DLL3-binding proteins and uses thereof

Abstract

The invention disclosed herein is directed to polypeptides that bind to DLL3 protein and their use for tumor / cancer diagnosis and treatment.

Description

[Technical field]

[0001] Submission of sequence listing as an ASCII text file The contents of the following ASCII text file submission are incorporated by reference in their entirety into this specification: Sequence Listing in Computer Readable Format (CRF) (Filename: A-2889-WO01-SEC_SeqListing.xml, Data Creation Date: December 8, 2022, Size: 73,728 bytes).

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 291,537, filed December 20, 2021, the contents of which are incorporated by reference herein in their entirety.

[0003] The invention disclosed herein relates to polypeptides that bind to Delta-like ligand 3 (DLL3) and their use for the diagnosis and treatment of tumors or cancer. [Background technology]

[0004] Delta-like ligand 3 (DLL3) is a type 1 transmembrane protein and a non-canonical Notch ligand. DLL3 is a promising target for the development of therapeutics due to its high expression on the cell surface of neuroendocrine tumors and minimal, mainly cytoplasmic localization in normal tissues. Owen et al., J Hematol Oncol., 12:61 (2019). Therapeutic agents against DLL3 have been developed in recent years for the treatment of DLL3-expressing tumors or cancers. See, for example, WO 2017 / 021349. Labeled (e.g., radiolabeled) small polypeptides are a class of pharmaceutical compounds used in tumor or cancer diagnosis and therapy. See, e.g., Christine Rangger and Roland Haubner, Pharmaceuticals, 13(2):22 (2020). Compared to antibodies, these polypeptides are non-immunogenic and exhibit fast diffusion and target localization. Additionally, peptides can be easily modified to improve metabolic stability and pharmacokinetics. Labeled small polypeptides also allow for non-invasive detection of tumor / cancer cells and monitoring of disease burden during or after treatment when used in tumor / cancer diagnosis. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Owen et al.,J Hematol Oncol.,12:61(2019) [Non-Patent Document 2] Christine Langger and Roland Haubner,Pharmaceuticals,13(2):22(2020) Summary of the Invention [Problem to be solved by the invention]

[0006] There have been no reports of polypeptides capable of binding to DLL3 and that can be used in cancer diagnosis and therapy. There is a need in the art for such compounds. [Means for solving the problem]

[0007] The invention disclosed herein is directed to polypeptides that bind to human DLL3 protein and their use in tumor / cancer diagnosis and therapy. It is demonstrated that the DLL3-binding peptides described herein can be dimerized and / or functionalized to conjugate 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.

[0008] Thus, in one aspect the invention provides a polypeptide comprising an amino acid sequence selected from C-X1-X2-X3-X4-X5-X6-X7-X8-C, where Xi 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).

[0009] In another aspect, the invention provides a polypeptide comprising an amino acid sequence selected from C-X1-X2-X3-X4-X5-X6-X7-X8-C, where Xi 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).

[0010] In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-23; any one of SEQ ID NOs: 1-7; any one of SEQ ID NOs: 39-61; or any one of SEQ ID NOs: 39-45.

[0011] In another aspect, the present invention provides a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 38. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 39 to 76, preferably SEQ ID NOs: 39 to 61, and more preferably SEQ ID NOs: 39 to 45.

[0012] In some embodiments, the amino acid sequences described herein further comprise the amino acid residue AETVEF or AETVE at the N-terminus of the amino acid sequence. In some embodiments, the polypeptides described herein are 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 aminated or amidated.

[0013] In some embodiments, the polypeptides described herein comprise a dimer of the amino acid sequence of SEQ ID NOs: 1-23, e.g., any one of the sequences for a homodimer or any two of the sequences for a heterodimer. In some embodiments, the polypeptides described herein comprise a dimer of the amino acid sequence of SEQ ID NOs: 1-7, or SEQ ID NOs: 24-38, or SEQ ID NOs: 39-76, or SEQ ID NOs: 39-61, or SEQ ID NOs: 39-45. In some embodiments, the dimer is a homodimer, e.g., any one of the sequences of SEQ ID NOs: 39-61 or SEQ ID NOs: 39-45. In some embodiments, the dimer comprises a first linker linking the two amino acid sequences.

