug that binds to the peptide-drug conjugate and is releasable under control.

[0160] Anticancer treatment: To date, chemotherapy has been the most common and frequently used in cancer treatment, either alone or in combination with other therapies. Currently available anticancer chemotherapies function by affecting specific molecular targets of proliferating cancer cells, resulting in the inhibition of essential intracellular processes such as DNA transcription, synthesis, and replication.

[0161] Unfortunately, because anti-cancer agents are designed to kill mammalian cells, they are highly toxic and thus harmful to normal proliferating cells, leading to debilitation and even lethal side effects. Other side effects include alopecia when hair follicles are attacked, myelosuppression due to toxicity to hematopoietic progenitor cells, and neutropenia. Therefore, the effectiveness of currently used anti-cancer agents is dose-limiting due to their toxicity to normal proliferating cells. The peptide-drug conjugates presented in the present application can be used in the treatment of any medical condition treatable by the peptide-drug conjugate by administering a therapeutically effective amount of the peptide-drug conjugate to a subject in need thereof. According to some embodiments of the present invention, the peptide-drug conjugate can also be used in the preparation of a pharmaceutical composition comprising the same and a pharmaceutically acceptable carrier and other desired materials. Therefore, the peptide-drug conjugate provided in the present application can be used as an active material in a method of treating any medical condition treatable by the peptide-drug conjugate by administering a therapeutically effective amount of the peptide-drug conjugate to a subject in need thereof.

[0162] The peptide-drug conjugates provided herein can be used for the treatment of medical conditions treatable by the administration of a bioactive agent (drug) in the form of a peptide-drug conjugate or an analog thereof in some embodiments of the present invention. According to some embodiments of the present invention, there is an advantage in using peptide-drug conjugates for the treatment of medical conditions treatable by the administration of a combination of drugs. In some embodiments, the medical condition includes cancer, more specifically, cancers of the type characterized by EGFR-positive cells. In the context of the present invention, EGFR-positive cells are cells with a high expression level of the epidermal growth factor receptor (EGFR), a transmembrane receptor protein that is activated by the binding of epidermal growth factor (EGF) or other related ligands, or a variant thereof. These receptors are involved in cell signaling pathways that regulate cell growth, differentiation, and survival. Overexpression or activation of EGFR or its variants has been associated with the development and progression of various cancers, and drugs targeting EGFR have been used in cancer therapy. EGFR-positive cells are frequently studied in cancer research to better understand the role of EGFR in tumorigenesis and to develop new drugs that specifically target these cells.

[0163] According to some embodiments of the present invention, the peptide-drug conjugates provided herein enable controlled, cell-specific anti-cancer activity. In some embodiments of the present invention, the medical condition is associated with malignant cells and tumors, collectively referred to as cancer herein.

[0164] The use of the conjugates according to embodiments of the present invention can optimize the balance between the desired anti-cancer activity of a certain anti-cancer agent and its undesirable side effects by quantitatively determining the actual amount of the drug released within the targeted cells.

[0165] In some embodiments, the functional portion of the peptide-drug conjugate provided in the present application is responsible for the peptide-drug conjugate being higher at the targeted site in the body compared to the non-targeted sites in the body, thereby reducing the undesirable side effects associated with the toxicity of the anti-cancer drug bound thereto, and is a cyclic peptide moiety. In addition, the linking portion that conjugates the anti-cancer agent to the cyclic peptide is cleaved under conditions that are more likely to be present at the target site than at the non-targeted site, and as a result, is selected such that the drug payload is released at a higher frequency at the targeted site compared to the non-targeted site.

[0166] In the context of some embodiments of the present invention, the term "cancer" means, but is not limited to, the following.Acute lymphoblastic, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, appendiceal cancer, basal cell carcinoma, bladder cancer, brain tumor, brainstem glioma, breast cancer, bronchial adenoma / carcinoid, Burkitt lymphoma, carcinoid tumor, cerebellar or cerebral astrocytoma, cervical cancer, cholangiocarcinoma, chondrosarcoma, chronic lymphocytic or chronic lymphocytic leukemia, chronic myelogenous or chronic myeloid leukemia, chronic myeloproliferative disorder, colorectal cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, epithelioma, esophageal cancer, Ewing sarcoma, extracranial primitive neuroectodermal tumor, extragonadal primitive neuroectodermal tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), cystic teratomatous tumor, brainstem glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, laryngeal cancer, leukemia, lip and oral cavity cancer, liposarcoma, lymphoma, male breast cancer, malignant mesothelioma, medulloblastoma, melanoma, Merkel cell skin cancer, mesothelioma, metastatic squamous neck cancer, oral cavity cancer, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma / plasma cell tumor, fungating polyposis, myelodysplastic syndrome / myeloproliferative disorder, nasal and paranasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, anaplastic glioma, oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, ovarian cancer, ovarian germ cell tumor, ovarian epithelial cancer (surface epithelial-stromal tumor), low malignant potential ovarian tumor, pancreatic cancer, paranasal and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary adenoma, plasma cell tumor, pleuropulmonary blastoma, primary carcinoma, primary central nervous system lymphoma, primary liver cancer, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenström macroglobulinemia and Wilms tumor.

[0167] According to some embodiments of the present invention, the peptide-drug conjugates provided in the present application can also be used in basic research that requires specific cancer cell targeting.

[0168] Chemical Definitions: The definitions of specific functional groups, chemical terms, and general terms used throughout this specification are described in more detail below. For the purposes of the present invention, chemical elements are identified according to the description on the inside and outside covers of the 75th edition of the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, and specific functional groups are generally defined as described in that publication. Further, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999, Smith and March March’s Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001, Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989, Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.

[0169] As used herein, the term "amine" or "amino" refers to both -NR'R'' end groups and -NR'-linking moieties, where R' and R'' are each independently hydrogen, alkyl, cycloalkyl, aryl, and these terms are as defined below.

[0170] Thus, the amine group can be a primary amine where both R' and R'' are hydrogen, a secondary amine where R' is hydrogen and R'' is alkyl, cycloalkyl or aryl, or a tertiary amine where each of R' and R'' is independently alkyl, cycloalkyl or aryl.

[0171] Alternatively, R' and R'' can each independently be hydrogen, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocycloaliphatic, amine, halo, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azide, sulfonamide, carbonyl, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine, as defined herein.

[0172] The term "alkyl" refers to saturated aliphatic hydrocarbons including straight-chain (unbranched) and branched-chain groups. Preferably, the alkyl group has 1 to 20 carbon atoms. In this specification, when a numerical range, for example, "1 to 20" is described, it means that this group (in this case, the alkyl group) can include 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 20 carbon atoms. More preferably, the alkyl is a medium-sized alkyl having 1 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkyl is a lower alkyl having 1 to 4 carbon atoms. The alkyl group may be substituted or unsubstituted. The substituted alkyl may have one or more substituents, and each substituent may independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocycloaliphatic, amine, halo, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiolhydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azide, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine, and hydrazine.

[0173] The alkyl group can be a terminal group (as defined above, this group is attached to a single adjacent atom), or a linking moiety (as defined above, connecting two or more moieties via at least 2 carbons in a chain). When the alkyl is a linking moiety, it is also referred to herein as "alkylene" and is, for example, methylene, ethylene, propylene, etc.

