DLL3 binding peptides and uses thereof

Abstract

The invention disclosed herein is directed to polypeptides that bind to DLL3 protein and uses thereof for the diagnosis and treatment of tumors / cancers.

Description

[Technical Field]

[0001] The invention disclosed herein relates to a polypeptide that binds to delta-like ligand 3 (DLL3) and its use in the diagnosis and treatment of tumors or cancer. [Previous Technology]

[0002] Delta-like ligand 3 (DLL3) is a type 1 transmembrane protein and a non-classical Notch ligand. DLL3 is a promising target for therapies because it is highly expressed on the cell surface of neuroendocrine tumors and has minimal localization in normal tissues, primarily in the cytoplasm. Owen et al., J Hematol Oncol. [Journal of Hematology and Oncology], 12:61 (2019). Therapies targeting DLL3 have been developed in recent years for the treatment of tumors or cancers that express DLL3. See, for example, WO 2017 / 021349.

[0003] Labeled (e.g., radiolabeled) small peptides are a class of pharmaceutical compounds used for the diagnosis and treatment of tumors or cancers. See, for example, Christine Rangger and Roland Haubner, Pharmaceuticals, 13(2):22 (2020). Compared to antibodies, these peptides are non-immunogenic and exhibit rapid diffusion and target localization. Furthermore, peptides can be readily modified to improve metabolic stability and pharmacokinetics. When used for tumor / cancer diagnosis, labeled small peptides also allow for noninvasive detection of tumor / cancer cells and monitoring of disease burden during or after treatment.

[0004] No peptides that can bind to DLL3 and be used for cancer diagnosis and treatment have been reported to date. Such compounds are needed in the art. [Summary of the Invention]

[0005] This invention discloses a polypeptide that binds to the human DLL3 protein and its use in the diagnosis and treatment of tumors / cancer. It demonstrates that the DLL3-binding peptides described herein can be dimerized and / or functionalized for conjugation with radioisotopes as DLL3-targeting radiotracers, allowing for non-invasive detection of DLL3-expressing tumor cells and monitoring of disease burden after treatment with DLL3-targeting molecules.

[0006] Therefore, in one aspect, the present invention provides a polypeptide comprising an amino acid sequence selected from C-X1-X2-X3-X4-X5-X6-X7-X8-C, wherein X1 is Y, H, T, K, S, W, D, E, L, N, Q or R; X2 is G, W, Y, M, T or V; X3 is D, N, Y, T, A, E, G or S; X4 is W, A, E, S, V, Y, D, G, N, P, Q, R or T; X5 is D, E, G, Y, N, W, K, R or S; X6 is E, D, G, N, T, A, Q or V; X7 is W, Y, V, E or S; and X8 is T, G, A or S (SEQ ID NO: 78); or alternatively, a polypeptide comprising the amino acid sequence CKWWGGAADEYTYSCGW (SEQ ID NO: 6).

[0007] In another aspect, the present invention provides a polypeptide comprising an amino acid sequence selected from C-X1-X2-X3-X4-X5-X6-X7-X8-C, wherein X1 is Y, H, T, W or N; X2 is G; X3 is D, N or T; X4 is W, A, S, N, R or T; X5 is D, E, G, Y, N or S; X6 is E, D or N; X7 is W, Y or E; and X8 is T (SEQ ID NO: 79).

[0008] In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 1-23, any one of SEQ ID NO: 1-7, any one of SEQ ID NO: 39-61, or any one of SEQ ID NO: 39-45.

[0009] In another aspect, the present invention provides a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 1-38. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 39-76, preferably SEQ ID NO: 39-61, and more preferably SEQ ID NO: 39-45.

[0010] In some embodiments, the amino acid sequence described herein further includes an amino acid residue AETVE or AETVE at the N-terminus of the amino acid sequence. In some embodiments, the polypeptide described herein is modified at the N-terminus, C-terminus, or both. In some embodiments, the amino acid residue at the N-terminus is acetylated. In other embodiments, the C-terminus of the polypeptide is amination or acetylation.

[0011] In some embodiments, the polypeptide described herein comprises a dimer of the amino acid sequences of SEQ ID NO: 1-23, for example, any one sequence of a homodimer or any two sequences of a heterodimer. In some embodiments, the polypeptide described herein comprises a dimer of the amino acid sequences of SEQ ID NO: 1-7, SEQ ID NO: 24-38, SEQ ID NO: 39-76, SEQ ID NO: 39-61, or SEQ ID NO: 39-45. In some embodiments, the dimer is a homodimer, for example, a homodimer of any sequence of SEQ ID NO: 39-61 or SEQ ID NO: 39-45. In some embodiments, the dimer comprises a first linker connecting two amino acid sequences.

[0012] In some embodiments, the polypeptide described herein further comprises a detectable agent. In some embodiments, the detectable agent includes a fluorescent agent (e.g., Cy5 dye, Alexa Fluor® 647 dye, or CF® 647 dye) or a radioisotope (e.g., 67Ga, 99mTc, 111In, 68Ga, 64Cu, 44Sc, 86Y, 89Zr, 18F, 125I, 123I, 124I, or 203Pb; or 47Sc, 114mIn, 177Lu, 90Y, 212 / 213Bi, 212Pb, 225Ac, 186 / 188Re, 67Cu, 131I, 227Th, 211At, or 90Y). In some embodiments, the detectable agent is linked to the polypeptide via a second linker and / or a chelating agent. In some embodiments, the chelating agent is DOTA, TETA, DFO, NOA, DTPA, HOPO, or Macropa.

[0013] In some embodiments, the first or second linker independently comprises a peptide linker or a non-peptide linker. In some embodiments, the first or second linker comprises a non-natural amino acid. In some embodiments, the first or second linker independently comprises a poly(ethylene glycol) (PEG) linker (e.g., PEG3, PEG4, PEG6, and bis-propargyl-PEG2). In some embodiments, the first linker further comprises hPra, Lys(N)3, trioxatridecan-succinamic acid (Ttds), Gly-Gly, or combinations thereof. In some embodiments, the second linker comprises a PEG linker. In some embodiments, the second linker further comprises a bicyclic [6.1.0]nonyne (BCN) group or a dibenzocyclooctyne (DBCO) group.

[0014] In some embodiments, the polypeptide described herein binds to DLL3 (e.g., human DLL3 expressed on the cell surface).

[0015] In another aspect, the present invention provides a pharmaceutical composition comprising a polypeptide as described herein.

[0016] In another aspect, the present invention provides a method for detecting DLL3 in a sample, the method comprising contacting the sample with the polypeptide or pharmaceutical composition described herein, and detecting DLL3 in the sample.

[0017] In some embodiments, the sample comprises cells. In some embodiments, the cells are in a subject, and the method includes administering the subject the polypeptide or pharmaceutical composition described herein and detecting DLL3 in the subject using an imaging technique (e.g., positron emission tomography (PET) scan). In some embodiments, the subject is a person suffering from a tumor or cancer expressing DLL3. In some embodiments, the tumor or cancer is small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), glioma, glioblastoma, melanoma, neuroendocrine prostate cancer, neuroendocrine pancreatic cancer, hepatoblastoma, large cell pulmonary neuroendocrine carcinoma, pancreatic neuroendocrine carcinoma, bladder neuroendocrine carcinoma, gastric neuroendocrine carcinoma, adrenal exocrine tumor, Merkel cell carcinoma, neuroblastoma, head and neck carcinoid or neuroendocrine carcinoma, head and neck paraganglioma, or cervical small cell neuroendocrine carcinoma.

[0018] In another aspect, the present invention provides a method for treating tumors or cancers exhibiting DLL3, and the method includes administering to a subject the polypeptide or pharmaceutical composition described herein. In some embodiments, the subject is a human being.

Implementation Method

[0022] Submit sequence list as ASCII text file

[0023] The following content, submitted as an ASCII text file, is incorporated herein by reference in its entirety: Computer-readable format (CRF) of sequence lists (filename: A-2889-WO01-SEC_SeqListing.xml, creation date: December 8, 2022, size: 73,728 bytes). Cross-references to related applications

[0024] This application claims the benefit of U.S. Provisional Application No. 63 / 291,537, filed on December 20, 2021. The contents of that application are incorporated herein by reference in their entirety.

[0025] The invention disclosed herein relates to peptides that bind to the human DLL3 protein and their use in the diagnosis and treatment of tumors / cancer. It has been demonstrated that the DLL3-binding peptides described herein can be dimerized and / or functionalized for conjugation with radioisotopes as DLL3-targeting radiotracers, allowing non-invasive detection of tumor cells expressing DLL3 and monitoring of disease burden after treatment with various methods (e.g., DLL3-targeted immunotherapy). Therefore, peptides are provided comprising an amino acid sequence of eight to thirteen (e.g., 8, 9, 10, 11, 12, or 13) amino acid residues, flanked by two cysteine ​​(Cys) residues. In some embodiments, the number of amino acid residues between the two Cys residues is eight, nine, or thirteen. In some embodiments, the peptide further comprises additional amino acid residues at the N-terminus or C-terminus or both of the amino acid sequence, for example, the peptide further comprises the N-terminal amino acid residues AETVE or AETVE. The peptides disclosed in this article were identified by screening Cys-bound peptide phage libraries for binding to DLL3 (e.g., human DLL3).

[0026] In one aspect, the present invention provides a polypeptide disclosed herein comprising an amino acid sequence selected from the following: a) C-X1-X2-X3-X4-X5-X6-X7-X8-C (SEQ ID NO: 80), wherein X1 is W, T, Y, H, S, D, K, L, N, Q, E, R or V; X2 is G, W, Y, L, V, T, E, H, M or P; X3 is D, N, T, Y, G, W, A, E, K, L, M, S or V; X4 is W, A, Y, G, S, E, Q, T, V, D, K, M, N, P or R; X5 is G, Y, D, E, W, N, K, R, S or T; X6 is G, E, D, N, W, T, A, K, Q, R, V or Y; X7 is W, Y, V, S, E, P, R, T; and X8 is T, G, S, V, A, M, Q or W.

[0027] In one aspect, the present invention provides a polypeptide disclosed herein comprising an amino acid sequence selected from the following: a) C-X1-X2-X3-X4-X5-X6-X7-X8-C, wherein X1 is Y, H, T, K, S, W, D, E, L, N, Q, or R; X2 is G, W, Y, M, T, or V; X3 is D, N, Y, T, A, E, G, or S; X4 is W, A, E, S, V, Y, D, G, N, P, Q, R, or T; X5 is D, E, G, Y, N, W, K, R, or S; X6 is E, D, G, N, T, A, Q, or V; X7 is W, Y, V, E, or S; and X8 is T, G, A, or S (SEQ ID NO: 78); or alternatively, b) A polypeptide comprising the amino acid sequence CKWWGGAADEYTYSCGW (SEQ ID NO: 6). Definition

[0028] As used herein, the term "peptide" or "polypeptide" refers to a molecule of two or more amino acids linked by peptide bonds. Polypeptides described herein typically contain 5 to 60 (e.g., 8 to 50) amino acids. Polypeptides can further form polymers, such as dimers, trimers, and higher oligomers, i.e., composed of more than one polypeptide molecule. The polypeptide molecules forming such dimers, trimers, etc., can be the same or different. Therefore, the corresponding higher-order structures of such polymers are called homodimers or heterodimers, homotrimers or heterotrimers, etc. The terms "peptide" and "polypeptide" also refer to naturally modified peptides / polypeptides, wherein the modification is achieved, for example, by post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. "Peptide" or "polypeptide," when mentioned herein, can also be chemically modified, for example, by, for example, acetylation, amination, or polyethylene glycol modification at the N-terminus and / or C-terminus, or can be cyclic (e.g., via disulfide bonds). Such modifications are well known in the art and are described below.

[0029] The term "amino acid" or "amino acid residue" typically refers to building blocks of proteins, such as amino acids selected from the group consisting of: alanine (Ala or A); arginine (Arg or R); aspartic acid (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamic acid (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His) Amino acids can be categorized as follows: (or H); isoleucine (He or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic, or rare amino acids may be used as needed. Generally, amino acids can be grouped into those with nonpolar side chains (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, Val); those with negatively charged side chains (e.g., Asp, Glu); those with positively charged side chains (e.g., Arg, His, Lys); or those with uncharged polar side chains (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0030] The terms "subjects in need" or "those in need of treatment" include those who already have a disorder as well as those who need to prevent a disorder. Subjects in need or "patients" include people and other mammalian subjects who receive preventive or therapeutic treatment.

[0031] The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Treatment includes the application or administration of the polypeptides described herein to the body, isolated tissues or cells of a patient who has a disease / disorder, symptoms of a disease / disorder, or a predisposition to a disease / disorder, with the aim of treating, curing, alleviating, reducing, altering, remedying, reducing, improving, or influencing the disease, symptoms of a disease, or a predisposition to a disease.

[0032] As used herein, the term "relief" means any improvement in the disease state of a patient suffering from a tumor or cancer or metastatic cancer as described below, achieved by administering the polypeptide according to the invention to a subject in need. Such improvement may also be considered as slowing or halting the progression of the patient's tumor or cancer or metastatic cancer.