[0014] In some embodiments, the polypeptides described herein further comprise a detectable agent. In some embodiments, the detectable agent comprises 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 attached to the polypeptide via a second linker and / or a chelator. In some embodiments, the chelator is DOTA, TETA, DFO, NOTA, DTPA, HOPO, or Macropa.

[0015] In some embodiments, the first linker or the second linker independently comprises a peptide linker or a non-peptide linker. In some embodiments, the first linker or the second linker comprises a non-natural amino acid. In some embodiments, the first linker or the 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, trioxatridecane-succinamic acid (Ttds), Gly-Gly, or a combination thereof. In some embodiments, the second linker comprises a PEG linker. In some embodiments, the second linker further comprises a bicyclo[6.1.0]nonyne (BCN) group or a dibenzocyclooctyne (DBCO) group.

[0016] In some embodiments, the polypeptides described herein bind to DLL3 (eg, human DLL3 expressed on the surface of a cell).

[0017] In another aspect, the present invention provides a pharmaceutical composition comprising a polypeptide described herein.

[0018] In another aspect, the present invention provides a method for detecting DLL3 in a sample, the method comprising contacting the sample with a polypeptide or pharmaceutical composition described herein and detecting DLL3 in the sample.

[0019] In some embodiments, the sample comprises cells. In some embodiments, the cells are in a subject, and the method comprises administering a polypeptide or pharmaceutical composition described herein to the subject and detecting DLL3 in the subject using an imaging technique (e.g., a positron emission tomography (PET) scan). In some embodiments, the subject is a human having a DLL3-expressing tumor or cancer. 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 lung 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.

[0020] In another aspect, the invention provides a method of treating a DLL3-expressing tumor or cancer, the method comprising administering to a subject a polypeptide or pharmaceutical composition described herein. In some embodiments, the subject is a human. [Brief description of the drawings]

[0021] [Figure 1] Binding of fluorescently labeled peptides PepSP1215 (left) and PepSP1216 (right) to cells expressing hDLL3 is shown. [Diagram 2] 1 shows huDLL3 expression in CHO cells engineered to express human DLL3 and negative control non-transfected CHO cells used in Example 4. [Diagram 3] Binding of biotin-labeled PepSP1462 to CHO cells expressing or not expressing DLL3 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The invention disclosed herein is directed to polypeptides that bind to human DLL3 protein and their use for tumor / cancer diagnosis and treatment. It is demonstrated that the DLL3-binding peptides described herein can be dimerized and / or functionalized for conjugation to radioisotopes as DLL3-targeted radiotracers to allow non-invasive detection of DLL3-expressing tumor cells and monitoring of disease burden following treatment with various modalities (e.g., DLL3-targeted immunotherapy). Thus, a polypeptide is provided that comprises an amino acid sequence of 8-13 (e.g., 8, 9, 10, 11, 12, or 13) amino acid residues flanked by two cysteine ​​(Cys) residues. In certain embodiments, the number of amino acid residues between the two Cys residues is 8, 9, or 13. In certain embodiments, the polypeptide further comprises additional amino acid residues at the N- or C-terminus or both of the amino acid sequence, for example, the polypeptide further comprises the amino acid residue AETVEF or AETVE at the N-terminus of the amino acid sequence. The polypeptides disclosed herein are identified by screening a Cys-restricted peptide phage library for binding to DLL3 (eg, human DLL3).

[0023] In one aspect, the present invention provides a peptide comprising a) C-X1-X2-X3-X4-X5-X6-X7-X8-C (SEQ ID NO: 80), where 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; and X4 is W, A, Y, G, S, E, Q, 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).