[0174] The term "alkenyl" refers to an unsaturated alkyl having at least 2 carbon atoms and at least one carbon-carbon double bond, as defined herein. The alkenyl may be substituted or unsubstituted by one or more substituents as described above for alkyl.

[0175] As defined herein, the term "alkynyl" or "alkyne" is an unsaturated alkyl having at least two carbon atoms and at least one carbon-carbon triple bond. As described above, alkynyl may be substituted with one or more substituents or may be unsubstituted.

[0176] The term "cycloalkyl" refers to a fully carbon monocyclic or fused ring (i.e., rings sharing adjacent pairs of carbon atoms) that does not have a completely conjugated π electron system in one or more of the rings. A cycloalkyl group may be substituted or unsubstituted. A substituted cycloalkyl may have one or more substituents, and each substituent may independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halo, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiolhydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azide, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine, and hydrazine. A cycloalkyl group may be a terminal group (as defined above, the group is attached to a single adjacent atom), or a linking moiety (as defined above, connecting two or more moieties at two or more of its positions).

[0177] The term "heteroalicyclic" represents a monocyclic or fused cyclic group having one or more atoms such as nitrogen, oxygen, and sulfur in the ring. The ring may also have one or more double bonds. However, the ring does not have a completely conjugated π - electron system. The heteroalicyclic group may be substituted or unsubstituted. The substituted heteroalicyclic group may have one or more substituents, and each substituent may independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halo, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiolhydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azide, sulfonamide, C - carboxylate, O - carboxylate, N - thiocarbamate, O - thiocarbamate, urea, thiourea, O - carbamate, N - carbamate, C - amide, N - amide, guanyl, guanidine, and hydrazine. The heteroalicyclic group may be a terminal group (as defined above, this group is attached to a single adjacent atom), or a linking moiety (as defined above, connecting two or more moieties at two or more positions). Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholine, etc.

[0178] The term "aryl" refers to a fully conjugated π - electron system having a monocyclic all - carbon or fused - ring polycyclic (i.e., rings sharing adjacent carbon - atom pairs) end - group. The aryl group may be substituted or unsubstituted. A substituted aryl may have one or more substituents, and each substituent may independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halo, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiolhydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azide, sulfonamide, C - carboxylate, O - carboxylate, N - thiocarbamate, O - thiocarbamate, urea, thiourea, N - carbamate, O - carbamate, C - amide, N - amide, guanyl, guanidine, and hydrazine. The aryl group may be an end - group (as defined above, the group is attached to a single adjacent atom), or a linking moiety (as defined above, connecting two or more moieties at two or more positions). Preferably, aryl is phenyl.

[0179] The term "heteroaryl" refers to a monocyclic or fused-ring (i.e., rings sharing adjacent atom pairs) end group having one or more atoms such as nitrogen, oxygen, and sulfur within the ring and having a more fully conjugated π-electron system. Examples of heteroaryl groups include, but are not limited to, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, and purine. The heteroaryl group may be substituted or unsubstituted. Substituted heteroaryl may have one or more substituents, and each substituent may independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halo, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiolhydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azide, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, O-carbamate, N-carbamate, C-amide, N-amide, guanyl, guanidine, and hydrazine. The heteroaryl group may be an end group (this phrase is as defined above, and this group is attached to a single adjacent atom), or a linking moiety (this phrase is as defined above, and it connects two or more moieties at two or more positions). Representative examples are pyridine, pyrrole, oxazole, indole, purine, and the like.

[0180] The term "alkaryl" refers to an alkyl as defined herein that is substituted by one or more aryl or heteroaryl groups. An example of alkaryl is benzyl.

[0181] The term "amine-oxide" represents an -N(OR’)(R’’) or -N(OR’)- group, where R’ and R’’ are as defined herein. This term refers to the -N(OR’)(R’’) group when the amine-oxide is a terminal group (as defined above), and to the -N(OR’)- group when the amine-oxime is a terminal group (as defined above).

[0182] As used herein, the term "acyl" refers to groups having the general formula -C(=O)R’, -C(=O)OR’, -C(=O)-O-C(=O)R’, -C(=O)SR’, -C(=O)N(R’)2, -C(=S)R’, -C(=S)N(R’)2, and -C(=S)S(R’), -C(=NR’)R’’, -C(=NR’)OR’’, -C(=NR’)SR’’, and -C(=NR’)N(R’’)2, where R’ and R’’ are each independently halo, substituted or unsubstituted hydroxyl, substituted or unsubstituted thiol, substituted or unsubstituted amine, substituted or unsubstituted acyl, cyclic or acyclic substituted or unsubstituted branched or unbranched aliphatic, cyclic or acyclic substituted or unsubstituted branched or unbranched heteroaliphatic, cyclic or acyclic substituted or unsubstituted branched or unbranched alkyl, cyclic or acyclic substituted or unsubstituted branched or unbranched alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aliphatic oxy, heteroaliphatic oxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphatic thioxy, heteroaliphatic thioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, monoaliphatic amino or dialiphatic amino, monoheteroaliphatic amino or diheteroaliphatic amino, monoalkylamino or dialkylamino, monoheteroalkylamino or diheteroalkylamino, monoarylamino or diarylamino, or monoheteroarylamino or diheteroarylamino, or two Rs X1The bases together form a 5- to 6-membered heterocyclic ring. Exemplary acyl groups include aldehyde (-CHO), carboxylic acid (-CO2H), ketone, acyl halide, ester, amide, imine, carbonate, carbamate, and urea. Acyl substituents include, but are not limited to, stable moieties (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thioxo, cyano, isocyano, amino, azide, nitro, hydroxyl, thiol, halo, aliphatic amino, heteroaliphatic amino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphatic oxy, heteroaliphatic oxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphatic thioxy, heteroaliphatic thioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, and acyloxy, etc., which may or may not be further substituted), any of the substituents described herein that result in the formation of.

[0183] As used herein, the term "aliphatic" or "aliphatic group" refers to an optionally substituted hydrocarbon moiety that may be linear (i.e., unbranched), branched, or cyclic ("carbocyclic"), and may be fully saturated, or may contain one or more unsaturated units, but is not aromatic. Unless otherwise specified, aliphatic groups contain from 1 to 12 carbon atoms. In some embodiments, aliphatic groups contain from 1 to 6 carbon atoms. In some embodiments, aliphatic groups contain from 1 to 4 carbon atoms, and in still other embodiments, aliphatic groups contain from 1 to 3 carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched alkyl groups, alkenyl groups, and alkynyl groups, and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0184] As used herein, the terms "heteroaliphatic" or "heteroaliphatic group" refer to an optionally substituted hydrocarbon moiety having from 1 to 5 heteroatoms in addition to carbon atoms, which may be linear (i.e., unbranched), branched, or cyclic ("heterocyclic"), and may be fully saturated or may contain one or more unsaturated units, but is not aromatic. Unless otherwise specified, a heteroaliphatic group contains from 1 to 6 carbon atoms, wherein from 1 to 3 carbon atoms are optionally and independently substituted with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, a heteroaliphatic group contains from 1 to 4 carbon atoms, wherein from 1 to 2 carbon atoms are optionally and independently substituted with heteroatoms selected from oxygen, nitrogen, and sulfur. In still other embodiments, a heteroaliphatic group contains from 1 to 3 carbon atoms, wherein 1 carbon atom is optionally and independently substituted with a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroaliphatic groups include, but are not limited to, linear or branched heteroalkyl, heteroalkenyl, and heteroalkynyl groups.