[0033] As used herein, the term "prevention" means the prevention of the occurrence or recurrence of a patient suffering from a tumor or cancer or metastatic cancer as specified below by administering the polypeptide according to the invention to a subject in need. The term "disease" means any condition that would benefit from treatment with the polypeptide or pharmaceutical composition described herein. This includes chronic and acute disorders or diseases, including those pathological conditions that make mammals susceptible to the diseases discussed.

[0034] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” also include plural indicators. Thus, for example, a reference to “a reagent” includes one or more of such different reagents, and a reference to “the method” includes reference to equivalent steps and methods known to those skilled in the art that can modify or replace the method described herein.

[0035] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or be able to determine, many equivalents of the specific embodiments of the invention described herein using only conventional testing. Such equivalents are intended to be covered by the invention.

[0036] The term “and / or” as used herein includes the meaning of “and”, “or” and “all or any other combination of elements connected by such terms”.

[0037] As used herein, the terms “about” or “approximately” mean within ±20%, preferably within ±15%, more preferably within ±10%, and most preferably within ±5% of a given value or range.

[0038] Throughout this specification and the following claims, unless the context otherwise requires, the word "comprise" and its variations such as "comprises" and "comprising" shall be understood to implicitly include the integer or step or the group of integers or steps, but not exclude any other integer or step or the group of integers or steps. When used herein, the term "comprise" may be replaced by the terms "containing" or "including," or sometimes by the term "having."

[0039] When used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting substantially of" does not exclude materials or steps that do not substantially affect the essential and novel features of the claim.

[0040] In each example herein, any one of the terms "comprising / including," "consistently comprising," and "composed of" may be replaced by any of the other two terms. DLL3 binding peptide

[0041] DLL3 is a non-canonical Notch ligand that acts autonomously to inhibit Notch signaling by binding to Notch in cis, thus blocking cell-to-cell interactions and internalization of Notch in target cells, a hallmark of canonical Notch signaling. The primary role of DLL3 is somatic cytogenesis during embryonic development. DLL3 knockout mice exhibit segmental defects in the development of the axial skeleton, skull, and neurons. Somatic pattern defects have also been observed in humans with certain germline DLL3 mutations, leading to a condition known as vertebral rib dysplasia (Bulman MP et al., Nature Genetics, 24, 438-441 (2000)).

[0042] The DLL3 lineage is a promising target for therapeutic development because it is highly expressed on the cell surface of neuroendocrine tumors / cancers and has minimal localization in normal tissues, primarily in the cytoplasm (Owen et al., J Hematol Oncol. [Journal of Hematology and Oncology], 12:61 (2019)). Neuroendocrine tumors / cancers typically originate in neuroendocrine cells and can occur in organs such as the lungs, appendix, small intestine, rectum, and pancreas.

[0043] This article provides a polypeptide that binds to human DLL3 protein (e.g., SEQ ID NO: 77) and its use in the diagnosis and treatment of tumors / cancers such as neuroendocrine tumors / cancers. In one aspect, the present invention provides a polypeptide disclosed herein comprising an amino acid sequence selected from the following: a) C-X1-X2-X3-X4-X5-X6-X7-X8-C, wherein X1 is Y, H, T, K, S, W, D, E, L, N, Q, or R; X2 is G, W, Y, M, T, or V; X3 is D, N, Y, T, A, E, G, or S; X4 is W, A, E, S, V, Y, D, G, N, P, Q, R, or T; X5 is D, E, G, Y, N, W, K, R, or S; X6 is E, D, G, N, T, A, Q, or V; X7 is W, Y, V, E, or S; and X8 is T, G, A, or S (SEQ ID NO: 78); or alternatively, b) a polypeptide comprising the amino acid sequence CKWWGGAADEYTYSCGW (SEQ ID NO: 78). 6) of the polypeptide.

[0044] In another aspect, the present invention provides a polypeptide comprising an amino acid sequence selected from C-X1-X2-X3-X4-X5-X6-X7-X8-C, wherein X1 is Y, H, T, W or N; X2 is G; X3 is D, N or T; X4 is W, A, S, N, R or T; X5 is D, E, G, Y, N or S; X6 is E, D or N; X7 is W, Y or E; and X8 is T (SEQ ID NO: 79).

[0045] In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 1-38, any one of SEQ ID NO: 1-23, any one of SEQ ID NO: 1-7, any one of SEQ ID NO: 39-61, or any one of SEQ ID NO: 39-45, as shown in Table 1 below. In some embodiments, the polypeptide described herein comprises an N-terminal amino acid residue AETVE or AETVE, for example, as shown in Table 1. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 39-76, preferably SEQ ID NO: 39-61, or more preferably SEQ ID NO: 39-45, as shown in Table 1. [Table 1] SEQ ID No. sequence 1 WWCNGNSENWTCTW 2 WGCTGTWGNETCWW 3 WDCWGNTDDWTCTW 4 YGCYGDRNDYTCTW 5 LGCHGDNSEWTCVW 6 WSCKWWGGAADEYTYSCGW 7 YGCTGDAYEYTCTW 8 WTCYWTEGVSGCRW 9 YWRCDWYEETVACGT 10 WSCHYSYWTVSCGT 11 YGCSMDWRGVTCAW 12 DYCLWAPDAWCQN 13 RGCTGNVYDWTCVW 14 WTCHGNADEWTCSW 15 DGCKGDWYEWTCWW 16 YGCRGGDGEWTCVW 17 DYCYGDVEEWTCVY 18 YGETSGWTCLW 19 YGCKWDWKGWTCAW 20 YGCSVNADGWTCSW 21 YGCYGEQEQWTCLW 22 YGCQYYGWDYTCAW 23 CONCLUSIONS 24 ETCWYLYGREWCVG 25 MRCYPWWYGPMCVR 26 WGCLGMGYDRTCWW 27 WTCWTVYGKSSCWD 28 YGCWWDAYGSTCTW 29 YGCDLWKGWTVCGY 30 RYCTHTQTYVQCQD 31 WGCNLDWYGWTCWW 32 WSCVVYGDNWTCSS 33 WSCQLGSGWWTCAW 34 WTCWETTEGYGCNW 35 WGCTGGAWEYTCWW 36 YGCSVTWGGYTCTW 37 YNCWYKMGWYVCWS 38 CRAYSNWFYIC 39 AETVEFWWCNGNSENWTCTW 40 AETVEFWGCTGTWGNETCWW 41 AETVEFWDCWGNTDDWTCTW 42 AETVEYGCYGDRNDYTCTW 43 AETVEFLGCHGDNSEWTCVW 44 AETVEFWSCKWWGGAADEYTYSCGW 45 AETVEYGCTGDAYEYTCTW 46 AETVEFWTCYWTEGVSGCRW 47 AETVEYWRCDWYEETVACGT 48 AETVEFWSCHYSYWTVSCGT 49 AETVEFYGCSMDWRGVTCAW 50 AETVEVDYCLWAPDAWGCQN 51 AETVEFRGCTGNVYDWTCVW 52 AETVEFWTCHGNADEWTCSW 53 AETVEFDGCKGDWYEWTCWW 54 AETVEFYGCRGGDGEWTCVW 55 AETVEFDYCYGDVEEWTCVY 56 AETVEYGCETYSGGWTCLW 57 AETVEFYGCKWDWKGWTCAW 58 AETVEYGCSVNADGWTCSW 59 AETVEFYGCYGEQEQWTCLW 60 AETVEFYGCQYYGWDYTCAW 61 AETVEFYGCWGNYNDYTCSW 62 AETVEFETCWYGREWCVG 63 AETVEFMRCYPWWYGPMCVR 64 AETVEFWGCLGMGYDRTCWW 65 AETVEFWTCWTVYGKSSCWD 66 AETVEFYGCWWDAYGSTCTW 67 AETVEFYGCDLWKGWTVCGY 68 AETVEFRYCTHTQTYVQCQD 69 AETVEFWGCNLDWYGWTCWW 70 AETVEFWSCVVYGDNWTCSS 71 AETVEFWSCQLGSGWWTCAW 72 AETVEFWTCWETTEGYGCNW 73 AETVEFWGCTGGAWEYTCWW 74 AETVEFYGCSVTWGGYTCTW 75 AETVFEYNCWYKMGWYVCWS 76 AETVEFCRAYSNWFYIC

[0046] In some embodiments, the DLL3-binding peptide described herein is modified at the N-terminus, C-terminus, or both, which may improve the peptide's stability and / or metabolism in vivo. In some embodiments, the N-terminal amino acid residue is acetylated. In some embodiments, the C-terminus of the peptide is modified, for example, by amination or acetylation (e.g., -NH2, -CONHR, or -CONH2). In some embodiments, the N-terminal amino acid residue is acetylated and the C-terminus of the peptide is amination or acetylation.

[0047] In some embodiments, the polypeptide described herein comprises a dimer of the amino acid sequences listed in Table 1. In some embodiments, the dimer system is a homodimer (e.g., a polypeptide comprising a specific pair of amino acid sequences listed in Table 1) or a heterodimer (e.g., a polypeptide comprising two different amino acid sequences listed in Table 1). In some embodiments, the polypeptide comprises a homodimer of any one of SEQ ID NO: 1-38, any one of SEQ ID NO: 3976, any one of SEQ ID NO: 39-61, or any one of SEQ ID NO: 39-45.

[0048] In some embodiments, peptides can be modified to improve target binding (e.g., pharmacokinetic properties), stability (e.g., resistance to peptidase digestion), and metabolism (e.g., reduced renal retention or hepatobiliary metabolism and clearance). For example, cyclization and modification of the N-terminus and / or C-terminus of a peptide can protect the peptide from exopeptidases and / or endopeptidases. Another strategy to improve endopeptidase stability is to use d-amino acids or non-natural amino acids, such as naphthylalanine, phenylglycine, leucine, and cyclohexylalanine. It may also be necessary to modify the N-terminus and / or C-terminus of the peptide (e.g., to form an amide at the C-terminus, such as -CONH2 or -CONHR) to link a detectable agent via a non-peptide linker such as a PEG linker containing a DBCO (dibenzocyclooctyne) group or a BCN (bicyclo[6.1.0]nonyne) group.

[0049] In some embodiments, the dimer includes a first linker that links two amino acid sequences.

[0050] In some embodiments, the polypeptide described herein further comprises a detectable agent. In some embodiments, the detectable agent is linked to the polypeptide via a second linker and / or a chelating agent. For example, the polypeptide described herein may have an XYZ configuration, wherein X is a detectable agent with or without a chelating agent (e.g., a radioisotope or fluorescent agent described herein), Y is a linker (e.g., a second linker), and Z is a polypeptide (e.g., a polypeptide comprising a dimer of the amino acid sequences listed in Table 1 linked by a first linker).

[0051] In various embodiments, the first linker and the second linker may be independently peptide linkers or non-peptide linkers. In some embodiments, the first linker and / or the second linker each independently comprise a peptide linker, such as a short peptide composed of some (e.g., 2-6) naturally occurring and / or non-naturally occurring amino acids. Naturally occurring residues are grouped based on common side chain characteristics. Exemplary groups include: (1) hydrophobic: leucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; (3) acidic: Asp, Glu; (4) basic: Asn, His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Examples of non-natural amino acids include, but are not limited to, D-amino acids, homoamino acids, β-homoamino acids, N-methylamino acids, and α-methylamino acids. In some embodiments, the peptide linker comprises or consists of lysine residues and / or two glycine residues (Gly-Gly).

[0052] In some embodiments, the first linker and / or the second linker each independently comprise a non-peptide linker. Examples of non-peptide linkers include, but are not limited to, poly(ethylene glycol) (PEG) linkers, β-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy)acetic acid (AEA), 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), trioxane-tetrazine-succinic acid (Ttds), fumonismethoxycarbonyl (Fmoc-)-Lys(N3)-OH, Lys(N3), high-propargyl glycine, and Fmoc-HPra-OH. These non-peptide linkers are known in the art and are commercially available from companies such as Pepscan and BroadPharm.

[0053] PEG linkers comprise a group of compounds that can be used for various purposes (e.g., biomarking). These linkers are generally classified as monodisperse or polydisperse. Monodisperse PEG linkers have a precise number of PEG units with specific chemical structures and precise molecular weights. In contrast, polydisperse PEG (also known as polymeric PEG or polyPEG) is a polymer with an average molecular weight. PEG linkers may also contain a variety of functional groups, such as azides, amines, alkynes, DBCO (dibenzocyclooctylene), BCN, TCO (trans-cyclooctene), NHS esters, and maleimides. Exemplary PEG linkers that can be used with the peptides disclosed herein include PEG linkers and PEG linkers containing multiple functional groups, such as PEG2, PEG3, PEG4, PEG6, bis-PEG (e.g., bis-PEG18, bis-PEG16, bis-PEG14), bis-propargyl-PEG (e.g., bis-propargyl-PEG6, bis-propargyl-PEG14, and bis-propargyl-PEG18), bis-PEG-NHS, BCN-PEG (e.g., PEG3-BCN), DBCO-PEG (e.g., PEG4-DBCO), and PEG-NHS-ester. These PEG linkers are commercially available from companies such as Peptide Scan, BioDer Pharmaceuticals, and JenKem Technology USA.