[0024] In one aspect, the present invention provides a compound represented by the formula: a) C-X1-X2-X3-X4-X5-X6-X7-X8-C, where 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; and X5 is D, E, G, Y, N , 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).

[0025] definition As used herein, the term "polypeptide" or "peptide" refers to a molecule of two or more amino acids linked by peptide bonds. A polypeptide as described herein typically contains 5-60 (e.g., 8-50) amino acids. A polypeptide may further form multimers, such as dimers, trimers and higher oligomers, i.e., may consist of two or more polypeptide molecules. The polypeptide molecules forming such dimers, trimers, etc. may be identical or non-identical. The corresponding higher structures of such multimers are consequently referred to as homo- or heterodimers, homo- or heterotrimers, etc. The terms "peptide" and "polypeptide" refer to naturally modified peptides / polypeptides, where the modification occurs, for example, by post-translational modification, such as glycosylation, acetylation, phosphorylation, etc. A "peptide" or "polypeptide", as referred to herein, may be modified by chemical modification, such as, for example, amidation, amination, or pegylation at the N- and / or C-terminus, or may be cyclic (e.g., via a disulfate bond). Such modifications are well known in the art and are described herein below.

[0026] The term "amino acid" or "amino acid residue" typically refers to amino acids selected from the group consisting of the building blocks of proteins, such as alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (Ile 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 if desired. In general, amino acids can be classified as having nonpolar side chains (e.g., Ala, Cys, Ile, Leu, Met, Phe, Pro, Val); negatively charged side chains (e.g., Asp, Glu); positively charged side chains (e.g., Arg, His, Lys); or uncharged polar side chains (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp and Tyr).

[0027] The term "subject in need" or "subject in need of treatment" includes subjects already with the disorder as well as subjects in which the disorder is to be prevented. A subject in need or a "patient" includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0028] The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Treatment includes the application or administration of a polypeptide as described herein to the body, isolated tissues or cells of a patient having a disease / disorder, a symptom of a disease / disorder, or a predisposition to a disease / disorder, with the intent to cure, heal, relieve, alleviate, alter, remedy, ameliorate, ameliorate or affect the disease, symptom of a disease, or predisposition to a disease.

[0029] As used herein, the term "amelioration" refers to any improvement in the disease state of a patient with a tumor or cancer or metastatic cancer as defined herein below, by administration of a polypeptide according to the present invention to a subject in need thereof. Such improvement can also be considered as a slowing down or halting of the progression of the tumor or cancer or metastatic cancer in the patient.

[0030] As used herein, the term "prevention" refers to the avoidance of onset or recurrence in a patient with a tumor or cancer or metastatic cancer as defined herein below, by administration of a polypeptide according to the present invention to a subject in need thereof. The term "disease" refers to any pathological condition that would benefit from treatment with a polypeptide or pharmaceutical composition as described herein. This includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disease in a subject.

[0031] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to a "reagent" includes one or more of such various reagents, and reference to a "method" includes reference to equivalent steps and methods known to those of skill in the art that may be modified for or substituted for the method described herein.

[0032] Unless otherwise indicated, the term "at least" preceding a series of elements shall be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

[0033] The term "and / or", wherever used in this specification, includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0034] As used herein, the term "about" or "approximately" means within ±20%, preferably within ±15%, more preferably within ±10%, and most preferably within ±5% of a given value or range.

[0035] Throughout this specification and the claims which follow, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprise" can also be replaced with the terms "containing" or "including" or, when used herein, the term "having."

[0036] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0037] In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with one of the other two terms.