[0185] The term "halo" represents a fluorine, chlorine, bromine, or iodine substituent.

[0186] The term "halide" refers to the anion of a halogen atom, i.e., F - , Cl - , Br - and I - .

[0187] The term "haloalkyl" refers to an alkyl group as defined above, further substituted with one or more halides.

[0188] The term "sulfate" refers to an -O-S(=O)2-OR' end group (wherein the term is as defined above), or an -O-S(=O)2-O- linking moiety (wherein the term is as defined above), wherein R' is as defined above.

[0189] The term "thiosulfate" refers to an -O-S(=S)(=O)-OR' end group or an -O-S(=S)(=O)-O- linking moiety (these terms being as defined above), where R' is as defined above.

[0190] The term "sulfite" refers to an -O-S(=O)-O-R' end group or an -O-S(=O)-O- group linking moiety (these terms being as defined above), where R' is as defined above.

[0191] The term "thiosulfite" refers to an -O-S(=S)-O-R' end group or an -O-S(=S)-O- group linking moiety (these terms being as defined above), where R' is as defined above.

[0192] The term "sulfinate" or "sulfinyl" refers to an -S(=O)-OR' end group or an -S(=O)-O- group linking moiety (these terms being as defined above), where R' is as defined above.

[0193] The term "sulfoxide" or "sulfinyl" refers to an -S(=O)R' end group or an -S(=O)- linking moiety (these terms being as defined above), where R' is as defined above.

[0194] The term "sulfonate" or "sulfonyl" refers to an -S(=O)2-R' end group or an -S(=O)2- linking moiety (these terms being as defined above), where R' is as defined herein.

[0195] The term "S-sulfonamide" refers to an -S(=O)2-NR'R'' end group or an -S(=O)2-NR'- linking moiety (these terms being as defined above), where R' and R'' are as defined herein.

[0196] The term "N-sulfonamide" refers to an R’S(=O)2-NR’’-terminal group or an -S(=O)2-NR’-linking moiety (these terms being as defined above), where R’ and R’’ are as defined herein.

[0197] The term "disulfide" refers to an -S-SR’-terminal group or an -S-S-linking moiety (these terms being as defined above), where R’ is as defined herein.

[0198] The term "phosphate" refers to an -O-P(=O)2(OR’)-terminal group or reactive group or an -O-P(=O)2(O)-linking moiety (these terms being as defined above), where R’ is as defined herein.

[0199] The term "phosphonate" refers to a -P(=O)(OR’)(OR’’)-terminal group or reactive group or a -P(=O)(OR’)(O)-linking moiety (these terms being as defined above), where R’ and R’’ are as defined herein.

[0200] The term "thiophosphonate" refers to a -P(=S)(OR’)(OR’’)-terminal group or a -P(=S)(OR’)(O)-linking moiety (these terms being as defined above), where R’ and R’’ are as defined herein.

[0201] As used herein, the term "carbonyl" or "carbonate" refers to a -C(=O)-R’-terminal group or a -C(=O)-linking moiety (these terms being as defined above), where R’ is as defined herein.

[0202] As used herein, the term "thiocarbonyl" refers to a -C(=S)-R’-terminal group or a -C(=S)-linking moiety (these terms being as defined above), where R’ is as defined herein.

[0203] As used herein, the term "oxo" represents the =O end group.

[0204] As used herein, the term "thioxo" represents the =S end group.

[0205] The term "oxime" represents the =N-OH end group or the =N-O linking moiety, and these terms are as defined above.

[0206] The term "hydroxyl" represents the -OH group.

[0207] As used herein, the term "aldehyde" refers to the -C(=O)-H group.

[0208] The term "acyl halide" represents the -(C=O)R'''' group, where, as defined above, R'''' is halo.

[0209] As used herein, the term "alkoxy" represents -O-alkyl, -O-cycloalkyl as defined above. The ether group -O- is also a possible linking moiety.

[0210] The term "aryloxy" represents both -O-aryl groups and -O-heteroaryl groups as defined herein.

[0211] As used herein, the term "disulfide" represents the -S-S- linking moiety, and in some cases is formed between two thiol groups.

[0212] As used herein, the terms "thio", "sulfhydryl" or "thiol" represent the -SH group.

[0213] The term "thioalkoxy" or "thioether" represents both -S-alkyl groups and -S-cycloalkyl groups as defined herein. The thioether group -S- is also a possible linking moiety.

[0214] The term "thioaryl oxy" represents both -S-aryl groups and -S-heteroaryl groups as defined herein. The thioaryl ether group -S-aryl- is also a possible linking moiety.

[0215] The term "cyano" or "nitrile" represents a -C≡N group.

[0216] The term "isocyanate" represents a -N=C=O group.

[0217] The term "nitro" represents a -NO2 group.

[0218] As used herein, the terms "carboxylate" or "ester" include C-carboxylates and O-carboxylates.

[0219] The term "C-carboxylate" represents a -C(=O)-OR' end group or a -C(=O)-O linking moiety (these terms are as defined above), where R' is as defined herein.

[0220] The term "O-carboxylate" represents an -OC(=O)R' end group or an -OC(=O)- linking moiety (these terms are as defined above), where R' is as defined herein.

[0221] As used herein, the term "thiocarboxylate" includes C-thiocarboxylates and O-thiocarboxylates.

[0222] The term "C-thiocarboxylate" represents a -C(=S)-OR' end group or a -C(=S)- linking moiety (these terms are as defined above), where R' is as defined herein.

[0223] The term "O-thiocarboxylate" represents an -OC(=S)R' end group or an -OC(=S)- linking moiety (these terms being as defined above), where R' is as defined herein.

[0224] As used herein, the term "carbamate" includes N-carbamates and O-carbamates.

[0225] The term "N-carbamate" represents an R''OC(=O)-NR'- end group or an -OC(=O)-NR'- linking moiety (these terms being as defined above), where R' and R'' are as defined herein.

[0226] The term "O-carbamate" represents an -OC(=O)-NR'R'' end group or an -OC(=O)-NR'- linking moiety (these terms being as defined above), where R' and R'' are as defined herein.

[0227] As used herein, the term "thiocarbamate" includes N-thiocarbamates and O-thiocarbamates.

[0228] The term "O-thiocarbamate" represents an -OC(=S)-NR'R'' end group or an -OC(=S)-NR'- linking moiety (these terms being as defined above), where R' and R'' are as defined herein.

[0229] The term "N-thiocarbamate" represents an R''OC(=S)NR'- end group or an -OC(=S)NR'- linking moiety (these terms being as defined above), where R' and R'' are as defined herein.

[0230] As used herein, the term "dithiocarbamate" includes N-dithiocarbamates and S-dithiocarbamates.