[0054] In some embodiments, the first linker comprises a peptide linker, a non-peptide linker, or a combination thereof. For example, in some embodiments, the first linker comprises one or more of PEG linkers (e.g., PEG2, PEG3, PEG4, PEG6), PEG linkers with functional groups (e.g., PEG azide), gly-gly, Ttds, Fmoc-Lys(N3)-OH, high-propargyl glycine, and Fmoc-HPra-OH. In some embodiments, the second linker comprises one or more of PEG linkers (e.g., PEG2, PEG3, PEG4, PEG6), bis-PEG linkers (e.g., bis-PEG16, bis-PEG18), and PEG linkers with functional groups (e.g., BCN-PEG, DBCO-PEG). In some embodiments, the first linker comprises one or more of PEG linkers (e.g., PEG2, PEG3, PEG4, PEG6), PEG linkers with functional groups (e.g., PEG azide), gly-gly, Ttds, Fmoc-Lys(N3)-OH, high-propargyl glycine, and Fmoc-HPra-OH, and the second linker comprises one or more of PEG linkers (e.g., PEG2, PEG3, PEG4, PEG6), bis-PEG linkers (e.g., bis-PEG16, bis-PEG18), and PEG linkers with functional groups (e.g., BCN-PEG, DBCO-PEG).

[0055] The detectable agent may be a radioactive isotope or a fluorescent agent. In some embodiments, the detectable agent is a radioactive isotope. Examples of radioactive isotopes include, but are not limited to, 11C, 18F, 44Sc, 47Sc, 51Cr, 52mMn, 58Co, 52Fe, 56Ni, 57Ni, 62Cu, 64Cu, 67Cu, 66Ga, 68Ga, 67Ga, 72As, 77As, 75Br, 76Br, 77Br, 82Br, 86Y, 89Zr, 90Y, 94mTc, 99mTc, 97Ru, 105Rh, 109Pd, 111Ag, 110mIn, 111In, 113mIn, 114mIn, 120I, 123 I, 124I, 125I, 131I, 117mSn, 121Sn, 127Te, 142Pr, 143Pr, 149Pm, 151Pm, 149Tb, 153Sm, 157Gd, 161Tb, 166Ho, 165Dy, 169Er, 1 69Yb, 175Yb, 172Tm, 177Lu, 186Re, 188Re, 191Pt, 197Hg, 198Au, 199Au, 201Tl, 203Pb, 211At, 212Bi, 213Bi, 212Pb, 225Ac, 227 Th and Al18F2+. In some embodiments, the radioactive isotopes are selected from 18F, 68Ga, 67Ga, 64Cu, 89Zr, 90Y, 99mTc, 111In, 123I, 124I, 131I, 177Lu, and Al18F2+. In some embodiments, the radioactive isotopes are selected from 64Cu, 67Ga, 68Ga, 99mTc, 111In, 123I, 131I, 90Y, 177Lu, 212Bi, 225Ac, and Al18F2+.

[0056] Different radioactive isotopes can emit different particles, such as alpha, beta, or gamma particles, which can be used for imaging and / or therapeutic purposes. Typically, radioactive isotopes that emit gamma rays are used for imaging purposes (e.g., diagnostic imaging), while radioactive isotopes that emit alpha or beta rays are used for therapeutic purposes. Exemplary techniques for imaging purposes include single-photon emission computed tomography (SPECT) and positron emission tomography (PET).

[0057] In some embodiments, the radioactive isotope is selected from 67Ga, 99mTc, 111In, 177Lu, 68Ga, 64Cu, 44Sc, 86Y, 89Zr, 18F, 125I, 123I, 124I, and 203Pb. In some embodiments, the radioactive isotope is 68Ga or 18F. In some preferred embodiments, the detectable isotope is 18F. These exemplary radioactive isotopes can be used for imaging purposes.

[0058] In some embodiments, the radioactive isotope is selected from 47Sc, 114mIn, 177Lu, 90Y, 212 / 213Bi, 212Pb, 225Ac, 186 / 188Re, 67Cu, 131I, 227Th, 211At, and 90Y. In some embodiments, the radioactive isotope is selected from the group consisting of 177Lu, 225Ac, 67Cu, and 212 / 213Bi. These exemplary radioactive isotopes can be used for therapeutic purposes.

[0059] In some embodiments, the detectable agent is a radioactive isotope (e.g., a metallic radioactive isotope) complexed with a chelating agent (also called a chelating reagent). The radioactive isotope / chelating agent complex can reduce radioactive loss and / or hydrolysis of the radioactive isotope and enable its delivery to the desired / intended site in the body. In some embodiments, the chelating agent is a bifunctional chelating agent, for example, having one functional / site that can bind the radioactive isotope and another functional / site that can bind the peptide directly or indirectly. Examples of chelating agents include, but are not limited to, NODASA, NODAGA, TETA, TRITA, TRAP, DTPA, CHX-DTPA, EDTA, CDTA, CPTA, DOTP, DOTPI, EGTA, HBED, TTHA, DTPA, DOTA, DOTAGA, NODA, HP-DOA3, CBTE2a, TE2A, TMT, DPDP, HYNIC, DFO, HEDTA, NOPO.MAG3, NCS-MP-NODA, NH2-MPAA-NODA, NODA, TRAP, DOTPI, DOTP, NOPO and TETA of DOTAGA, NODA, NODA, DTPA, CHX-DTPA, NODA, and their functionalizations. In some embodiments, the chelating agents are DOTA, TETA, NOTA, NETA, TACN-TM, DTPA, 1B4M-DTPA, CHX-A00-DTPA, TRAP (PRP9) NOPO, H2dedpa, H4octapa, H2azapa and H5decapa, HBED, SHBED, BPCA, CP256, PCTA, deferoxamine (DFO), HEHA and PEPA. Chelating agents suitable for radioisotopes are known in the art and can be readily selected by those skilled in the art; see, for example, Christine Rangger and Roland Haubner, Pharmaceuticals 2020, 13, 22; doi:10.3390 / ph13020022; Eric W. Price and Chris Orvig, Chem. Soc. Rev. 2014, 43, 260-290. In some embodiments, the chelating agent is DOTA (e.g., for 111In, 177Lu, or 213Bi), TETA (e.g., for 64Cu / 67Cu), DFO (e.g., for 89Zr), NOA (e.g., for 68Ga or 64Cu / 67Cu), DTPA (e.g., for 111In), HOPO (e.g., for 89Zr or 227Tr), or Macropa (e.g., for 225Ac).

[0060] In some embodiments, the detectable agent is a fluorescent agent. Fluorescent agents are known in the art and are commercially available. Examples of fluorescent agents include, but are not limited to, Cy3 dyes, Cy5 dyes, fluorescein isothiocyanate (FITC), anthranilyl, 2-aminobenzoyl (Abz), 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), carboxytetramethylrosin (TAMRA), 5-(dimethylamino)naphthyl-1-sulfonyl (dansyl), 5-[(2-aminoethyl)amino]naphthyl-1-sulfonic acid (EDANS), and 7-methoxycoumarinyl-4-acetylated (Mca). Fluorescently labeled peptides can be prepared by modifying isolated peptides or by incorporating labeling during solid-phase synthesis using methods known in the art.

[0061] The polypeptides described herein can be prepared by methods known in the art, such as solid-phase peptide synthesis. See, for example, P. Lloyd-Williams, F. Albericio and E. Girald; Chemical Approaches to the Synthesis of Peptides and Proteins, CRC Press, 1997.

[0062] Peptides can be directly labeled by reacting a radioactive isotope with the peptide for direct labeling in a single step. See, for example, Williams J. et al., Bioconjugate Chem., 32(7): 1242-1254 (2021). In some embodiments, the carrier molecule can be labeled with a radioactive isotope, purified (if desired), and then reacted with the peptide to produce a labeled peptide in a two-step reaction. See, for example, Yu S., Biomed Imaging Interv J., 2(4): e57 (Oct-Dec, 2006). In some embodiments, peptide dimerization and / or labeling with a detectable agent (e.g., a radioactive isotope or a fluorescent agent) is performed using "click chemistry" known in the art, such as Huisgen 1,3-dipolar cycloaddition of azides and terminal alkynes. See, for example, Hein, Christopher D. et al., Pharm Res. [Pharmaceutical Research] Oct 2008; 25(10): 2216–2230. doi:10.1007 / s11095-008-9616-1; Kolb, HC et al., Angew. Chem. Int. Ed. [Applied Chemistry International Edition] 2001, 40, 2004–2021. Click chemistry refers to chemical reactions between reagent pairs (click chemistry tools) that react with each other under mild conditions and are effectively inert to naturally occurring functional groups such as amines. Reagents used in click chemistry are known in the art and are commercially available from companies such as Bioder Pharmaceuticals and AlphaThera. In some embodiments, peptide dimerization and / or labeling with a detectable agent (e.g., a radioisotope or a fluorescent agent) is performed using Scheme I and / or Scheme II and / or Scheme III (Embodiment 3) disclosed herein.

[0063] The polypeptides disclosed herein can bind DLL3. In some embodiments, the polypeptide binds human DLL3, for example, human DLL3 expressed in recombinant cells such as those expressing huDLL3 or in cells of patient cancer cells expressing DLL3. The amino acid sequence of human DLL3 (SEQ ID NO: 77) is listed below. In some embodiments, the polypeptide comprises any amino acid sequence listed in Table 1; in some embodiments, the polypeptide comprises a dimer of any amino acid sequence listed in Table 1; in some embodiments, the polypeptide is modified at the N-terminus, C-terminus, or both ends of the peptide. In some embodiments, the dimer system is a homodimer.

[0064] Human DLL3 (SEQ ID NO: 77) MVSPRMSGLLSQTVILALIFLPQTRPAGVFELQIHSFGPGPGGAPSPCSARLPCRLFFRVCLKP GLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREEL GDQIGGPAWSLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTACTRLCRP RSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSC LSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCF NGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFA GPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCY AHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLLPPALGLLVAAGVAG AALLLVHVRRRGHSQDAGSRLLLAGTPEPSVHALPDALNNLRTQEGSGDGPSSSVDWNRPEDV DPQGIYVISAPSIYAREVATPLFPPLHTGRAGQRQHLLFPYPSSILSVK Compositions and Formulations

[0065] The present invention also provides a pharmaceutical composition comprising a polypeptide as described herein.

[0066] As used herein, the term "pharmaceutical composition" refers to a composition suitable for administration to subjects or patients, preferably human subjects or patients. Particularly preferred pharmaceutical compositions of the present invention comprise one or more polypeptides of the present invention. Preferably, the pharmaceutical composition further comprises suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives, and / or adjuvants. The acceptable components of the pharmaceutical composition are preferably non-toxic to the recipient at the dosage and concentration used. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0067] Excipients that can be used in pharmaceutical compositions include gentianic acid, maleic acid, β-cyclodextrin, α-cyclodextrin, ascorbic acid, thioglycerol, glutathione, tartaric acid, nicotinamide, ascorbic acid, FeCl3, glutamic acid, methylene diphosphonic acid, β-hydroxypropyl cyclodextrin, xanthine, aspartic acid, calcium chloride, mannitol, calcium gluconate, sodium succinate, boric acid, sodium carbonate, sodium chloride, or combinations thereof.

[0068] In some embodiments, the pH of the pharmaceutical composition is in the range of about pH 4.5 to about pH 8.0, for example, about pH 5.0 to about pH 7.5, which can be achieved by using one or more buffers such as those known in the art. Exemplary buffers include acetate buffer, phosphate buffer, and citrate buffer.

[0069] In some embodiments, the polypeptide contained in the pharmaceutical composition comprises a polypeptide of a detectable agent such as a radioactive isotope or a fluorescent agent. In some embodiments, the polypeptide includes radioactive isotopes such as 67Ga, 99mTc, 111In, 68Ga, 64Cu, 44Sc, 86Y, 89Zr, 18F, 125I, 123I, 124I, or 203Pb. In some embodiments, the polypeptide comprises 68Ga or 18F. In some embodiments, the polypeptide includes radioactive isotopes such as 47Sc, 114mIn, 177Lu, 90Y, 212 / 213Bi, 212Pb, 225Ac, 186 / 188Re, 67Cu, 131I, 227Th, 211At, or 90Y.

[0070] In embodiments where the pharmaceutical composition comprises a polypeptide containing a radioactive isotope, the pharmaceutical composition may comprise one or more agents, such as N-terapeptide-α-phenylnitrone (PBN), ethanol, sodium ascorbate, and gentianic acid. Containing such agents can stabilize and protect the radioactive isotope. Such agents can be used in amounts that are non-toxic to the recipient at the employed dosage and concentration.

[0071] In some embodiments, the pharmaceutical composition is a liquid composition. In some embodiments, the pharmaceutical composition is a solid composition, such as a lyophilized composition. In some embodiments, the pharmaceutical composition (e.g., a liquid composition or a reconstituted lyophilized composition) is suitable for intravenous administration. Diagnosis and Treatment

[0072] The peptides disclosed herein, such as those containing a detectable agent, can be used to detect DLL3 (e.g., human DLL3) in a sample. The sample may be cells expressing DLL3, such as recombinant cells expressing DLL3 or tumors or cancer cells expressing DLL3. Therefore, this document discloses a method for detecting DLL3 in a sample, comprising contacting a peptide containing the detectable agent as described above or a pharmaceutical composition containing the peptide with a sample and detecting DLL3 in the sample.