[0038] DLL3-binding polypeptides DLL3 is a non-canonical Notch ligand that functions cell-autonomously to inhibit Notch signaling in cells, binding to Notch in cis and thus blocking cell-cell interaction and internalization of Notch in target cells, which is a hallmark of canonical Notch signaling. The main role of DLL3 is somitogenesis during embryonic development. Mice with DLL3 knockout show somitic defects in the axial skeleton as well as cranial and neural development. Somatic patterning defects are also seen in humans with certain germline DLL3 mutations, resulting in a condition called spondylocostal dysostosis (Bulman MP et al. Nature Genetics 24:438-441 (2000)).

[0039] DLL3 is a promising target for therapeutic development due to its high cell surface expression in neuroendocrine tumors / cancers and minimal, mainly cytoplasmic localization in normal tissues (Owen et al., J Hematol Oncol., 12:61 (2019)). Neuroendocrine tumors / cancers typically begin in neuroendocrine cells and can arise in organs such as the lung, appendix, small intestine, rectum, and pancreas.

[0040] Provided herein are polypeptides that bind to human DLL3 protein (e.g., SEQ ID NO: 77) and uses thereof for the diagnosis and treatment of tumors / cancers, such as neuroendocrine tumors / cancers. In one aspect, the present invention provides a polypeptide comprising: a) C-X1-X2-X3-X4-X5-X6-X7-X8-C, where 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; and X5 is D, E, G, Y, N, 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).

[0041] In another aspect, the invention provides a polypeptide comprising an amino acid sequence selected from C-X1-X2-X3-X4-X5-X6-X7-X8-C, where Xi 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).

[0042] In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-38, any one of SEQ ID NOs: 1-23, any one of SEQ ID NOs: 1-7, any one of SEQ ID NOs: 39-61, or any one of SEQ ID NOs: 39-45 as shown in Table 1 below. In some embodiments, the polypeptide described herein comprises, for example, amino acid residues AETVEF or AETVE at the N-terminus of the amino acid sequence as shown in Table 1. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 39-76, preferably SEQ ID NOs: 39-61, or more preferably SEQ ID NOs: 39-45 as shown in Table 1.

[0043] [Table 1-1]

[0044] [Table 1-2]

[0045] [Table 1-3]

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

[0047] In some embodiments, the polypeptides described herein comprise dimers of the amino acid sequences listed in Table 1. In some embodiments, the dimers are homodimers (e.g., polypeptides comprising a pair of particular amino acid sequences listed in Table 1) or heterodimers (e.g., polypeptides comprising two different amino acid sequences listed in Table 1). In some embodiments, the polypeptides comprise homodimers of any of SEQ ID NOs: 1-38, or any one of SEQ ID NOs: 39-76, or any one of SEQ ID NOs: 39-61, or any one of SEQ ID NOs: 39-45.

[0048] In some embodiments, the polypeptides 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 hepatic biliary metabolism and clearance). For example, cyclization and modifications at the N- and / or C-terminus of the peptide can protect the peptide from exopeptidases and / or endopeptidases. Another strategy for improving stability against endopeptidases is to use d-amino acids or unnatural amino acids, such as naphthylalanine, phenylglycine, norleucine and cyclohexylalanine. Modifications at the N- and / or C-terminus of the peptide (e.g., formation of amides at the C-terminus, such as -CONH2 or -CONHR) may also be required to attach detectable agents via non-peptide linkers, such as PEG linkers containing DBCO (dibenzocyclooctyne) or BCN (bicyclo[6.1.0]nonyne) groups.

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

[0050] In some embodiments, the polypeptides described herein further comprise a detectable agent. In some embodiments, the detectable agent is attached to the polypeptide via a second linker and / or a chelator. For example, the polypeptides described herein may have the following structure: XYZ, where X is a detectable agent with or without a chelator (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 and second linkers can be, independently, peptide linkers or non-peptide linkers. In some embodiments, the first and / or second linkers each independently comprise a peptide linker, e.g., a short peptide consisting of several (e.g., 2-6) naturally and / or non-naturally occurring amino acids. Naturally occurring residues are categorized into groups based on common side chain properties. Exemplary groups include: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophobic: Cys, Ser, Thr; (3) acidic: Asp, Glu; (4) basic: Asn, His, Lys, Arg; (5) residues that affect 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, beta-homoamino acids, N-methyl amino acids, alpha-methyl amino acids. In some embodiments, the peptide linker comprises or consists of a lysine residue and / or two glycine residues (Gly-Gly).