[0231] The term "S-dithiocarbamate" represents an -SC(=S)-NR’R’’ end group or an -SC(=S)NR’- linking moiety (these terms being as defined above), where R’ and R’’ are as defined herein.

[0232] The term "N-dithiocarbamate" represents an R’’SC(=S)NR’- end group or an -SC(=S)NR’- linking moiety (these terms being as defined above), where R’ and R’’ are as defined herein.

[0233] The term "urea", also referred to herein as "ureido", represents an -NR’C(=O)-NR’’R’ end group or an -NR’C(=O)-NR’’- linking moiety (these terms being as defined above), where R’ and R’’ are as defined herein, and R’’’ is as defined herein for R’ and R’’.

[0234] The term "thiourea", also referred to herein as "thioureido", represents an -NR’-C(=S)-NR’’R’’’ end group or an -NR’-C(=S)-NR’’- linking moiety, where R’, R’’ and R’’’ are as defined herein.

[0235] As used herein, the term "amide" includes C-amides and N-amides.

[0236] The term "C-amide" represents a -C(=O)-NR’R’’ end group or a -C(=O)-NR’- linking moiety (these terms being as defined above), where R’ and R’’ are as defined herein.

[0237] The term "N-amide" represents an R’C(=O)-NR’’- end group or an R’C(=O)-N linking moiety (these terms being as defined hereinabove), where R’ and R’’ are as defined hereinabove.

[0238] The term "imine" is interchangeably referred to as "Schiff base" in the art, represents an -N=CR'- linkage, where R' is as defined herein or is hydrogen. As is well known in the art, Schiff bases are typically formed by reacting an aldehyde or ketone with an amine-containing moiety, such as an amine, hydrazine, hydrazide, etc., and these terms are as defined herein. The term "aldimine" refers to a -CH=N- imine derived from an aldehyde. The term "ketimine" refers to a -CR'=N- imine derived from a ketone.

[0239] The term "hydrazone" refers to an -R'C=N-NR''- linkage, where R' and R'' are as defined herein.

[0240] The term "semicarbazone" refers to a linkage formed in the condensation reaction between an aldehyde or ketone and semicarbazide. The semicarbazone linkage derived from a ketone is -R'C=NNR''C(=O)NR'''- and the linkage derived from an aldehyde is -CR'=NNR''C(=O)NR'''- where R' and R'' are as defined herein and R''' is as defined for R'.

[0241] As used herein, the term "lactone" refers to a cyclic ester, i.e., the intramolecular condensation product of an alcohol group -OH and a carboxylic acid group -COOH within the same molecule.

[0242] As used herein, the term "lactam" refers to a cyclic amide as defined herein. A lactam having two carbon atoms other than carbonyl and a total of four ring atoms is called a β-lactam, a lactam having three carbon atoms other than carbonyl and a total of five ring atoms is called a γ-lactam, a lactam having four carbon atoms other than carbonyl and a total of six ring atoms is called a δ-lactam, and so on.

[0243] The term "guanidyl" represents an R’R’’NC(=N)-terminal group or an -R’NC(=N)-linking moiety (these terms being as defined above), where R’ and R’’ are as defined herein.

[0244] The term "guanidine" represents an -R’NC(=N)-NR’’R’’’ terminal group or an -R’NC(=N)-NR’’-linking moiety (these terms being as defined above), where R’, R’’ and R’’’ are as defined herein.

[0245] The term "hydrazine" represents an NR’-NR’’R’’’ terminal group or an -NR’-NR’’-linking moiety (these terms being as defined above), where R’, R’’ and R’’’ are as defined herein.

[0246] As used herein, the term "hydrazide" represents a -C(=O)-NR’-NR’’R’’’ terminal group or a -C(=O)-NR’-NR’’-linking moiety (these terms being as defined above), where R’, R’’ and R’’‘ are as defined herein.

[0247] The term "hydroxylamine", as used herein, refers to either an -NHOH group or an -ONH2.

[0248] As used herein, the term "azo" or "diazo" represents an -N=N-R’ terminal group or an -N=N linking moiety (these terms being as defined above), where R’ is as defined herein.

[0249] As used herein, the term "azide" is -N=N + =N(-N3) and represents a terminal group.

[0250] The term "triazine" refers to a heterocyclic ring that is similar to a 6-membered benzene ring but in which 3 carbons are replaced by nitrogen atoms. The three isomers of triazine are distinguished from one another by the positions of their nitrogen atoms and are called 1,2,3-triazine, 1,2,4-triazine, and 1,3,5-triazine. Other aromatic nitrogen heterocycles include pyridine, which has one ring nitrogen atom, diazine, which has two nitrogen atoms within the ring, and tetrazine, which has four ring nitrogen atoms.

[0251] The term "triazole" refers to either one of a pair of isomeric compounds of the formula C2H3N3 having a 5-membered ring of two carbon atoms and three nitrogen atoms, namely 1,2,3-triazole and 1,2,4-triazole.

[0252] As used herein, the term "aziridine" refers to a 3-membered heterocyclic ring having one amine group and two methylene groups and having a reactive group of the formula -C2H3NH.

[0253] As used herein, the term "thiohydrazide" represents a -C(=S)-NR'-NR''R''' end group or a -C(=S)-NR'-NR''- linking moiety (these terms being as defined above), where R', R'' and R''' are as defined herein.

[0254] As used herein, the term "methyleneamine" represents a -NR'-CH2-CH=CR''R''' end group or a -NR'-CH2-CH=CR''- linking moiety (these terms being as defined above), where R', R'' and R''' are as defined herein.

[0255] As used herein, the term "diene" refers to a -CR'=CR''-CR'''=CR''''- group, where R' is as defined above and R'', R''' and R'''' are as defined for R'.

[0256] As used herein, the term "dienophile" typically refers to a reactive group that reacts with a diene via a Diels-Alder reaction mechanism, and thus a dienophile is typically a double bond or alkenyl.

[0257] As used herein, the term "epoxy" refers to a reactive group that is a three-membered heterocycle having one oxygen and two methylene groups and having the molecular formula -C2H3O.

[0258] As used herein, the phrase "covalent bond" refers to one or more pairs of electrons shared between atoms in the form of a chemical bond.

[0259] It is expected that many related cyclic peptides having affinity for EGFR and variants thereof will be developed between the filing of this application and the expiration of the patent right. However, the scope of the phrase "cyclic peptide" is not intended a priori to include all such new technologies.

[0260] It should be understood that certain features of the invention described in connection with separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, the multiple features of the invention described in connection with a single embodiment for brevity may also be provided separately, or in any suitable partial combination, or as appropriate, for any other described embodiment of the invention. Specific features described in connection with various embodiments should not be considered essential features of that embodiment unless the embodiment would be inoperable without that element.

[0261] The various embodiments and aspects of the invention described herein and claimed in the claims are experimentally supported in the following examples.

Examples

[0262] Examples are referred to below. These examples illustrate some embodiments of the invention in a non-limiting manner together with the above description.