[0073] In some embodiments, the polypeptide comprises any one of SEQ ID NO: 1-23, any one of SEQ ID NO: 1-7, any one of SEQ ID NO: 39-76, any one of SEQ ID NO: 39-61, or any one of SEQ ID NO: 39-45 listed in Table 1, and a detectable agent. In some embodiments, the polypeptide comprises a dimer of the amino acid sequence of SEQ ID NO: 1-38 or SEQ ID NO: 39-76, and a detectable agent. In some embodiments, the dimer system is a homodimer; for example, the polypeptide comprises a homodimer of any one of SEQ ID NO: 1-38 or any one of SEQ ID NO: 39-76. In some embodiments, the polypeptide comprises a homodimer of any one of SEQ ID NO: 39-61. In some embodiments, the dimer system heterodimer, for example, the polypeptide, comprises any two different sequences of SEQ ID NO: 1-38 or any two different sequences of SEQ ID NO: 39-76. In some embodiments, the homodimer or heterodimer comprises a first linker linking two amino acid sequences. Suitable linkers for use as the first linker are as described above.

[0074] In some embodiments, the detectable agent is a radioactive isotope such as 67Ga, 99mTc, 111In, 68Ga, 64Cu, 44Sc, 86Y, 89Zr, 18F, 125I, 123I, 124I, or 203Pb. In some embodiments, the radioactive isotope is 67Ga or 18F. In some embodiments, the radioactive isotope is 18F. In some embodiments, the radioactive isotope is linked to the polypeptide via a second linker, a chelating agent, or a combination thereof. Suitable chelating agents and linkers that can be used as the second linker are as described above.

[0075] In some embodiments, the detectable agent is a fluorescent agent, such as Cy3 agent, Cy5 dye, fluorescein isothiocyanate (FITC), o-aminobenzoyl, 2-aminobenzoyl (Abz), 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), carboxytetramethylrosin (TAMRA), 5-(dimethylamino)naphthyl-1-sulfonyl (dansylsulfonyl), 5-[(2-aminoethyl)amino]naphthyl-1-sulfonic acid (EDANS), or 7-methoxycoumarinyl-4-acetyl (Mca). In some embodiments, the fluorescent agent is linked to the polypeptide via a second linker, as described above.

[0076] In some embodiments, the sample comprises cells expressing DLL3, such as human DLL3. In some embodiments, the cell line is a recombinant cell expressing human DLL3. In some embodiments, the cell line is a tumor or cancer cell expressing DLL3, such as cells obtained from a cancer patient.

[0077] Methods that can be used to detect peptides in a sample include those known in the art. For example, when the detectable agent is a fluorescent agent, flow cytometry can be used, and when the detectable agent is a radioactive isotope, imaging methods such as PET or SPECT can be used.

[0078] In some embodiments, the cells are in the subject's body, and the method includes administering a polypeptide or a pharmaceutical composition containing a polypeptide to the subject and detecting DLL3 in the subject using imaging techniques. The polypeptide or a pharmaceutical composition containing a polypeptide may be administered to the subject via parenteral administration. In some embodiments, the polypeptide or a composition thereof is administered via intravenous administration. In some embodiments, the subject is a person suffering from a cancer or tumor expressing DLL3, such as a cancer of neuroendocrine origin. In some embodiments, the tumor or cancer is lung cancer such as small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC), glioma, glioblastoma, melanoma, prostate cancer such as neuroendocrine prostate cancer, neuroendocrine pancreatic cancer, hepatoblastoma, large cell pulmonary neuroendocrine carcinoma, pancreatic neuroendocrine carcinoma, bladder neuroendocrine carcinoma, gastric neuroendocrine carcinoma, adrenal exocrine tumor, Merkel cell carcinoma, neuroblastoma, head and neck carcinoid or neuroendocrine carcinoma, head and neck paraganglioma, or cervical small cell neuroendocrine carcinoma. In some embodiments, the tumor or cancer is prostate cancer (e.g., neuroendocrine prostate cancer) or lung cancer (e.g., small cell lung cancer). In some embodiments, the imaging technique is positron emission tomography (PET).

[0079] The polypeptides disclosed herein, such as those containing radioactive isotopes, can be used to treat tumors or cancers in a subject. Radioactive isotopes that can be used for cancer treatment include those that emit alpha or beta particles. In one aspect, this document also provides a method for treating tumors or cancers exhibiting DLL3, the method comprising administering to a subject in need a polypeptide containing one of the radioactive isotopes described herein or a pharmaceutical composition containing one of the polypeptides described herein.

[0080] In some embodiments, the polypeptide comprises any one of SEQ ID NO: 1-23, any one of SEQ ID NO: 1-7, any one of SEQ ID NO: 39-76, any one of SEQ ID NO: 39-61, or any one of SEQ ID NO: 39-45 listed in Table 1, and a radioactive isotope. In some embodiments, the polypeptide comprises a dimer of the amino acid sequence of SEQ ID NO: 1-38 or SEQ ID NO: 39-76, and a radioactive isotope. In some embodiments, the dimer system is a homodimer; for example, the polypeptide comprises a homodimer of any one of SEQ ID NO: 1-38 or any one of SEQ ID NO: 39-76. In some embodiments, the polypeptide comprises a homodimer of any one of SEQ ID NO: 39-61. In some embodiments, the dimer system heterodimer, for example, the polypeptide, comprises any two different sequences of SEQ ID NO: 1-38 or any two different sequences of SEQ ID NO: 39-76. In some embodiments, the homodimer or heterodimer comprises a first linker linking two amino acid sequences. Suitable linkers for use as the first linker are as described above.

[0081] In some embodiments, the radioisotope is 47Sc, 114mIn, 177Lu, 90Y, 212 / 213Bi, 212Pb, 225Ac, 186 / 188Re, 67Cu, 131I, 227Th, 211At, or 90Y. In some embodiments, the radioisotope is linked to the polypeptide via a second linker, a chelating agent, or a combination thereof. In some embodiments, the radioisotope is linked to the polypeptide via a chelating agent. Suitable chelating agents and linkers for use as the second linker are as described above.

[0082] In some embodiments, the subject is a person with a tumor or cancer expressing DLL3, such as a neuroendocrine cancer. In some embodiments, the tumor or cancer is lung cancer such as SCLC or NSCLC, glioma, glioblastoma, melanoma, prostate cancer such as neuroendocrine prostate cancer, neuroendocrine pancreatic cancer, hepatoblastoma, large cell pulmonary neuroendocrine carcinoma, pancreatic neuroendocrine carcinoma, bladder neuroendocrine carcinoma, gastric neuroendocrine carcinoma, adrenal exocrine tumor, Merkel cell carcinoma, neuroblastoma, head and neck carcinoid or neuroendocrine carcinoma, head and neck paraganglioma, or cervical small cell neuroendocrine carcinoma. In some embodiments, the tumor or cancer is prostate cancer or lung cancer; in some embodiments, the tumor or cancer is neuroendocrine prostate cancer or small cell lung cancer.

[0083] In some embodiments, the polypeptide or a pharmaceutical composition comprising the polypeptide is administered to the subject (e.g., a human patient) via parenteral administration. In some embodiments, the polypeptide or a pharmaceutical composition comprising the polypeptide is administered to the subject via intravenous administration.

[0084] If the drug composition has been lyophilized, the lyophilized material is first reconstituted in a suitable liquid before administration. The lyophilized material can be reconstituted in, for example, water for injection (BWFI), physiological saline, phosphate-buffered saline (PBS), or the same formulation in which the peptide was previously in the lyophilized form.

[0085] In another embodiment, this document discloses a kit containing the polypeptides disclosed herein, such as those containing detectable agents that can be used for diagnostic or therapeutic purposes. In some embodiments, the detectable agent is a radioisotope. In some embodiments, the kit further includes instructions on how to use the polypeptide (e.g., in the form of a leaflet or instruction manual). The kit may also include means for administering the polypeptide or its pharmaceutical composition, such as a syringe, pump, infusion set, etc., means for reconstituted polypeptide, and / or means for diluted polypeptide.

[0086] In the context of this invention, the term "kit" refers to two or more components (one of which corresponds to a polypeptide or pharmaceutical composition of this invention) packaged together in a container, acceptor, or other container. Thus, a kit can be described as a group of products and / or devices sufficient to achieve a specific objective, which can be sold as a single unit.

[0087] As is apparent from the context of the citation, all identified patents and other publications are expressly incorporated herein by reference in their entirety or in relevant portions, for the purpose of describing and disclosing methods, such as those described in such publications, that may be used in conjunction with the information described herein.

[0088] The invention disclosed herein is further illustrated by the following examples. Example 1. Selection and screening of DLL3 binding peptides from a phage library.

[0089] Selection was performed using two Cys-bound peptide phage libraries, 9-CYS and 12-C8C, displayed on the pIII major coat protein of M13. As shown in Table 2, four different selection strategies were employed, combining multiple rounds of selection with the use of recombinant DLL3 protein and three different stable transfected cell lines expressing human DLL3 at different levels on the surface. [Table 2] Choose A Choose B Select B1 (12C8C only) Choose C R1: Protein R1: Protein R1: Protein R1: Protein R2: Protein R2: HEK293-DLL3 F8 R2: HEK293-huDLL3 A6 R2: CHO-huDLL3 R3: Protein R3: HEK293-DLL3 F8 R3: HEK293-huDLL3 A6 R3:CHO-huDLL3 R4: HEK293-DLL3 F8 R4: HEK293-huDLL3 A6 R4:CHO-huDLL3 Cell line DLL3 receptor density HEK293-huDLL3 F8 3000 Low HEK293-huDLL3 A6 127000 high CHO-huDLL3 13600 medium

[0090] The first round of selection was universal for all four strategies using recombinant human DLL3 (rDLL3). 10 μg of rDLL3-Fc was incubated with protein A Dynabeads (Life Technologies) to immobilize the protein onto beads. The beads were then washed twice with PBS buffer and incubated with pre-blocked phages. Approximately 10¹¹ phages from the library were used in this round of selection. In short, the phage particles were blocked in buffer at room temperature and then incubated with the immobilized protein. At the end of incubation, the beads were collected with a magnet and washed five times with PBS buffer. The bound phages were eluted with 100 mM triethylamine (TEA) and then used to infect TG1 bacteria. After 1 hour at 37°C, the bacteria were inoculated onto 2xTY / Amp / Glu plates and incubated overnight at 30°C.

[0091] On the second day, colonies were collected from 10 ml of culture medium in the plate and used to rescue selected phages. The collected bacteria were inoculated into 50 ml of 2XTY / Amp / Glu and grown at 37°C until OD 600 nm = 0.5, then infected with K07 helper phages with an MOI of approximately 10 (10 phages / cell). The infected bacteria were centrifuged, resuspended in 2XTY / Amp / Kan, and grown overnight at 25°C. On the second day, the supernatant was clarified by centrifugation, and the infected bacteria were then blocked in PBS containing 4% milk.

[0092] Parental cell lines (CHO and HEK 293) and DLL3 overexpressing cell lines were isolated, counted, and allocated at 10⁷ cells / tube. Cells were blocked in PBS containing 3% milk and incubated with pre-blocked phages. DLL3 overexpressing cells and phages were incubated for one hour, and unbound phages were removed by washing in PBS. Phages were then eluted by resuspending the cells in 500 µl of 100 mM HCl for 10 minutes. The eluted phages were used to infect 10 ml of TG1 intermediate logarithmic culture. The bacteria were then inoculated onto 2xTY / Amp / Glu plates and incubated overnight at 30°C. This process was repeated twice in four rounds of selection.

[0093] The binding of the phage library obtained after each round of selection to rDLL3-Fc was tested by phage ELISA, and reverse selection was performed on hIgG-Fc. In short, the protein was immobilized on a NUNC-Maxisorp ELISA plate. The plate was then washed and blocked. 100 μl of pre-blocked phage was added to the blocked plate and incubated at room temperature for 1 hour. Anti-M13 HRP conjugated mAb was added, and the plate was read at A370 nm on a Multiskan Ascent (Thermo) ELISA reader after the addition of TMB (Sigma).

[0094] The binding of the phage library to cells overexpressing human DLL3 on its surface was also screened. Cells were isolated, counted, and resuspended at 1.8 × 10⁶ cells / ml in FACS buffer containing 2% FBS. Pre-blocked phages were then added to the cells and incubated at room temperature for 1 hour. After washing, 1 μg of α-M13-FITC (strain MM05T, Sino Biologics) was added to the cells and incubated for another 1 hour. The cells were then washed and resuspended in FACS buffer. Fluorescence was obtained using a flow cytometer (FACS Canto / Becton Dickinson).

[0095] After each round of selection, a single phage strain was selected and grown. Bacterial cultures were infected with helper phages with an MOI of approximately 10 and incubated at 37°C. The infected bacteria were centrifuged, resuspended, and grown overnight at 25°C. The supernatant was clarified by centrifugation the next day. Aliquots of the supernatant were used for phage ELISA assays using the same method described above, or for FACS staining of DLL3-expressing cells.

[0096] The binding of a phage strain four times the background density to DLL3 on the surface of cells expressing DLL3 was further tested by cell ELISA. In short, parental cells and DLL3-overexpressing cells were plated on 96-well plates one day prior to the assay. Cells were then fixed and washed before adding the pre-blocked phage as described above. Cells were then washed and anti-M13-HRP was added. The signal was measured at A370 nm using a Multiskan Ascent (Thermo Fisher Scientific) ELISA reader. Phages binding to DLL3-expressing cells were selected for sequencing the displayed peptide.