[0052] In some embodiments, the first and / or second linker each independently comprises a non-peptide linker. Examples of non-peptide linkers include, but are not limited to, poly(ethylene glycol) (PEG) linker, beta-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy)acetic acid (AEA), 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), trioxatridecane-succinamic acid (Ttds), fluorenylmethoxycarbonyl (Fmoc)-Lys(N3)-OH, Lys(N3), homopropargylglycine, 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 include a group of compounds that are useful for various purposes (e.g., biolabeling). These linkers can generally be classified as monodisperse or polydisperse. Monodisperse PEG linkers have a precise number of PEG units with a defined chemical structure and a precise molecular weight. In contrast, polydisperse PEG (also known as polymeric PEG or polyPEG) is a polymer with an average molecular weight. PEG linkers can also contain various functional groups, such as azide, amine, alkyne, DBCO (dibenzocyclooctyne), BCN, TCO (trans-cyclooctene), NHS ester, maleimide. Exemplary PEG linkers useful for the polypeptides disclosed herein include PEG linkers and PEG linkers containing various 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 Pepscan, BroadPharm 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 a PEG linker (e.g., PEG2, PEG3, PEG4, PEG6), a functionalized PEG linker (e.g., PEG azide), gly-gly, Ttds, Fmoc-Lys(N3)-OH, homopropargylglycine, and Fmoc-HPra-OH. In some embodiments, the second linker comprises one or more of a PEG linker (e.g., PEG2, PEG3, PEG4, PEG6), a bis-PEG linker (e.g., bis-PEG16, bis-PEG18), and a functionalized PEG linker (e.g., BCN-PEG, DBCO-PEG). In some embodiments, the first linker comprises one or more of a PEG linker (e.g., PEG2, PEG3, PEG4, PEG6), a functionalized PEG linker (e.g., PEG azide), gly-gly, Ttds, Fmoc-Lys(N3)-OH, homopropargylglycine, and Fmoc-HPra-OH, and the second linker comprises one or more of a PEG linker (e.g., PEG2, PEG3, PEG4, PEG6), a bis-PEG linker (e.g., bis-PEG16, bis-PEG18), and a functionalized PEG linker (e.g., BCN-PEG, DBCO-PEG).

[0055] The detectable agent may be a radioisotope or a fluorescent agent. In some embodiments, the detectable agent is a radioisotope. Examples of radioisotopes 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, 123I, 124I, 125I, 126I, 127I, 128I, 129I, 130I, 131I, 132I, 133I, 134I, 135I, 136I, 137I, 138I, 139I, 140I, 141I, 142I, 143I, 144I, 145I, 146I, 147I, 148I, 149I, 150I, 151I, 152I, 153I, 154I, 155I, 156I, 157I, 158I, 159I, 160I, 161I, 162I, 163I, 164I, 165I, 166 Examples of such elements include 24I, 125I, 131I, 117mSn, 121Sn, 127Te, 142Pr, 143Pr, 149Pm, 151Pm, 149Tb, 153Sm, 157Gd, 161Tb, 166Ho, 165Dy, 169Er, 169Yb, 175Yb, 172Tm, 177Lu, 186Re, 188Re, 191Pt, 197Hg, 198Au, 199Au, 201Tl, 203Pb, 211At, 212Bi, 213Bi, 212Pb, 225Ac, 227Th, and Al18F2+. In some embodiments, the radioisotope is selected from 18F, 68Ga, 67Ga, 64Cu, 89Zr, 90Y, 99mTc, 111In, 123I, 124I, 131I, 177Lu, and Al18F2+. In some embodiments, the radioisotope is selected from 64Cu, 67Ga, 68Ga, 99mTc, 111In, 123I, 131I, 90Y, 177Lu, 212Bi, 225Ac, and Al18F2+.