[0263] Example 1 Materials and Methods Cells and Culture Conditions: Lung cancer cell lines (H1299, H1975), glioblastoma cell lines (DKMG, also known as CVCL_1173 cell line), myeloid leukemia cell line (K562), breast cancer cell line (MDA-435), normal mammary cell line (MCF-10A), and embryonic cell line (HEK-293) were cultured in RPMI 1640 medium or DMEM medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% penicillin / streptomycin (all from Biological Industries, Israel). The cells were maintained at 37 °C in a 5% CO2 humidified atmosphere. H1299 cells, H1975 cells, and DKMG cells were characterized by overexpression of EGFR on the cell surface. DKMG cells are known to express two types of EGFR, namely wild-type (WT) and another type with a mutation in the extracellular domain of the receptor (EGFRvIII). H1975 cells (also known as NCI-H1975 or CRL-5908) have a mutation in the internal domain of EGFR (L858R / T790M).

[0264] Positive in vitro Biopanning Selection of Specific Peptides: A heptameric peptide phage display library (Ph.D.-C7C phage display library kit, New England Biolabs, USA) was used for in vitro biopanning experiments. All cell lines were individually incubated with the phage of the stock library. K562 cells that do not express EGFR were used for negative biopanning selection. Adherent cells (H1299, H1297, and DKMG) were seeded in 6-well plates at a density of 10 5 cells / ml. When the cultures reached 80% confluence, the first well was treated with the phage library (10 11It was incubated at 37°C for 1 hour with gentle stirring together with (pfu / 10 μl). The medium containing unbound phage was collected, transferred to a second similar well, and incubated again. This procedure was repeated in the third well as well. The cells in the third well were washed 4 times with 0.5% Tween-20 in PBS. Next, 0.5 ml of elution buffer (0.2 M glycine-HCl, pH 2.2) was added, incubated at 4°C for 10 minutes, and the pH was neutralized by adding 75 μl of 1 M Tris-HCl buffer, pH 9.0. The supernatant containing cell surface-bound phage was collected. The cells were then lysed by incubating with 2 ml of 30 mM Tris-HCl and 1 mM EDTA, pH 8.0, at 4°C for 1 hour. The medium was collected and centrifuged at 1,500 rpm for 5 minutes, and the supernatant containing intracellular phage was transferred to a new tube. The internalized and surface-bound phage were amplified according to the manufacturer's instructions, an equal amount of phage was retained for sequencing (see below), and the rest was subjected to two additional rounds of biopanning. For K562 suspended cells, the same scheme was used, except that cell washing was performed by centrifugation at 1,800 rpm for 5 minutes at RT.

[0265] DNA Preparation and Sequencing: Following biopanning, DNA was extracted from not only the first and third rounds of phage but also the original phage pool according to the manufacturer's instructions. The library was prepared for NGS, and sequencing was performed by Hylabs Pty Ltd. using the MiSeq technology (Rehovot, Israel).

[0266] Analysis of NGS Data: For the analysis of DNA sequencing data, specific scripts were written. The data processing workflow included translation, creation of a report list sorted by the number of repeats of the amino acid sequence, and sorting of the list by removing sequences internalized by K562 cells or not present in the original phage display library of the original Ph.D. C7C. From this workflow, 11 peptides were selected, from which two sublists were created: first, a list of peptides internalized by all three cell lines H1299, H1975, and DKMG, and second, a list of peptides internalized by each cell line alone.

[0267] Solid-phase polymerization of peptides: Eleven heptamer S-S crosslinked cyclic peptides (a total of 9 amino acids including terminal cysteine in each sequence) were synthesized using standard Fmoc protocols by solid-phase peptide synthesis on Rink amide resin (loading amount 0.4 - 0.8 mmol / g, 100 - 200 mesh, Sigma-Aldrich) as free peptides or conjugates via a γ-aminobutyric acid GABA linker with fluorescein-5-isothiocyanate (FITC) or camptothecin (CPT, (S)-(+)-camptothecin). Cyclization between the two cysteine amino acids of the peptide was carried out in 2 hours using I2 (10 equivalents) in DMF / H2O (4:1) prior to the conjugation step.

[0268] Conjugation of peptides to FITC and CPT: Using PyBop (2 equivalents) as a coupling agent and DIPEA (6 equivalents) as a base in DMF, the Fmoc-deprotected cyclic peptide on the resin was coupled to Fmoc-GABA-OH linker (2 equivalents) in 2 hours. Fmoc was removed (20% piperidine in DMF, 2 × 15 minutes), the resin was washed (3 × 3 minutes with DMF, 3 × 3 minutes with DCM), and the deprotected peptidyl resin was reacted as follows. For FITC conjugation, with FITC (2 equivalents) in DMF for 2 hours in the presence of DIPEA (6 equivalents) For the CPT conjugation, it was carried out with (S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-4-yl (4-nitrophenyl) carbonate (CPT-OPNF) (2 equivalents) in DMF for 2 hours in the presence of DIPEA (6 equivalents) and DIMAP (0.5 equivalents).

[0269] The resin was washed (3×3 minutes with DMF, 3×3 minutes with DCM), dried, and cleaved by treatment with cold TFA cocktail (95% TFA, 2.5% TIS, 2.5% H2O) for 2 hours. Further, the solution of PDC was evaporated (N2), and the crude product was purified using preparative HPLC (ECOM preparative system including dual UV detection), column: Phenomenex Gemini® 10μm RP18 110Å, LC 250×21.2mm, 25°C, eluent A (0.1% FA in water) and eluent B (CH3CN). A typical elution was a gradient from 100% A to 100% B at a flow rate of 25 ml / min for 35 minutes.

[0270] The P6-FITC conjugate was (yield 42%, purity 95%) LC-MS: RT = 8.66 minutes, MS: (ESI-MS) calculated m / z: 1448.5, measured: 725.26 [M+2H] 2+ The P6-CPT conjugate was (yield 50%, purity 27%) LC-MS: RT = 4.79 minutes, MS: (ESI-MS) calculated m / z: 1433.55, measured: 717.9 [M+2H] 2+ The P9-CPT conjugate was (yield 45%, purity 60%) LC-MS: RT = 5.21 minutes, MS: (ESI-MS) calculated m / z: 1473.52, measured: 737.8 [M+2H] 2+ .

[0271] [Chemical formula]

[0272] Stability study of peptide drug conjugates: The peptide-FITC conjugate and the peptide-CPT conjugate were incubated in 1 ml of DMEM or RPMI complete growth medium at 37 °C for 0, 0.6, 3, 7, 24, 48, and 72 hours. At each time point, 10 μl of the sample was taken out and mixed with 25 μl of ACN / H20 (1:1, v / v). The sample was subjected to centrifugation at 1,400 rpm for 15 minutes. The supernatant was collected, filtered, and analyzed by liquid chromatography-mass spectrometry (LC-MS).