[0097] Then, testing was performed on single phage strains that showed specific binding to rDLL3-Fc to DLL3 stably expressed on the cell surface to identify strains that recognize the native form of the protein. DNA sequencing of the display peptide was performed on all phages capable of recognizing the cell.

[0098] In summary, 3400 individual plants were analyzed. Of these, 1718 were bound to the recombinant DLL3 protein, and 387 of these were also bound to the DLL3 protein on the surface of cell lines that stably express DLL3.

[0099] Sequence analysis identified a total of 38 unique sequences, listed in Table 1 (SEQ ID NO: 39-76). Of these, 37 were derived from library 12-C8C, and 1 from library 9-CYS. Example 2. Synthesis, modification, and characterization of DLL3-binding peptides.

[0100] Materials and Methods

[0101] Peptide Synthesis: Peptide synthesis was performed using a Liberty Blue microwave synthesizer (CEM Corp.) via standard Fmoc stepwise solid-phase synthesis (SPPS). Synthesis was carried out at a scale of 150 μmol using Rink amide AM resin Novabiochem 0.29 mmol / g. Each amino acid (0.2 M DMF solution) was amided 8-fold excess using equimolar amounts of DIC (0.5 M) and Oxyma (1 M) DMF solution as activators. Unless otherwise specified, amino acids were used with standard side-chain protecting groups. Cysteine ​​required for disulfide bridges was amided on the solid phase to Fmoc-Cys(Trt)-OH. Aspartic acid was coupled as Fmoc-Asp(OMpe)-OH to minimize the formation of aspartic imine, and after the introduction of Asp-(OMpe)-OH, Fmoc deprotection was performed at room temperature with a 20% piperidine DMF solution. Except for Fmoc-His(Trt)-OH (50°C), single and double coupling were performed at 90°C for 2 minutes under microwave irradiation. Diacetylation was performed on all Fmoc-Arg(Pbf)-OH and the first three amino acids (AET) at the N-terminus. The peptides were cleaved for 1.5 hours at room temperature using a solution of 87.5% TFA, 5% H₂O, 2.5% TIS, and 5% phenol, followed by precipitation with cold tertiary butyl methyl ether. After centrifugation, the peptide precipitate was washed with diethyl ether, dried, dissolved in a 0.1% TFA solution of (1:1) H₂O / ACN, and lyophilized.

[0102] Analytical Characterization: Crude and purified peptides were analyzed by ultra-high performance liquid chromatography (UPLC-UV-MS) with UV and mass spectrometry detection. Analysis was performed on a Waters Acquity UPLC system equipped with an analytical Waters BEH130 C4 column (2.1 × 100 mm, 1.7 µm, at 45°C). Detection was performed by UV absorbance at 214 nm. Mass analysis was performed on a Waters SQ detector with electroionization in positive ion detection mode, with a mass-to-charge ratio scan range of 400–1800. Analytical analysis was performed using a linear gradient of a binary mixture of H₂O (A) containing 0.1% TFA and acetonitrile (B) containing 0.1% TFA. The linear gradients of B used were 20%B-20%B (1 min), 20%B-60%B (4 min), and 60%B-80%B (0.2 min); flow rate: 0.4 mL / min; temperature: 45°C.

[0103] N-terminal acetylation: At the end of sequence assembly, the resin was acetylated using a DMF solution of 10 molar equivalents of acetic anhydride. It is believed that blocking the reactivity of the N-terminal amino group of the peptide by acetylation can enable a direct synthetic strategy for attaching a detectable agent (e.g., fluorescence of a radiolabeled moiety) to the peptide.

[0104] C-terminal amide or amination: C-terminal amide or amination is performed during solid-phase synthesis using methods known in the art.

[0105] Cyclization: Cyclization is thought to restrict the conformation of peptide binders to improve their activity against targets. If desired, the peptide is cyclized between two cysteine ​​groups via a disulfide bridge to form a cyclic structure and purified using the methods described herein. The peptide was incubated overnight in 10% DMSO, 90% Tris 0.1M pH 8 (final peptide concentration 1 mg / mL) to form a disulfide. The reaction was monitored by UPLC on a BEH130 C4 Acquity Waters column (2.1 × 100 mm, 1.7 µm) with gradients of 20%B-20%B (1 min), 20%B-60%B (4 min), and 60%B-80%B (0.2 min); flow rate: 0.4 mL / min; temperature: 45°C. The reaction was quenched with TFA, and DMSO was added until the peptide was completely dissolved, followed by purification.

[0106] HPLC Purification: Reverse-phase HPLC of the cyclized peptide was performed using a preparative HPLC Waters system with a C4 (Waters DeltaPak 200 × 20 mm, 300 Å, 15 μm) column and an appropriate linear gradient of gradually increasing concentrations of acetonitrile-water solution, 0.1% TFA (15%B-30%B over 20 min; 20%B-35%B over 20 min; 25%B-40%B over 20 min; flow rate 80 mL / min). Fractions containing the desired product were combined and lyophilized. Characterization was performed using linear gradients of acetonitrile aqueous solution: 20%B-20%B (1 min), 20%B-40%B (4 min), 40%B-80%B (0.2 min); 25%B-25%B (1 min), 25%B-45%B (4 min), 45%B-80%B (0.2 min); 30%B-30%B (1 min), 30%B-50%B (4 min), 50%B-80%B (0.2 min).

[0107] The sequences and structures of exemplary cyclic peptides are shown in Table 3 below. [Table 3] PepSP ID Sequences and Structures* PepSP1146 PepSP1147 PepSP1148 PepSP1149 PepSP1150 PepSP1151 PepSP1152 PepSP1153 PepSP1154 PepSP1155 PepSP1156 PepSP1157 PepSP1158 PepSP1159 PepSP1160 PepSP1161 PepSP1162 PepSP1163 PepSP1164 PepSP1165 PepSP1166 PepSP1167 PepSP1168 PepSP1169 PepSP1170 PepSP1171 PepSP1172 PepSP1173 PepSP1174 PepSP1175 PepSP1176 PepSP1177 PepSP1178 PepSP1179 PepSP1180 PepSP1181 PepSP1182 PepSP1183 PepSP1213 PepSP1214 PepSP1269 *: All peptides are cyclic, with a disulfide bond between the two Cys residues.

[0108] Combined kinetic determination using biolayer interferometry (BLI) and surface plasma resonance (SPR)

[0109] Binding kinetics were determined using biolayer interferometry (BLI). The recombinant DLL3 extracellular domain (rhDLL3-Fc) was biotinylated using the EZ-Link Sulfo-NHS-LC-LC-Biotin kit, according to the manufacturer's protocol (via Pierce, Thermo Fisher Scientific). The recombinant protein was incubated with excess NHS-LC-LC biotin ester at a 1:3 (protein:biotin) molar ratio at 25°C for 90 min. Unreacted biotin ester was removed by buffer exchange on a 0.5 ml Zeba rotary desalting column 7K MWCO (via Pierce, Thermo Fisher Scientific).

[0110] Biomolecular interaction analysis was performed using an Octet Red 96e instrument (Forte Bio). Biotinylated hDLL3 was diluted at 12.5 µg / ml in 1x kinetic buffer (Forte Bio) and captured for 20 min at a maximum of 8–10 nm on a streptavidin infusion and readout biosensor (Forte Bio), followed by blocking the sensor surface with 10 µg / ml biocytokinin for 60 s (using Invitrogen, Thermo Fisher Scientific). For assays, the peptide was diluted to final concentrations of 20, 4, and 0.8 µM in binding assay buffer (1x kinetic buffer supplemented with 5% DMSO), and binding to DLL3 was assessed using 90 s association followed by 120 s dissociation. Peptides showing positive binding were selected and tested within a dynamic range (10 x dissociation constant (KD) to 0.1 x KD), with dissociation increased to 300 s for better assessment of affinity and binding kinetic parameters (e.g., kon, koff, and KD). Sensing maps were analyzed using Data Analysis HT 11.1 software (Ford Biosciences): specific binding was obtained by subtracting the binding from the negative control. Kinetic parameters were measured using a global fit based on the 1:1 Langmuir binding isotherm equation.

[0111] The dissociation rate of peptides was screened using a Biacore T200 instrument (Cytiva, Sweden). Biotinylated rhDLL3-Fc was diluted at 12.5 µg / ml in HBS-P+ 1X buffer (Cytiva, Sweden) and then captured up to 6000 RU on an PBS-P+ 1X sensor chip (Cytiva, Sweden). The peptide was diluted at 300 nM in PBS-P+ 1X buffer (Cytiva, Sweden) supplemented with 1% DMSO and 0.1% BSA, and the ligand was injected at 30 µl / min for 5 minutes, followed by dissociation for 1 hour. The sensor map was analyzed using Biacore T200 evaluation software 3.0 (Cytiva, Sweden): specific binding was obtained by subtracting nonspecific binding to the naked biosensor and the zero analyte concentration prepared by binding assay buffer (dual reference). The kinetic parameters of koff were measured using a global fitting method based on the 1:1 Langmuir combined with the isothermal equation.

[0112] Flow Cytometry

[0113] HEK293-huDLL3, CHO-huDLL3, and their corresponding parental cell lines were separated using 2.5 mM EDTA and resuspended at 1.8 × 10⁶ cells / ml in FACS buffer (1XPBS, 2% FBS), dispensed in 500 μl test tubes, and centrifuged at 1300 rpm for 5 min in a Heraeus Multifuge X3R centrifuge. To measure the binding of fluorescently labeled peptides to hDLL3 on the cell surface, cells were resuspended in 500 μl FACS buffer in the presence of 100 nM AF647-labeled peptides (e.g., PepSP1215 or PepSP1216). In the competition assay, cells were resuspended in 500 μl of FACS buffer containing 30 nM peptide (e.g., PepSP1215), with increments of unlabeled peptides (e.g., PepSP1146, PepSP1270, PepSP1271, or PepSP1272) added from 500 μM to 4 nM. After incubation at room temperature for 1 hour, cells were washed once with FACS buffer, centrifuged, and resuspended in FACS buffer / 1% formaldehyde. Samples were obtained on a FACS ARIA flow cytometer (Becton Dickinson) and data were analyzed using FACS Express software.

[0114] Result

[0115] DLL3 binding properties: 35 peptides were tested. Of these, 23 peptides (PepSP1146, 1147, 1151, 1155, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1170, 1171, 1173, 1176, 1177, 1178, and 1182) showed dose-response saturation binding, which fit well according to the 1:1 Langmuir binding model. The remaining 12 showed poor binding (3) or heterogeneous binding (9) patterns. Generally, peptides with Kd in the micromolar range (e.g., greater than 1 μM) are considered poor binders, while peptides with Kd in the single-digit to double-digit nM range are considered good binders. Peptides that did not fit well to the 1:1 Langmuir binding model were considered to have heterogeneous binding. Twenty-three peptides were retested in a wider dose-response curve, ranging from approximately 10 x KD to 0.1 x KD, and kinetic parameters were determined using a global fitting formula based on the 1:1 Langmuir model. Affinity values ​​were obtained as koff to kon ratios. The characterized peptides showed overall affinity below 1 μM, and seven peptides (PepSP1146, 1178, 1163, 1161, 1147, 1171, and 1182) showed affinity in the double-digit nM range with residence times ranging from 60 to 180 seconds. These seven peptides were retested in three independent experiments. Data for the 23 peptides and PepSP1213, 1214, and 1269 are summarized in Table 4 below. [Table 4]: Binding Properties PEP ID KD(nM) RT* (seconds) 1146 68.9 ± 27.6 184.5 ± 7.8 1161 42.5 ± 10 87.8 ± 6 1178 146.8 ± 46.7 75.9 ± 9 1163 68.7 ± 18.4 166.8 ± 19.5 1171 70 ± 19 110.1 ± 32.4 1182 75.8 ± 15.5 96.6 ± 3.8 1147 59.0 ± 3.4 65.6 ± 6.8 1151 125 167 1155 155.2 177 1157 123 89 1158 643 31 1159 900 6 1160 494.5 47 1161 43 87.8 1162 159 51 1163 69 166.8 1164 125 125 1165 263 66 1166 144.6 48 1167 399 47 1168 568 44 1170 165.6 43 1173 105.6 101 1176 207 67 1177 236.5 52 1213 7498 1.36 1214 34.5 233 1269 181.1 20 RT: Detention Time

[0116] Peptides were functionalized at the N-terminus or C-terminus to evaluate their effects on DLL3 binding. N-terminal acetylation of peptides is ideal because it blocks the reactivity of the N-terminal amino group, which allows for a more direct synthetic process for further peptide modification (e.g., partial conjugation with radiolabeled groups). Surface plasma resonance (SPR) studies showed that acetylation of the N-terminal sequence or acetylation or amination of the C-terminus did not interfere with the peptide's binding properties.

[0117] Additionally, all N-terminal amino acids (AETVEF or AETVE) common to the unique sequence were deleted to assess their effect on binding. It was found that amino acids can play a role in the binding of some peptides to DLL3; however, not all peptides require amino acids to bind DLL3. See, for example, binding data for PepSP1213 and PepSP1214, and binding data for PepSP1161 and 1269. Example 3. Labeling, dimerization, and characterization of DLL3-binding peptides.