[0056] Different radioisotopes can emit different particles, such as alpha, beta or gamma particles, which can be used for imaging and / or therapeutic purposes. Typically, gamma emitting radioisotopes are used for imaging purposes (e.g., diagnostic imaging), while alpha or beta emitting radioisotopes 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 radioisotope is selected from 67Ga, 99mTc, 111In, 177Lu, 68Ga, 64Cu, 44Sc, 86Y, 89Zr, 18F, 125I, 123I, 124I, and 203Pb. In some embodiments, the radioisotope is 68Ga or 18F. In some preferred embodiments, the detectable agent is 18F. These exemplary radioisotopes may be used for imaging purposes.

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

[0059] In some embodiments, the detectable agent is a radioisotope (e.g., a metallic radioisotope) complexed with a chelator (also referred to as a chelating agent). The radioisotope / chelator complex may reduce radiation loss and / or hydrolysis of the radioisotope and allow for its delivery to the desired / intended site in vivo. In some embodiments, the chelator is a bifunctional chelator, having one functional group / site that can bind, for example, a radioisotope and another functional group / site that can bind directly or indirectly to a polypeptide. Examples of chelators 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, NOTA, HP-DOA3, CBTE2a, TE2A, TMT, DPDP, HYNIC, DFO, HEDTA, NOPO, MAG3, NCS-MP-NODA, NH2-MPAA-NODA, DOTA, NODA, TRAP, DOTPI, DOTP, NOPO, and DOTAGA, NOTA, DTPA, CHX-DTPA, TETA of NODA and functionalized versions thereof. In some embodiments, the chelator is 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, desferrioxamine (DFO), HEHA and PEPA. Suitable chelators for radioisotopes are known in the art and can be easily 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.Endocr Rev.2014;43:260-290).In some embodiments, the chelator is DOTA (e.g., for 111In, 177Lu, or 213Bi), TETA (e.g., for 64Cu / 67Cu), DFO (e.g., for 89Zr), NOTA (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 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), carboxytetramethylrhodamine (TAMRA), 5-(dimethylamino)naphthalene-1-sulfonyl (dansyl), 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (EDANS), and 7-methoxycoumarinyl-4-acetyl (Mca). Fluorescently labeled peptides can be prepared using methods known in the art by modifying isolated peptides or by incorporating labels during solid phase synthesis.

[0061] The polypeptides described herein can be produced 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] Polypeptides can be directly labeled by reacting a radioisotope with the polypeptide for direct labeling using a single step. See, e.g., Williams J. et al., Bioconjugate Chem., 32(7):1242-1254 (2021). In some embodiments, a carrier molecule can be labeled with a radioisotope, purified (if necessary), and then reacted with the polypeptide to produce a labeled polypeptide in a two-step reaction. See, e.g., Yu S., Biomed Imaging Interv J., 2(4):e57 (Oct-Dec 2006). In some embodiments, peptides are dimerized and / or labeled with a detectable agent (e.g., a radioisotope or a fluorescent agent) using "click chemistry" known in the art, such as Huisgen 1,3-dipolar cycloaddition of an azide and a terminal alkyne. See, for example, Hein, Christopher D. et al., Pharm Res. 2008 October; 25(10):2216-2230. doi:10.1007 / s11095-008-9616-1; Kolb, HC et al., Angew. Chem. Int. Ed. 2001, 40, 2004, 2021. Click chemistry refers to a chemical reaction between pairs of reagents (click chemistry tools) that react with each other under mild conditions and are effectively inert to naturally occurring functional groups, such as amine groups. Reagents for click chemistry are known in the art and are commercially available from companies such as BroadPharma and AlphaThera. In some embodiments, peptides are dimerized and / or labeled with a detectable agent (e.g., a radioisotope or a fluorescent agent) using Scheme I, and / or Scheme II, and / or Scheme III (Example 3) disclosed herein.