[0273] Detection of EGFR positive / EGFRvIII expression by flow cytometry: H1299 adherent cells and DKMG adherent cells were collected after trypsinization, resuspended in flow cytometry staining buffer (FCSB, Biological Ind, Israel), and centrifuged at 1,500 rpm for 5 minutes. K562 suspended cells were directly suspended in FCSB. 10 6 Cells / ml of the population were then incubated with anti-human EGFR-APC (3 μl) antibody in the dark at 4 °C for 1 hour or with mouse anti-human EGFRvIII primary (3 μl), followed by washing twice with FCSB. Next, 3 μl of APC labeled with anti-mouse Ig antibody was added, and the cells were incubated in the dark at 4 °C for 20 minutes. After washing, the cells were resuspended in 200 μl of FCSB and analyzed with a Cytoflex cell analyzer from Beckmann coulter. For each sample, 5×10 4 Cells were tested. FlowJo software was used for data analysis.

[0274] Binding analysis of the peptide-FITC conjugate: Cells at 80-90% confluence were incubated with 2.5 μM of the FITC-conjugated peptide in RPMI-5% FBS at 37 °C for 1 hour. The cells were scraped from the culture flask and washed twice with 2 ml of FCSB. The cells were analyzed as previously reported for the internalization of the peptide-FITC conjugate.

[0275] The cells were cultured overnight in a 6-well plate at 80 - 90% confluence, and the medium was replaced with 1 ml of fresh medium containing 5% FBS and 2.5 μM of FITC-conjugated peptide. The plate was incubated at 37 °C for 1, 2, or 3 hours. After incubation, the cells were collected with trypsin, washed twice with FCSB, and resuspended in 200 μl of FCSB. Analysis was performed using a FACS cell analyzer.

[0276] Confocal microscope: H1299 cells, DKMG cells, and HEK-293 cells were seeded onto 12-well glass-bottom black plates at a density of 5.0×10 4 , 1.0×10 5 , and 2.5×10 4 cells / well, respectively. The cells were cultured in complete growth medium at 37 °C in 5% CO2 for 24 hours. After incubation, the medium was removed, and the cells were washed twice with PBS Ca 2+ Mg 2+ . Then, 0.5 ml of fresh medium containing 25 μM of FITC-labeled P6 or P9 peptide was added. The stained cells were incubated at 37 °C in 5% CO2 for 0 and 3 hours, washed twice with PBS, and stained with Hoechst 33258 (16.23 mM, 0.5 ml, 37 °C, 5% CO2) for 10 minutes. The cells were washed twice with PBS and fixed with 4% paraformaldehyde (PFA). Next, fluorescence images were recorded with a Zeiss LSM700 confocal laser microscope at a magnification of ×200.

[0277] Competitive binding assay: The H1299 cell line and the DKMG cell line were cultured in a 6-well plate in complete RPMI growth medium overnight until 80% confluent. Pre-incubation was carried out at 4°C for 30 minutes with a blocking buffer consisting of PBS, pH 7.4, containing 5 mg / ml BSA. Next, the cells were washed twice with PBS and incubated with 2.5 μM FITC-labeled peptide with or without 80 nM EGF at 4°C for 1 hour. Thereafter, the cells were incubated at 37°C for 15 minutes in 5% CO2 to internalize the peptide. The cells were washed twice by centrifugation at 1,500 rpm for 5 minutes and analyzed by flow cytometry.

[0278] Peptide docking: The three-dimensional structure of the peptide was predicted using the PEPstrMOD server. The EGFR structure (Protein Data Bank ID 1IVO) was used for the docking simulation of the peptide to its receptor. This structure represents the extracellular region of human EGFR complexed with the EGF ligand. The ligand was removed from the structure before the docking simulation. Rigid-body docking without bias (search without omission of all possible binding sites and binding poses) was performed using three servers: HDOCK, LZerD, and ZDOCK. Next, the optimal binding poses obtained from each of the three servers were optimized using the local docking protocol of the RosettaDock server, which identifies low-energy conformations by optimizing the rigid-body orientation and side-chain conformations. The top 3 local docking poses (9 in total) of each optimization run were minimized using UCSF Chimera, and the peptide-EGFR binding energy was determined using PRODIGY.

[0279] Cytotoxicity of the peptide-CPT conjugate: Cell proliferation in the presence of PDC was measured using a commercially available XTT assay kit (Cell Proliferation Kit, XTT-based, Biological Industries, Israel). Cells (10 4Cells / well) were seeded into a 96-well plate and incubated overnight in complete growth medium. The cells were washed and then cultured for 24 hours, 48 hours, and 72 hours in 100 μl of fresh medium containing various concentrations of drugs or peptides. XTT solution was added to each well and the plate was further incubated at 37 °C for 2 - 3 hours. The optical density in the wells was measured at both 480 nm and 680 nm using a TECAN Infinite M200 ELISA reader. All tests were performed in 3 sets and each experiment was repeated 3 times.

[0280] In vivo targeting: The animal protocol was approved by the Animal Experimentation Committee of Ariel University, State of Israel. Nude mice received an injection of H1299 cells in the right flank and an injection of K562 cells in the left flank, so each mouse had dual H1299 and K562 grafts. When the tumors reached 100 mm 3 in size, 100 μg of the FITC-labeled cyclic peptide in some embodiments of the present invention was injected via the tail vein of the mice and imaged with an IVIS® Spectrum CT 8 hours and 24 hours after the injection. Immediately thereafter, the mice were sacrificed for ex vivo imaging of the organs.

[0281] Statistical analysis All experiments were performed in 3 sets and the results were expressed as mean ± standard deviation. The data were analyzed using GraphPad Prism 6.0 (GraphPad Software, Inc., California, USA). Statistical analysis was performed using a two-way ANOVA test. A P-value < 0.05 was considered to indicate a statistically significant difference.

[0282] Example 2 Results Evaluation of cell surface expression of EGFRWT and EGFRvIII by flow cytometry: Before using H1299 cells, H1975 cells, DKMG cells, and K562 cells for biopanning, the expression of EGFRWT and EGFRvIII was confirmed by flow cytometry analysis. As expected, flow cytometry analysis showed that H1299 cells, H1975 cells, and DKMG cells expressed EGFRWT (65%, 55%, and 92% respectively), while only DKMG cells expressed EGFRvIII (82%). K562 cells did not express either of these receptors and were used as a negative control.

[0283] Selection of specific peptides: Peptides that specifically bind to and internalize into EGFR-positive cells were isolated using a Ph.D-C7C peptide phage library. For each cell line, biopanning was performed for 3 rounds, and both surface phage and internalized phage were recovered. DNA from the phage pool was extracted from the first and third rounds and sequenced by NGS and translated. To select target-specific peptides, the inventors first demonstrated that all peptide sequences occurred twice in the original phage display library. Next, peptides derived from EGFR-negative K562 cells were removed from the list. After this step, 617,503 sequences remained.

[0284] Finally, two groups of peptides were constructed. The first included sequences present in all three EGFR-positive cell lines and contained approximately 416 sequences. The second was divided into three subgroups, one for each cell line. The subgroup for H1299 contained approximately 1,666 sequences, the subgroup for H1975 contained approximately 10,437 sequences, and the subgroup for DKMG contained approximately 981 sequences. From this list, the top 11 S-S cross-linked cyclic peptide sequences were selected based on the counts (the largest read numbers) in the sequencing data.