[0118] Materials and Methods

[0119] The following lists various exemplary reagents (e.g., linkers, click chemistry reagents, fluorescent agents) used in this series of experiments. These reagents are commercially available.

[0120] Linker / spacer region: trioxane-succinylamine (Ttds), Gly-Gly-Ttds, Gly-Gly-Ttds-K, Gly-Gly-Ttds-K(PEG4), Gly-Gly-Ttds-K(PEG3), Gly-Gly-Ttds-K(PEG4-PEG3), Gly-Gly-Ttds-K(Ttds-Ttds), Gly-Gly-Ttds-K(PEG4-DBCO), Gly-Gly-Ttds-K(Ttds-Ttds-PEG3), PEG4-DBCO, PEG linkers such as PEG3, PEG4, PEG6, bis-propargyl-PEG6, bis-propargyl-PEG14 and bis-propargyl-PEG18.

[0121] Click on chemical reagents: High propargyl glycine (hPra), Fmoc-HPra-OH, Fmoc-Lys(N3)-OH

[0122] Fluorescent agents: AFDye™ 647 dibenzocyclooctylene (DBCO), Cy5 DBCO

[0123] PepSP1396 and PepSP1342: At the end of the assembly of PepSP1324, Fmoc-N-amino-PEG4-acid (CAS 557756-85-1) was amided on the resin with 4 times excess using an equimolar amount of DIC and HOAt DMF solution as an activator, followed by Fmoc deprotection (20% piperidine DMF solution, 3 × 3 min) to obtain PepSP1396. Similarly, at the end of PepSP1324 assembly, Fmoc-Ttds-OH (CAS 17208914-4) was acetylated on the resin with a 4-fold excess using an equimolar amount of DIC and HOAt DMF solution as an activator, followed by Fmoc deprotection (20% piperidine DMF solution, 3 × 3 min); this treatment was repeated for the second Fmoc-Ttds-OH present in the sequence to obtain PepSP1342.

[0124] Fluorescent labeling: PepSP 1146 and 1171 were used for labeling and dimerization experiments. At the start of synthesis, the non-natural amino acid Fmoc-Lys(N3)-OH (CAS 159610-89-6) was amide-coated onto resin using standard methods, followed by amide-coated linker Fmoc-Ttds-OH, and peptides were synthesized using the same procedure described above. Next, the peptides were extended at the C-terminus with linker (Ttds) and Lys(N3) groups to generate peptide precursors PepSP1273 and PepSP1274 (see Table 5 below). The peptide precursors were then labeled with fluorescent agents, for example, using copper-free click chemistry with AlexaFluor647DBCO, to prepare PepSP1215 and PepSP1216.

[0125] Specifically, in order to label with AlexaFluor647, purified peptide precursors (final peptide concentration 30 mg / ml) were conjugated to AFDye™ 647 DBCO by incubation with 1.3 equivalents of Alexa-DBCO (dissolved in DMSO) (click Chemical Tools Catalog No. 1302). The overnight reaction was monitored by UPLC on a BEH300 C4 Acquity Waters column (2.1 × 100 mm, 1.7 μm) with gradients of 20%B-20%B (1 min), 20%B-90%B (4 min), 90%B-90%B (0.2 min), and 25%B-25%B (1 min), 25%B-45%B (4 min), and 45%B-95%B (0.3 min) (eluent: A = H2O + 0.1% TFA; B = CH3CN + 0.1% TFA); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45°C; MS: Waters Acquity ESI+, single quadrupole. The reaction was then diluted with DMSO and loaded onto a reverse-phase HPLC system using a Delta Pak C4 200 × 25 mm 300A 15 μm column with a linear gradient of 15%B-15%B-30%B over 20 min (eluent: A = H2O + 0.1% TFA; B = CH3CN + 0.1% TFA; flow rate: 50 mL / min; λ: 214 nm). Characterization was performed on a BEH300 C4 Acquity Waters 2.1 × 100 mm, 1.7 μm column with gradients of 25%B-25%B (1 min), 25%B-45%B (4 min), and 45%B-80%B (0.2 min).

[0126] Dimerization

[0127] It is believed that peptide dimerization can improve the binding properties of peptides due to, for example, increased affinity. To this end, peptide dimers were synthesized and tested. The precursor PepSP1273 was homodimerized using three linkers, namely dipropyne-PEG6, dipropyne-PEG14, and dipropyne-PEG18, each linker having two terminal alkyne functional groups, via copper-catalyzed click chemistry to prepare homodimers PepSP1270, 1271, and 1272. Specifically, 1 part of the peptide azide precursor (PepSP1273) was incubated with 0.5 molar equivalents of dipropyne-PEGx (x being 6, 14, or 16), 3 molar equivalents of CuSO4, and 5 molar equivalents of sodium ascorbate. The reaction was carried out in DMSO with the salt dissolved in water (water content < 10%), resulting in a final peptide concentration of 30 mg / mL. The reaction, completed after 5 min, was monitored by UPLC on a BEH300 C4 Acquity Waters column (2.1 × 100 mm, 1.7 µm) with gradients of 30%B-30%B (1 min), 30%B-50%B (4 min), and 50%B-90%B (0.2 min) (eluents: A = H₂O + 0.1% TFA; B = CH₃CN + 0.1% TFA); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45°C; MS: Waters Acquity ESI+, single quadrupole. The reaction was diluted with DMSO and TFA and loaded onto a preparative HPLC Waters system under the following conditions.

[0128] PepSP1270: Gradient: 20%B-20%B (5 min), 20%B-40%B over 25 min; Column: X Bridge C4 150 × 19 mm 300A 5 μm; Eluent: A = H2O + 0.1% TFA; B = CH3CN + 0.1% TFA; Flow rate: 20 mL / min; λ: 214 nm; PepSP1271: Gradient: 25%B-25%B (5 min), 25%B-45%B over 25 min; Column: Daisogel C4 200 × 20 mm 200A 5 μm; Eluent: A = H2O + 0.1% TFA; B = CH3CN + 0.1% TFA; Flow rate: 15 mL / min; λ: 214 nm nm; PepSP1272: Gradient: 20%B-20%B (5 min), 20%B-40%B over 25 min; Column: Delta Pak C4 200 × 25 mm 300A 15 μm; Eluent: A = H2O + 0.1% TFA; B = CH3CN + 0.1% TFA; Flow rate: 50 mL / min; λ: 214 nm.

[0129] To improve dimerization and labeling reactions, it is necessary to incorporate into the same linker molecule: a) the optimal form and b) the ability to directly conjugate with a radioactive moiety (e.g., 18F) for imaging or therapeutic purposes. To achieve this, trifunctionalized linkers were designed and synthesized using Scheme I as shown below.

[0130] Scheme I. Synthesis of propargyl trifunctionalized linkers for dimerization and labeling reactions

[0131] Starting with linker PEG18, trifunctionalized linkers were synthesized according to Scheme I and partially functionalized with DBCO or BCN. Then, using copper-free click chemistry, a radioactive isotope (e.g., 18F) was conjugated to the peptide using a linker with a DBCO or BCN motif. PepSP1384: The peptide azide precursor PepSP1274 (1 equivalent) was incubated with 0.5 equivalents of bis(propargyl)-PEG18, 2 equivalents of CuSO4, and 2 equivalents of sodium ascorbate. The reaction was carried out in DMSO with the salts dissolved in water (water content < 10%), resulting in a final peptide concentration of 20 mg / ml. The reaction, which was completed after 5 min, was monitored by UPLC on a BEH300 C4 Acquity Waters column (2.1 × 100 mm, 1.7 µm) with gradients of 30%B-30%B (1 min), 30%B-50%B (4 min), and 50%B-90%B (0.2 min) (eluent: A = H2O + 0.1% TFA; B = CH3CN + 0.1% TFA); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45°C; MS: Waters Acquity ESI+, single quadrupole. The reaction was diluted with DMSO and TFA and loaded onto a reverse-phase HPLC system using a Delta Pak C4 200 × 25 mm 300A 15 μm column with a linear gradient of 25%B–40%B over 20 min (eluent: A = H₂O + 0.1% TFA; B = CH₃CN + 0.1% TFA; flow rate: 50 mL / min; λ: 214 nm). Analysis and characterization were performed on a BEH300 C4 Acquity Waters 2.1 × 100 mm, 1.7 μm column with gradients of 30%B–30%B (1 min), 30%B–50%B (4 min), and 50%B–90%B (0.2 min); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45°C; MS: Waters Acquity ESI+, single quadrupole. Similarly, PepSP1321 and PepSP1324 were synthesized.

[0132] PepSP1343, PepSP1344, and PepSP1371 dimers were prepared using PepSP1318 as a precursor. Specifically, PepSP1318 was dimerized with a suitable PEG linker and a propargyl-tri-functionalized linker (see Scheme I) to prepare PepSP1343. PepSP1344 and PepSP1371 were prepared from PepSP1343 and a suitable linker derived with a DBCO or BCN group (PEG4-DBCO or PEG3-BCN).

[0133] Specifically, for PepSP1343, PepSP1318 (prep) (1 equivalent) was incubated with 0.6 equivalents of propargyl-trifunctionalized linker (synthesized according to Scheme III described below), and 1.9 equivalents of CuSO4 and 1.9 equivalents of sodium ascorbate were added to the solution. The reaction was carried out in DMSO with the salt dissolved in water (water content < 10%), and the final peptide concentration was 20 mg / ml. The reaction, which was completed after 5 min, was monitored by UPLC on a BEH300 C4 Acquity Waters column (2.1 × 100 mm, 1.7 µm) with gradients of 30%B-30%B (1 min), 30%B-50%B (4 min), and 50%B-90%B (0.2 min) (eluent: A = H2O + 0.1% TFA; B = CH3CN + 0.1% TFA); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45°C; MS: Waters Acquity ESI+, single quadrupole. The reaction was quenched with TFA, diluted with DMSO, and loaded onto a reverse-phase HPLC system using a Delta Pak C4 25×200 mm 300A 15µm column and a linear gradient: 25%B - 25%B (5 min), followed by 25%B - 40%B over 20 min (eluent: A = H2O + 0.1% TFA; B = CH3CN + 0.1% TFA; flow rate: 50 mL / min; λ: 214 nm). The analysis and characterization were performed on a BEH300 C4 Acquity Waters 2.1 × 100 mm, 1.7 μm column with gradients of 30%B - 30%B (1 min), 30%B - 50%B (5 min), and 50%B - 90%B (0.2 min); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45°C; MS: Waters Acquity ESI+, single quadrupole.

[0134] For PepSP1344, the dimer precursor PepSP1343 was incubated with 1.1 equivalents of DBCO-PEG4-NHS ester (BioDx Pharmaceuticals, MW: 649.7 Da) and 1% DIPEA was added. The reaction was carried out in DMSO to a final peptide concentration of 20 mg / ml. The reaction, which was completed after 1 hour, was monitored by UPLC on a BEH300 C4 Acquity Waters column (2.1 × 100 mm, 1.7 µm) with gradients of 35%B-35%B (1 min), 35%B-55%B (4 min), and 55%B-90%B (0.2 min) (eluents: A = H2O + 0.1% TFA; B = CH3CN + 0.1% TFA); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45°C; MS: Waters Acquity ESI+, single quadrupole. The reaction was quenched with TFA, diluted with DMSO, and loaded onto a reverse-phase HPLC system using a Waters Delta Pak C4 (200 × 25 mm 300A 15 μm) column and a linear gradient: 25% B-40% B over 20 min (eluent: A = H2O + 0.1% TFA; B = CH3CN + 0.1% TFA; flow rate: 50 mL / min; λ: 214 nm). Analytical characterization was performed on a BEH300 C4 Acquity Waters (2.1 × 100 mm, 1.7 µm) column with gradients of 35% B-35% B (1 min), 35% B-55% B (4 min), and 55% B-90% B (0.2 min) (eluents: A = H₂O + 0.1% TFA; B = CH₃CN + 0.1% TFA); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45°C; MS: Waters Acquity ESI+, single quadrupole. Similarly, PepSP1371 was synthesized using PepSP1343 as a precursor and BCN-PEG3-NHS ester (BioDer Pharmaceuticals, MW: 494.53 Da).

[0135] Further work was carried out to develop a new synthetic route that could be used as an alternative to Scheme I described above. The new scheme (Scheme II) is compatible with disulfide bridge formation and conjugation with radioactive ligands. First, using Scheme II, the precursor compounds PepSP1321 and PepSP1324 were synthesized, both of which have a spacer group Gly-Gly-Ttds at the C-terminus, which provides greater flexibility for the final dimer. Other precursor compounds, PepSP1396 and PepSP1342, having linkers GG-Ttds-K(PEG4)-CONH2 and GG-Ttds-K(Ttds-Ttds)-CONH2, respectively, were also synthesized.

[0136] Precursor compounds were used to synthesize compounds PepSP1462, PepSP1487, PepSP1488, and PepSP1489, which can be conjugated with radioactive isotopes such as 18F. Specifically, PepSP1462 was prepared using PepSP1324 and the reagent PEG4-DBCO; PepSP1487 was prepared using PepSP1324 and the NHS-trifunctionalized linker (Scheme II); PepSP1488 was prepared using PepSP1396 and the NHS-trifunctionalized linker (Scheme II); and PepSP1489 was prepared using PepSP1342 and the NHS-trifunctionalized linker (Scheme II).