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

[0064] Human DLL3 (SEQ ID NO: 77) [ka]

[0065] Compositions and Formulations The present invention further provides pharmaceutical compositions comprising the polypeptides described herein.

[0066] As used herein, the term "pharmaceutical composition" relates to a composition suitable for administration to a subject or patient, preferably a human subject or patient. Particularly preferred pharmaceutical compositions of the present invention comprise one or more polypeptides of the present invention. Preferably, the pharmaceutical composition further comprises a suitable blend of one or more (pharmaceutical effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives and / or adjuvants. Acceptable components of a pharmaceutical composition are preferably non-toxic to a recipient at the dosages and concentrations employed. Pharmaceutical compositions of the present invention include, but are not limited to, liquid compositions, frozen compositions and lyophilized compositions.

[0067] Excipients that may be used in the pharmaceutical composition include gentisic acid, maleic acid, beta-cyclodextrin, alpha-cyclodextrin, ascorbic acid, thioglycerol, glutathione, tartaric acid, niacinamide, ascorbic acid, FeCl3, glutamic acid, methylene diphosphonic acid, beta-hydroxypropylcyclodextrin, 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 ranges from about pH 4.5 to about pH 8.0, e.g., from about pH 5.0 to about pH 7.5, which may be achieved by using one or more buffers, e.g., those known in the art. Exemplary buffers include acetate buffers, phosphate buffers, and citrate buffers.

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

[0070] In embodiments in which the pharmaceutical composition comprises a polypeptide comprising a radioisotope, the pharmaceutical composition may include one or more agents, such as N-tert-butyl-α-phenylnitrone (PBN), ethanol, sodium ascorbate, and gentisic acid. The inclusion of such agents may stabilize and protect the radioisotope. Such agents may be used in amounts that are non-toxic to the recipient at the dosages and concentrations employed.

[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.

[0072] Diagnosis and Treatment The polypeptides disclosed herein, e.g., those comprising a detectable agent, can be used to detect DLL3 (e.g., human DLL3) in a sample. The sample can be a cell expressing DLL3, e.g., a recombinant cell expressing DLL3 or a DLL3-expressing tumor or cancer cell. Thus, disclosed herein is a method of detecting DLL3 in a sample, comprising contacting the sample with a polypeptide comprising a detectable agent as described above or a pharmaceutical composition comprising the polypeptide, and detecting DLL3 in the sample.

[0073] In some embodiments, the polypeptide comprises any one of SEQ ID NOs: 1-23; any one of SEQ ID NOs: 1-7; any one of SEQ ID NOs: 39-76; any one of SEQ ID NOs: 39-61 or any one of SEQ ID NOs: 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 NOs: 1-38 or SEQ ID NOs: 39-76, and a detectable agent. In some embodiments, the dimer is a homodimer, e.g., the polypeptide comprises a homodimer of any one of SEQ ID NOs: 1-38 or a homodimer of any one of SEQ ID NOs: 39-76. In some embodiments, the polypeptide comprises a homodimer of any one of SEQ ID NOs: 39-61. In some embodiments, the dimer is a heterodimer, e.g., the polypeptide comprises any two different sequences of SEQ ID NOs: 1-38 or any two different sequences of SEQ ID NOs: 39-76. In some embodiments, the homodimer or heterodimer comprises a first linker linking the two amino acid sequences. Suitable linkers for use as the first linker are described above.