[0285] For sequence analysis, a script for data processing was developed that included the following. 1. Translation of each three-base codon to the amino acid sequence at the protein level from the DNA sequence. 2. Aggregation of the number of occurrences of each array (peptide) in the array file. 3. Creation of a report list sorted by the number of occurrences and then sorted alphabetically. 4. Exclusion of peptides that do not appear in the original phage display library kit (e.g., Ph.D.-C7C phage display library kit #E8120, USA, New England Biolabs INC). The heptapeptide phage display library is a combinatorial library of random peptides with disulfide-constrained loops, while the Ph.D.-12 phage display peptide library and the Ph.D.-7 phage display peptide library are linear peptides. This exclusion step was used as a positive control to screen for sequences created by sequencing errors that do not appear in the original library. 5. Exclusion of peptides that appeared in samples of EGFR-negative cells (K562 cell line). This exclusion step was used as a negative control to screen for sequences showing non-specific binding that bound to and internalized in EGFR-negative cells.

[0286] This data processing resulted in the creation of two groups of peptides, namely, the first consisting of sequences present in all three cell lines of interest, and the second consisting of three subgroups containing sequences specific to each cell line.

[0287] Binding and internalization of the selected peptides: The binding and internalization ability of the cyclic peptides provided in this application was analyzed using fluorescein-5-isothiocyanate (FITC, a fluorescent probe for labeling amines) as a detectable moiety (label). Eleven selected FITC-labeled cyclic peptides, P1 to P11 (each 2.5 μM), were tested on K562 cells, H1299 cells, and DKMG cells as described above. The concentration was selected based on a preliminary dose-dependent study. The total number of cells to which the peptide binds but does not internalize was calculated by subtracting the number of peptide-internalizing cells from the total number of positive cells.

[0288] Flow cytometry analysis showed that the FITC-labeled peptides P2 - P6, P8, P9, and P11 selectively bind to H1299 cells and DKMG cells, but not to K562 cells. Furthermore, the FITC-labeled P3, P6, P8, P9, and P11 peptides were internalized only into EGFR-positive cells. The FITC-labeled P3 - P5, and P9 showed higher internalization activity against the DKMG cell line that expresses both EGFR-positive and EGFRvIII mutations. Table 3 shows the peptide specificity per cell line (referencing the cell source where the peptide was first identified).

[0289]

Table 3

[0290] Competitive binding of peptides to EGFR: To demonstrate that the internalizing peptides target the EGFR receptor, a competitive binding assay with the natural ligand EGF was performed. A fluorescence-activated cell sorting (FACS) assay of the competitive uptake of labeled cyclic peptides by H1299 cells (30,000 cells) in the absence or presence of 80 nM EGF protein showed that EGF was successful in competing with the peptide and significantly reduced the binding of the FITC-labeled cyclic peptide.

[0291] Evaluation of peptide target specificity for EGFR expressed by cells by flow cytometry: The target specificity of the FITC-labeled P4 - P6, P8, and P9 peptides was investigated in three cell lines not used in biopanning, namely, normal kidney HEK-293 cells and normal breast MCF-10A cells, and breast cancer MDA-435 cells. To demonstrate EGFR WT expression by these cell lines, the inventors stained the cells with an APC-labeled monoclonal anti-human EGFR antibody and performed flow cytometry analysis. The EGFR WT expression by HEK-293 cells and MCF-10A cells was negligible (less than 10%), while 97% of MDA-435 cells were EGFR WTIt was positive.

[0292] The interactions of FITC-labeled P4 - P6, P8, and P9 with three cell lines were analyzed by flow cytometry. All cyclic peptides investigated had low binding ability to EGFR-negative HEK-293 cells, but P6 and P9 showed distinct internalization into high EGFR-expressing MDA-435 cells, while P8 had low specificity and showed significant binding to both MCF-10A and MDA-435 cell lines.

[0293] Evaluation of the target specificity of peptides for EGFR expressed by cells using a fluorescence microscope: EGFR of FITC-labeled P6 and P9 peptides compared with EGFR-negative HEK-293 normal cells wt To obtain additional validation data on specificity for H1299 lung cancer cell line and EGFRvIII DKMG glioblastoma cell line, the inventors used a confocal scanning microscope. Cells were incubated with 25 μM of FITC-labeled P6 and P9, the cell nuclei were stained with Hoechst-293, the cells were fixed with 4% paraformaldehyde, and the cells were harvested immediately after addition and 3 hours later. Clearly, all cells emitted a blue fluorescence signal of Hoechst-293, demonstrating their viability. A clear correlation between the green FITC signal and cell type was also established. After 3 hours, strong signals were observed from FITC-P6-stained H1299 cells and DKMG cells expressing EGFR, but low-intensity signals from HEK-293 cells lacking these receptors. Symmetrically, FITC-P9 exhibited high binding affinity and specificity for DKMG (strong green signal). The intensity of FITC-P6 on H1299 and DKMG was undetectable at 0 hour, but FITC-P9 showed distinct uptake on DKMG, which increased significantly after 3 hours.

[0294] Peptide docking: To identify the binding site within the receptor, unbiased rigid body docking simulations were performed between the cyclic peptide and the EGFR receptor. The docking simulations showed that most of the structure to be docked binds to the cavity between domains I and III of the receptor. Similar results were obtained from the LZerD docking server and the ZDOCK docking server. After identification of the binding site, optimization of the binding pose was performed using the RosettaDock server. The poses when each peptide docked optimally from each server were subjected to a local docking protocol with side chain flexibility. The top three of the locally docked structures obtained (for each peptide) were then minimized, and the binding energy of the peptide to the receptor was determined using PRODIGY. According to the docking results, the peptide binds to the EGFR receptor near the EGF binding site. P6 and P9 showed substantial overlap with EGF, while P11 bound deeper within the cavity (towards domain II) and had less overlap with EGF. The PROGIDY binding energies were -13.0, -11.9, and -11.3 kcal mol -1 for each of the peptides P6, P9, and P11, respectively.

[0295] Synthesis of peptide CPT conjugate: Taking all of the above results together, the peptides with the broadest binding and internalization within the cell line panel, and the most promising peptides defined by docking in terms of competition ability were selected for further study. Two of the most promising peptide candidates, P6 and P9, were conjugated with camptothecin (CTP). CPT, an alkaloid, is a DNA topoisomerase I (Topo I) inhibitor with an IC 50 of 679 nM. CPT exhibits a strong antitumor effect against colorectal cancer, breast cancer, lung cancer, and ovarian cancer, and regulates hypoxia-inducible factor 1α (HIF-1α) activity by altering the microRNA (miRNA) expression pattern of human cancer cells.

[0296] First, the resin-bound intermediates P6 and P9 with a deprotected GABA N-terminus were obtained after removing Fmoc from their precursors (20% piperidine in NMP, 10 mL). Apparently, in their free form, especially CPT is very potent, but has low solubility and generates off-target cytotoxicity. Due to these factors, it was excluded from clinical development. The inventors generally hypothesized that the conjugation of CPT to a targeting peptide would improve its pharmacological properties. Thus, loading activated CPT onto the unbound N-terminal amines of the obtained P6 and P9 led to the creation of biodegradable carbamate linkages. Finally, all of the conjugates synthesized on the resin were cleaved from the solid support, precipitated by the addition of cold diethyl ether, isolated, lyophilized, and identified as peptide conjugates P6-CTP and P9-CPT by LC-MS and HRMS. Scheme 1 below provides the synthesis of the P6-CTP conjugate and the P9-CPT conjugate.