[0137] Specifically, for PepSP1462, the precursor PepSP1324 (1 equivalent) was dissolved in DMSO, and 10 equivalents of DIPEA were added to the solution. Then, 1.1 equivalents of DBCO-PEG4-NHS (BioDx Pharmaceuticals, MW: 649.7 Da) was dissolved in DMSO and slowly added dropwise to the solution containing the peptide precursor to obtain a final peptide concentration of 30 mg / mL. The reaction was monitored by UPLC-MS on a BEH300 C4 Acquity Waters column (2.1 × 100 mm, 1.7 µm) with gradients of 30%B-30%B (1 min), 30%B-50%B (4 min), and 50%B-90%B (0.2 min) (eluent: A = H2O + 0.1% TFA; B = CH3CN + 0.1% TFA); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45°C; MS: Waters Acquity ESI+, single quadrupole. After 30 minutes, the reaction was completed and the sample was loaded onto a reverse-phase HPLC system using a DeltaPak C4 column, 40 × 200 mm, 300 Å, 15 μm, and a linear gradient: 15%B - 15%B (5 min), 15%B - 35%B (20 min), 35%B - 40%B (5 min) (eluent: A = H2O + 0.05% NH3; B = CH3CN; flow rate: 80 mL / min; λ: 214 nm). The analysis and characterization were performed on a BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm column with gradients of 30%B - 30%B (1 min), 30%B - 50%B (4 min), and 50%B - 90%B (0.2 min); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45°C; MS: Waters Acquity ESI+, single quadrupole.

[0138] For PepSP1487, 1488, and 1489, the precursors PepSP1324, 1396, and 1342 (1 equivalent) were dissolved in DMSO, and 10 equivalents of DIPEA were added to the solution. Then, 0.5 equivalents of NHS-trifunctionalized linker (Scheme 4) were dissolved in DMSO and slowly added dropwise to the solution containing the peptide precursors to obtain a final peptide concentration of 1 mg / mL. The reaction was analyzed by UPLC-MS on a BEH300 C4 Acquity Waters column (2.1 × 100 mm, 1.7 µm) with gradients of 30%B-30%B (1 min), 30%B-50%B (4 min), and 50%B-90%B (0.2 min) (eluents: A = H2O + 0.1% TFA; B = CH3CN + 0.1% TFA); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45°C; MS: Waters Acquity ESI+, single quadrupole. One night later, the reaction was completed. It was diluted 1:2 with H2O + 0.05% NH3 and loaded onto a reverse-phase HPLC system using an XBridge Protein BEH C4 column, 30×150 mm, 300 Å, 5 μm, and a linear gradient: 5%B - 5%B (5 min), 5%B - 25%B (20 min), 25%B - 30%B (5 min) (eluent: A = H2O + 0.05% NH3; B = CH3CN; flow rate: 80 mL / min; λ: 214 nm). The analysis and characterization were performed on a BEH300 C4 Acquity Waters 2.1 × 100 mm, 1.7 μm column with gradients of -30%B - 30%B (1 min), -30%B - 50%B (4 min), and -50%B - 90%B (0.2 min); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45°C; MS: Waters Acquity ESI+, single quadrupole.

[0139] DBCO derivatives PepSP1462, PepSP1487, PepSP1488 and PepSP1489 were finally conjugated with fluoroethyl azide (FEA) to obtain the final fluorinated compounds PepSP1538, PepSP1581, PepSP1582 and PepSP1583, as described below.

[0140] Synthesis of the monomeric fluoroethyl azide (FEA) derivative PepSP1538: One equivalent of the DBCO peptide precursor PepSP1462 was dissolved in DMSO at a final concentration of 20 mg / mL, and 1.2 equivalents of FEA were added to the reaction mixture. The reaction was monitored by UPLC-MS on a BEH300 C4 Acquity Waters column (2.1 × 100 mm, 1.7 µm) with gradients of 30%B-30%B (1 min), 30%B-60%B (4 min), and 60%B-90%B (0.2 min) (eluents: A = H2O + 0.1% TFA; B = CH3CN + 0.1% TFA); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45°C; MS: Waters Acquity ESI+, single quadrupole. After the reaction was complete, it was diluted with H2O + 0.05% NH3 at a ratio of 1:2 and loaded onto a reverse-phase HPLC system using a C4, 25 × 200 mm, 15 μm, 300A Waters Deltapak column and a linear gradient: 20%B - 20%B (5 min) to 35%B (20 min) (eluent: A = H2O + 0.05% NH3; B = CH3CN; flow rate: 80 mL / min; λ: 214 nm). Analytical characterization was performed on a BEH300 C4 Acquity Waters 2.1 × 100 mm, 1.7 μm column with gradients of 30%B - 30%B (1 min), 30%B - 60%B (4 min), and 60%B - 90%B (0.2 min); eluents: A: H2O + 0.1% TFA; B: AcN + 0.1% TFA; flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45°C; MS: Waters Acquity ESI+, single quadrupole.

[0141] Synthesis of dimer FEA derivatives PepSP1581, PepSP1582 and PepSP1583: 1 equivalent of dimer DBCO peptide precursor (PepSP1487, 1488 or 1489) was dissolved in DMSO at a final concentration of 1 mg / ml, and 1.2 equivalents of FEA were added to the reaction mixture. The reaction was monitored by UPLC-MS on a BEH300 C4 Acquity Waters column (2.1 × 100 mm, 1.7 µm) with gradients of 30%B-30%B (1 min), 30%B-60%B (4 min), and 60%B-90%B (0.2 min) (eluent: A = H2O + 0.1% TFA; B = CH3CN + 0.1% TFA); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45°C; MS: Waters Acquity ESI+, single quadrupole. After the reaction was complete, it was diluted with H2O + 0.05% NH3 at a ratio of 1:2 and loaded onto a reverse-phase HPLC system using a C4, 20 × 150 mm, 5 μm, 300A Waters XBridge column and a linear gradient: 10%B - 10%B (5 min) to 25%B (20 min) (eluent: A = H2O + 0.05% NH3; B = CH3CN; flow rate: 80 mL / min; λ: 214 nm). Analytical characterization was performed on a BEH300 C4 Acquity Waters 2.1 × 100 mm, 1.7 μm column with gradients of 30%B - 30%B (1 min), 30%B - 60%B (4 min), and 60%B - 90%B (0.2 min); eluents: A: H2O + 0.1% TFA; B: AcN + 0.1% TFA; flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45°C; MS: Waters Acquity ESI+, single quadrupole.

[0142] Scheme II. Synthesis of trifunctional linkers for dimerization and labeling reactions.

[0143] Scheme III below shows the synthesis scheme of propargyl-trifunctionalized linker.

[0144] The following steps were performed to synthesize the propargyl trifunctionalized linker (Scheme III). Step 1: 1.3 equivalents of Boc-Gly-OH (4530-20-5) were dissolved in DMF containing 1.3 equivalents of HATU, and after 5 minutes, the mixture was added to 1 equivalent of NH-bis(PEG4-acid) and DIPEA (3 equivalents) dissolved in DMF. The coupling reaction was completed after 15 minutes. The reaction was monitored by UPLC-MS. Step 2: HATU and DIPEA (2.3 equivalents) were added to the reaction mixture, stirred at room temperature, and after 5 minutes, 2 equivalents of propargyl-PEGx-amine dissolved in DMF were added. The reaction was stirred and monitored by UPLC-MS, then quenched with AcOH and concentrated to dryness under high vacuum. Step 3: The crude material was dissolved in TFA / H2O 95:5 and stirred at room temperature for 10 minutes, then concentrated to dryness. The crude material was purified by rapid RP chromatography using a Luknova C18 column (gradient (%B): 0% for 4 CV, 0% to 35% for 10 CV, 35% for 3 CV). A: H2O + 0.1% TFA; B: ACN + 0.1% TFA; λ: 214 nm; the collected fractions were lyophilized. UPLC-MS: Acquity BEH C18, 2.1 × 100 mm, 1.7 μm, 130A, flow rate: 0.4 mL / min; gradient (%B): 20% over 1 min, 20% to 70% over 4 min; A: H2O + 0.1% TFA; B: ACN + 0.1% TFA; λ: 214 nm).

[0145] Scheme 4 below illustrates the synthesis of NHS-trifunctionalized linkers.

[0146] The following steps were performed to synthesize the NHS-trifunctionalized linker (Scheme 4): Step 1: 1 equivalent of NH-bis(PEG3-acid) HCl salt (Borde Pharmaceuticals, 425.47 Da) and 1.2 equivalents of Boc-Gly-OSu (3392-07-2) were dissolved in DCM and 2 equivalents of DIPEA were added; the reaction was monitored by UPLC-MS and completed after 15 minutes. Purification was performed by rapid chromatography using a Luknova SuperSep HP 25 g column (gradient: (%B): 0% CV for 2, 0% to 15% CV for 8, 15% CV for 2). A: DCM + 0.2% acetic acid; B: MeOH + 0.2% acetic acid; λ: 206 nm; the collected fractions were lyophilized. UPLC-MS: Acquity BEH C18, 2.1 × 100 mm, 1.7 μm, 130A, flow rate: 0.4 mL / min; gradient (%B): 5%B-5%B (1 min), 5%B-95%B (4 min); A: H2O + 0.1% TFA; B: CH3CN + 0.1% TFA; λ: 214 nm). Step 2: The purified product was dissolved in TFA / DCM 20:80, stirred for 10 min, and then concentrated to dryness. The product was dissolved in DCM, 3 equivalents of DIPEA were added, followed by 1 equivalent of DBCO-PEG4-NHS ester (Bode Pharmaceuticals, MW: 649.7 Da), stirred for 15 min, and monitored by UPLC-MS. Step 3: Add 3 equivalents of TEA to 1 equivalent of the product dissolved in DCM; add 3 equivalents of N,N'-disuccinimidyl carbonate (Borde Pharmaceuticals, 256.17 Da) dissolved in DMF to react in a DCM:DMF 1:1 ratio; stir the reaction for 1 hour and monitor by UPLC-MS. Load the fraction onto a reverse-phase HPLC and purify using a Waters XBridge C18 (50 × 150 mm, 130 A, 5 μm) column with the following linear gradient: 30%B-30%B (5 min), 30%B-50%B (20 min); eluent: A = H2O + 0.1% TFA; B = CH3CN + 0.1% TFA; flow rate: 80 mL / min; λ: 214 nm; freeze-dry the collected fractions.Analytical characterization was performed on an Acquity BEH C18, 2.1 × 100 mm, 1.7 μm, 130A, at a flow rate of 0.4 mL / min; gradient (%B): 5-B - 5%B (1 min), 5-B - 95%B (4 min); A: H₂O + 0.1% TFA; B: CH₃CN + 0.1% TFA; λ: 214 nm; temperature: 45°C; MS: Waters Acquity ESI+, single quadrupole.

[0147] The sequences and structures of the labeled peptides and / or dimerized peptides are shown in Table 5 below. [Table 5] Sequences and structures of labeled peptides and dimerized peptides PEP ID Sequences and Structures PepSP1273 PepSP1215 PepSP1274 PepSP1216 PepSP1270 PepSP1271 PepSP1272 PepSP1318 PepSP1343 PepSP1344 PepSP1371 PepSP1384 PepSP1321 PepSP1324 PspSP1396 PspSP1342 PspSP1462 PspSP1487 PspSP1488 PspSP1489 PepSP1538 PepSP1581 PepSP1582 PepSP1583

[0148] Using cells expressing human DLL3, the binding of peptides containing various linkers and / or fluorescently labeled peptides to DLL3 was tested by BLI, SPR, or flow cytometry. The binding properties of the peptides are summarized in Table 6 below. As shown in the table below, the addition of linkers and / or fluorescent agents did not negatively affect the binding to DLL3. Fluorescently labeled peptides were also found to specifically bind to the DLL3 protein expressed on the surface of cells (CHO cells); see Figure 1. [Table 6]. Binding Characteristics PepSP ID Kd RT 1215 11.6 681 1274 21.4 254 1321 36.8 369 1324 29±16 189.3 ± 73 1342 42±8 198.9 ± 41.1

[0149] Testing the binding of peptide dimers to DLL3. As shown in Table 7 below, dimerization significantly increased the stability of the complex, resulting in a 30 to 100-fold more stable dissociation rate. Dimers prepared using the bis-PEG18 linker showed the greatest improvement compared to the corresponding monomers. Dimeric peptides PepSP1270, 1271, and 1272 were also able to interact better with native huDLL3 on the surface of cells (e.g., CHO cells) than the corresponding monomer PepSP1146, as evidenced by their higher efficiency in competitively binding AF647-labeled PepSP1146. The peptide dimerized via the bis-PEG18 linker (PepSP1172) showed the most efficient binder. The data in Table 7 below are kinetic parameters measured from curve fitting of the peptide's SPR sensing plot. [Table 7]. Binding properties of peptide dimers Pep-ID connector KD(nM) kon (1 / Ms) koff (1 / s) IC50 PepSP1270 Bis-PEG6 3 7.43E+04 2.4E-04 15.3 PepSP1271 Bis-PEG14 3 1.04E+05 2.8E-04 10 PepSP1272 Bis-PEG18 0.61 9.0E+04 5.5E-05 7.2 PepSP1146 n / a 8.7E+04 5.4E-03 8.7E+04 76

[0150] Binding data for the dimer peptides PepSP1344, 1371, 1384, 1462, 1487, 1488, and 1489 are shown in Table 8 below. The data in the table were measured by curve fitting of the peptide's SPR sensing plot. The data show that dimerization significantly improves binding with DLL3. [Table 8]. Binding properties of the selected dimer peptides with their corresponding monomeric peptides. Pep ID KD(nM) Kon (1 / Ms) koff (1 / s) PepSP1146 68.9 8.7E+04 5.4E-03 PepSP1344 6 1.8E+05 1.0E-03 PepSP1371 2.4 3.0E+05 5.7E-04 PepSP1171 70 1.4E+05 9.7E-03 PepSP1384 4.0 1.8E+05 6.6E-04 PepSP1462 6.3 2.3E+05 1.4E-03 PepSP1487 5.5 7.6E+04 4.2E-04 PepSP1488 2.6 1.2E+05 3.1E-04 PepSP1489 3.3 1.1E+05 3.7E-04

[0151] Four fluorescently labeled conjugates (PepSP1538, PepSP1581, PepSP1582, and PepSP1583) were tested at a single site by SPR binding assays of rhDLL3-Fc, and the complex stability (koff) of each molecule was measured. The results showed that the peptides acting as tight rhDLL3-Fc binders had stable complex half-lives (Table 9). [Table 9] Binding properties of labeled peptides Pep ID koff ± SE (1 / s) PepSP1538 3.9 ± 0.05 E-03 PepSP1581 3.4 ± 0.02 E-05 PepSP1582 3.6 ± 0.09 E-05 PepSP1583 3.2 ± 0.08 E-05 Example 4. Binding and cross-reactivity of DLL3-binding peptides

[0152] Materials and Methods: Cells expressing DLL3 used in the experiments included CHO cells expressing DLL3 from humans (hu), rhesus macaques (cyno), mice (mu), and rats. CHO DHFR- cells and CHO cells expressing human FLT3 were used as negative controls. DLL3 target expression was analyzed by flow cytometry. DLL3-expressing CHO cells or DLL3-negative CHO cells listed above were suspended in FACS buffer (1x PBS + 1% fetal bovine serum) and incubated at 4°C with 10 mg / ml AMG 757 for 40 min. Cells were then washed twice in FACS buffer and incubated with an anti-human IgG Fcg antibody labeled with allophycocyanin at 4°C for 20 min. DLL3 cell surface expression was analyzed using an LSR Fortessa flow cytometer (Bettson-Dickinson) and FACSDiva software (Bettson-Dickinson). The table below summarizes the percentage of CHO cells expressing DLL3 from different species. cell lines CHO huDLL3 CHO cynoDLL3 CHO muDLL3 High CHO rtDLL3 Low CHO rtDLL3 CHO DHFR- CHO huFTL3 DLL3 positive cells (%) 99.7 98.4 99.5 98.6 98.6 12.3 3.3

[0153] The peptide PepSP1462 identified in this study was labeled with biotin. The biotin-labeled peptide was detected using an anti-streptavidin antibody conjugated with AF488. The AF488-labeled peptide was also detected directly.

[0154] The binding of the labeled PepSP1462 peptide to DLL3-expressing cells was tested by flow cytometry. The binding specificity of the labeled peptide to DLL3 was assessed using DLL3-negative cell lines. DLL3-expressing and DLL3-negative cells were incubated at 4°C for 30 min in the absence or presence of 100 μM PepSP1462-biotinylated peptide. Cells were then washed twice with FACS buffer and incubated with an anti-streptavidin antibody conjugated to AF488 (Thermo Fisher). As a control, cells were incubated only with an anti-streptavidin-Alexa Fluor 488 antibody. Cells were then washed twice with FACS buffer and resuspended in FACS buffer containing 0.5 μg / ml propidium iodide. Cells were analyzed by flow cytometry using LSR Fortessa and FACSDiva software (Bottom-Dickinson) or Flow Jo (Flow Jo LLC).

[0155] Figure 3 shows that PepSP1462, labeled with biotin and detected with anti-streptavidin-AF488 antibody, binds to CHO cells expressing DLL3 from humans, mice, rhesus monkeys, or rats, but not to CHO cells untransfected with DLL3 (CHO huFLT3). The binding of the labeled peptide to DLL3 expressed on cells is shown as a histogram shift to the right of the negative control cell line. These data also indicate that PepSP1462 cross-reacts with DLL3 from other species.

[0156] This specification will be understood most thoroughly based on the teachings of the references cited herein. The embodiments described herein provide an illustration of implementations of the invention and should not be construed as limiting the scope of the invention. It will be readily apparent to those skilled in the art that the invention encompasses many other embodiments. All disclosures, patents, and sequences referenced in this disclosure are incorporated herein by reference in their entirety. Where material incorporated by reference conflicts or is inconsistent with this specification to some extent, this specification shall supersede any such material. Any reference cited herein is not an admission that such reference is prior art to this invention.

[0157] Those skilled in the art will recognize, or be able to determine, many equivalents of the specific embodiments of the invention described herein using only conventional experiments. Such equivalent embodiments are intended to be covered by the following embodiments. [Simplified Explanation of the Diagram]

[0019] [Figure 1] shows the binding of fluorescently labeled peptides PepSP1215 (left) and PepSP1216 (right) to cells expressing hDLL3.

[0020] Figure 2 shows the huDLL3 expression in CHO cells engineered to represent human DLL3 and in the negative control untransfected CHO cells used in Example 4.

[0021] Figure 3 shows the binding of biotin-labeled PepSP1462 to CHO cells that express or do not express DLL3. [Biomaterial Storage]

[0159] None

Claims

1. A polypeptide comprising an amino acid sequence selected from the following: a) C-X1-X2-X3-X4-X5-X6-X7-X8-C (SEQ ID NO: 78), wherein X1 is Y, H, T, K, S, W, D, E, L, N, Q, or R; X2 is G, W, Y, M, T, or V; X3 is D, N, Y, T, A, E, G, or S; X4 is W, A, E, S, V, Y, D, G, N, P, Q, R, or T; X5 is D, E, G, Y, N, W, K, R, or S; X6 is E, D, G, N, T, A, Q, or V; X7 is W, Y, V, E, or S; and X8 is T, G, A, or S; or b) SEQ ID NO:

6.

2. The polypeptide as claimed in claim 1, wherein a) X1 is Y, H, T, W or N; X2 is G; X3 is D, N or T; X4 is W, A, S, N, R or T; X5 is D, E, G, Y, N or S; X6 is E, D or N; X7 is W, Y or E; and X8 is T.

3. The polypeptide as claimed in claim 1, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 1-23.

4. The polypeptide as claimed in claim 1 or 2, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 1-7.

5. The polypeptide as claimed in any one of claims 1-4, wherein the amino acid sequence further comprises an N-terminal amino acid residue AETVE or AETVE of the amino acid sequence.

6. The polypeptide as claimed in claim 5, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 39-61.

7. The polypeptide as claimed in claim 5 or 6, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 39-45.

8. A polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 1-38.

9. The polypeptide as claimed in claim 8, wherein the amino acid sequence further comprises an amino acid residue AETVE or AETVE at the N-terminus of the amino acid sequence.

10. The polypeptide as claimed in claim 9, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 39-76, preferably SEQ ID NO: 39-61, and more preferably SEQ ID NO: 39-45.

11. The polypeptide as claimed in any one of claims 1-10, wherein the polypeptide is modified at the N-terminus, C-terminus, or both.

12. The polypeptide as claimed in claim 11, wherein the amino acid residue at the N-terminus is acetylated.

13. The polypeptide as claimed in claim 11 or 12, wherein the C-terminus of the polypeptide is amide- or amination-treated.

14. The polypeptide as claimed in any one of claims 1-13, wherein the polypeptide comprises a dimer of an amino acid sequence.

15. The polypeptide as claimed in claim 14, wherein the dimer system is a homodimer.

16. The polypeptide as claimed in claim 14 or 15, wherein the dimer comprises a first linker linking two amino acid sequences.

17. The polypeptide as claimed in any one of claims 1-16, wherein the polypeptide further comprises a detectable agent.

18. The polypeptide as claimed in claim 17, wherein the detectable agent is linked to the polypeptide via a second linker, a chelating agent, or a combination thereof.

19. The polypeptide as described in any one of claims 16-18, wherein the first linker or the second linker is independently a peptide linker or a non-peptide linker.

20. The polypeptide as claimed in claim 19, wherein the first linker or the second linker comprises a non-natural amino acid.

21. The polypeptide as claimed in claim 20, wherein the first linker or the second linker independently comprises a polyethylene glycol (PEG) linker.

22. The polypeptide as claimed in claim 21, wherein the PEG linker comprises PEG2, PEG3, PEG4, PEG6, bis-PEG18, bis-PEG16, bis-PEG14, bis-PEG12, bis-propargyl-PEG2, bis-propargyl-PEG6, bis-propargyl-PEG14, bis-propargyl-PEG18, or combinations thereof.

23. The polypeptide as claimed in any one of claims 16-22, wherein the first linker further comprises hPra, Lys(N)3, trioxane-tetrazine-succinamine (Ttds), Gly-Gly, or combinations thereof.

24. The polypeptide as claimed in any one of claims 18, 19 or 21, wherein the second linker comprises a bicyclic [6.1.0]nonyne (BCN) group or a dibenzocyclooctyne (DBCO) group.

25. The polypeptide as claimed in claim 18, wherein the chelating agent is DOTA, TETA, DFO, NOA, DTPA, HOPO, or Macropa.

26. The polypeptide as claimed in claim 17, wherein the detectable agent comprises a fluorescent agent or a radioisotope.

27. The polypeptide as claimed in claim 26, wherein the fluorescent agent is Cy3, Cy5, fluorescein isothiocyanate (FITC), o-aminobenzoyl, 2-aminobenzoyl (Abz), 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), carboxytetramethylrosin (TAMRA), 5-(dimethylamino)naphthyl-1-sulfonylurea (dansylsulfonylurea), 5-[(2-aminoethyl)amino]naphthyl-1-sulfonic acid (EDANS), or 7-methoxycoumarinyl-4-acetylurea (Mca).

28. The polypeptide as claimed in claim 26, wherein the radioisotope is 67Ga, 99mTc, 111In, 68Ga, 64Cu, 44Sc, 86Y, 89Zr, 18F, 125I, 123I, 124I or 203Pb.

29. The polypeptide as claimed in claim 28, wherein the radioisotope is 18F.

30. The polypeptide as claimed in claim 26, wherein the radioisotope is 47Sc, 114mIn, 177Lu, 90Y, 212 / 213Bi, 212Pb, 225Ac, 186 / 188Re, 67Cu, 131I, 227Th, 211At or 90Y.

31. The polypeptide as described in any one of claims 1-30, wherein the polypeptide binds DLL3.

32. The polypeptide as claimed in claim 31, wherein the polypeptide binds to human DLL3 expressed on the cell surface.

33. A pharmaceutical composition comprising a polypeptide as described in any one of claims 1-32.

34. The pharmaceutical composition as claimed in claim 33, wherein the composition further comprises N-tert-butyl-α-phenylnitrone (PBN), ethanol, sodium ascorbate, gentianic acid, or combinations thereof.

35. The pharmaceutical composition as described in claim 33 or 34, wherein the pH of the composition is from 4.5 to 8.

0.

36. A method for detecting DLL3 in a sample, the method comprising contacting the sample with a polypeptide as described in any one of claims 17-32 or a pharmaceutical composition as described in any one of claims 33-35, and detecting DLL3 in the sample.

37. The method as described in claim 36, wherein the sample contains cells representing DLL3.

38. The method as described in claim 36 or 37, wherein the DLL3 is DLL3.

39. The method of claim 38, wherein the cell is in a subject, and the method includes administering the polypeptide or the pharmaceutical composition to the subject and detecting DLL3 in the subject using imaging techniques.

40. The method as described in claim 39, wherein the subject is a person suffering from a tumor or cancer that expresses DLL3.

41. The method as described in claim 39 or 40, wherein the tumor or cancer is small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), glioma, glioblastoma, melanoma, neuroendocrine prostate cancer, neuroendocrine pancreatic cancer, hepatoblastoma, large cell pulmonary neuroendocrine carcinoma, pancreatic neuroendocrine carcinoma, bladder neuroendocrine carcinoma, gastric neuroendocrine carcinoma, adrenal exocrine tumor, Merkel cell carcinoma, neuroblastoma, head and neck carcinoid or neuroendocrine carcinoma, head and neck paraganglioma or cervical small cell neuroendocrine carcinoma.

42. The method as described in any one of claims 39-41, wherein the imaging technique is positron emission tomography (PET) scanning.

43. A method for treating a tumor or cancerous disease exhibiting DLL3, the method comprising administering to a subject in need a polypeptide as described in claim 30 or a pharmaceutical composition as described in any one of claims 33-35.

44. The method as described in claim 43, wherein the subject is a human being.

45. The method as described in any one of claims 38-44, wherein the administration is intravenous.

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