[0297]

Chemical formula

[0298] Stability of the peptide-drug conjugate: The stabilities of P6-CPT and P9-CPT were measured at 37 °C for 0 h, 0.5 h, 3 h, 7 h, 24 h, 48 h, and 72 h in RPMI and DMEM complete growth media. Degradation of the conjugate and release of CPT were analyzed by LC-MS. The stabilities and drug releases of these conjugates were similar. The half-life of conjugate degradation was 6 - 7 h.

[0299] Nevertheless, the stabilities of both conjugates were potentially sufficient to provide potent anti-cancer activity. Thus, next, the cytotoxicity of these conjugates was tested in cancer cell lines.

[0300] Effects of P6-CPT and P9-CPT: Using two protocols, the cytotoxicity of peptide-drug conjugates (PDCs) was tested against H1299 cells and DKMG cells in a concentration range (0.5 - 50 μM). In the first protocol, the cells were pre-incubated with P6, P9, P6-CPT, and P9-CPT for 6 hours, the medium was removed, and fresh drug-free complete medium was added for an additional 24 hours (denoted as 6h24h) or 48 hours (denoted as 6h48h). In the second protocol, the cells were continuously exposed to PDCs for 24 hours, 48 hours, and 72 hours. IC 50 was calculated, and the results are shown in Table 4 and explained below.

[0301] H1299: According to the first protocol, P6 and P9 did not show cytotoxicity against H1299 cells even after 48 hours of pre-incubation. On the other hand, although P6-CPT and P9-CPT exerted cytotoxic effects after 48 hours, their IC 50 was higher than the cytotoxic effect of CPT alone (see Table 4). However, in the second protocol with continuous exposure, the cytotoxicity of P6-CPT and P9-CPT was observed 24 hours after exposure. The IC 50 of these PDCs was 2 - 3 times lower than that of free CPT (Table 4).

[0302] DKMG: After 24 hours of pre-incubation with P9 and P9-CPT, according to the first protocol, there was no effect on DKMG cell viability. The cytotoxic effect of P9-CPT on DKMG was observed after 48 hours, but its IC 50 concentration was 4 times higher than that of free CPT alone (Table 4). After 24 hours of continuous incubation (second protocol), the P9-CPT conjugate was observed to be more toxic (IC 50 = 6.76 μM) compared to free CPT (IC 50 = 25 μM). After 48 hours and 72 hours of incubation (second protocol), P9-CPT showed a cytotoxic effect similar to that of free CPT.

[0303] Since the peptide does not bind to and internalize into EGFR-negative cells, the cytotoxic effect of the peptide CPT conjugate was not tested in HEK normal cells.

[0304] Table 4 shows the IC 50 values (μM) of P6, P6-CPT, P9, and P9-CPT against H1299 cells and DKMG cells, and these values were calculated using non-linear regression.

[0305] [Table 4]

[0306] In vivo targeting: Mice bearing H1299 and K562 were intravenously injected with FITC-labeled P6 or P11 peptide. The signal from the targeted tumor was determined for each animal compared to other tissues.

[0307] The accumulation of FITC-labeled P6 or P11 in H1299 tumors compared to K562 tumors indicated that the peptide could distinguish EGFR-positive tumors from EGFR-negative tumors in vivo. No significant accumulation of P6 and P11 peptides was seen in other organs. One case of liver metastasis was found, which was also positive for FITC.

[0308] The present invention has been described in conjunction with its specific embodiments, but it is obvious that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, all such alternatives, modifications, and variations are intended to be included within the spirit and broad scope of the appended claims.

[0309] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In addition, the citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that chapter headings are used, such headings shall not necessarily be construed as limiting.

[0310] Furthermore, any priority documents of this application are hereby incorporated by reference in their entirety.

Claims

1. A cyclic peptide having two terminal cysteine ​​residues and a disulfide bond between the terminal cysteine ​​residues, and having a sequence selected from the group consisting of the following. CLRWRFGRC (Sequence ID 1), CSAETVESC (Sequence ID 2), CVRWRRFGRC (Sequence ID 3), CLAVEVRPC (SEQ ID NO: 4), CPNDSYHQC (Sequence ID 5), CHVPGSYIC (Sequence ID 6), CWHSLSLAC (Sequence ID 7), CSALWASHC (Sequence ID 8), CVNAMQSYC (Sequence ID 9), CNWLSRTEC (Sequence ID 10), and CAQYTPGRC (Sequence ID 11, and any C-terminal amidates, salts, hydrates, or solvates thereof.

2. The cyclic peptide according to claim 1, further comprising a portion consisting of a bioactive agent bound thereto.

3. The cyclic peptide according to claim 2, wherein the bioactive agent is selected from the group consisting of drugs, cytotoxic agents, imaging agents, diagnostic agents, and labeling agents.

4. The cyclic peptide according to claim 1, for use in targeted drug delivery to cells overexpressing EGFR and EGFRvIII variants.

5. A conjugate comprising a portion consisting of the cyclic peptide described in Claim 1 and a portion consisting of a bioactive agent.

6. The conjugate according to claim 5, wherein the portion comprising the cyclic peptide and the portion comprising the bioactive agent are linked by a connecting portion, and the connecting portion is biodegradable.

7. The conjugate according to claim 6, wherein the connecting portion includes a spacer portion.

8. The conjugate according to claim 6 or 7, wherein the connecting portion is selected from the group consisting of a γ-aminobutyric acid (GABA) portion, a glutathione portion, a lysine portion, a succinic acid portion, a 2-amino-5-(carbamoylamino)pentanoic acid (PABA) portion, a citrulline portion, a valine-citrulline-PABA portion, and any combination thereof.

9. The cyclic peptide according to claim 2 or 3, wherein the biological agent is a cytotoxic agent selected from the group consisting of camptothecin (CTP), doxorubicin (DOX), monomethyl auristatin F (MMFA), and 7-ethyl-10-hydroxycamptothecin (SN38).

10. The conjugate according to claim 5, wherein the biological agent is a cytotoxic agent selected from the group consisting of camptothecin (CTP), doxorubicin (DOX), monomethyl auristatin F (MMFA), and 7-ethyl-10-hydroxycamptothecin (SN38).

11. A pharmaceutical composition comprising the conjugate described in Claim 5 and a pharmaceutically acceptable carrier, diluent, or excipient, packaged in packaging material, and clearly marked on the inside or surface of the packaging material as being for the treatment of a medical condition.

12. The pharmaceutical composition according to claim 11, wherein the medical condition is associated with cells overexpressing EGFR and EGFRvIII variants, and the medical condition is treatable with the bioactive agent.

13. The pharmaceutical composition according to claim 11 or 12, wherein the biological agent is a cytotoxic agent.

14. The pharmaceutical composition according to claim 11 or 12, wherein the medical condition is cancer.

15. The conjugate according to claim 5, for use as a diagnostic agent in imaging or detection techniques for diagnosing diseases associated with cells overexpressing EGFR and EGFRvIII variants.

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