[0074] In some embodiments, the detectable agent is a radioisotope, such as Ga, Tc, In, Ga, Cu, Sc, Y, Zr, F, I, I, I, or Pb. In some embodiments, the radioisotope is Ga or F. In some embodiments, the radioisotope is F. In some embodiments, the radioisotope is linked to the polypeptide via a second linker, a chelator, or a combination thereof. Suitable chelators and linkers that can be used as the second linker are described above.

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

[0076] In some embodiments, the sample comprises a cell that expresses DLL3, e.g., human DLL3. In some embodiments, the cell is a recombinant cell that expresses human DLL3. In some embodiments, the cell is a DLL3-expressing tumor or cancer cell, e.g., a cell obtained from a cancer patient.

[0077] Methods that can be used to detect polypeptides in a sample include those known in the art.For example, if the detectable agent is a fluorescent agent, flow cytometry can be used, while if the detectable agent is a radioisotope, imaging methods such as PET or SPECT can be used.

[0078] In some embodiments, the cell is in a subject, and the method includes administering to the subject a polypeptide or a pharmaceutical composition comprising the polypeptide, and detecting DLL3 in the subject using an imaging technique. The polypeptide or a composition comprising the polypeptide may be administered to the subject parenterally. In some embodiments, the polypeptide or a composition thereof is administered intravenously. In some embodiments, the subject is a human having a DLL3-expressing cancer or tumor, 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 lung 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.

[0079] The polypeptides disclosed herein, for example those containing radioisotopes, can be used to treat tumors or cancer in subjects. Radioisotopes useful for cancer treatment include those that emit alpha or beta particles. In one aspect, the disclosure also provides a method of treating DLL3-expressing tumors or cancer disease, comprising administering to a subject in need thereof a polypeptide containing a radioisotope as described herein or a pharmaceutical composition containing a polypeptide as described herein.

[0080] In some embodiments, the polypeptide comprises any one of SEQ ID NOs: 1-23; any one of SEQ ID NOs: 1-7; any one of SEQ ID NOs: 39-76; any one of SEQ ID NOs: 39-61 or any one of SEQ ID NOs: 39-45 listed in Table 1, and a radioisotope. In some embodiments, the polypeptide comprises a dimer of the amino acid sequence of SEQ ID NOs: 1-38 or SEQ ID NOs: 39-76, and a radioisotope. In some embodiments, the dimer is a homodimer, e.g., the polypeptide comprises a homodimer of any one of SEQ ID NOs: 1-38 or a homodimer of any one of SEQ ID NOs: 39-76. In some embodiments, the polypeptide comprises a homodimer of any one of SEQ ID NOs: 39-61. In some embodiments, the dimer is a heterodimer, e.g., the polypeptide comprises any two different sequences of SEQ ID NOs: 1-38 or any two different sequences of SEQ ID NOs: 39-76. In some embodiments, the homodimer or heterodimer comprises a first linker linking the two amino acid sequences. Suitable linkers for use as the first linker are 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 attached to the polypeptide via a second linker, a chelator, or a combination thereof. In some embodiments, the radioisotope is attached to the polypeptide via a chelator. Suitable chelators and linkers for use as the second linker are described above.

[0082] In some embodiments, the subject is a human with a DLL3-expressing tumor or cancer, such as a cancer of neuroendocrine origin.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 lung 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 small cell cervical neuroendocrine carcinoma.In some embodiments, the tumor or cancer is prostate cancer or lung cancer, and 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 a subject (e.g., a human patient) via parenteral administration. In some embodiments, the polypeptide or a pharmaceutical composition comprising the polypeptide is administered to a subject via intravenous administration.

[0084] If the pharmaceutical composition is lyophilized, the lyophilized material is first reconstituted in a suitable liquid prior to administration, for example, bacteriostatic water for injection (BWFI), saline, phosphate buffered saline (PBS), or the same formulation in which the polypeptide was present prior to lyophilization.

[0085] In further embodiments, the present disclosure provides kits that include the polypeptides disclosed herein, such as those that include a detectable agent useful for diagnostic or therapeutic purposes. In some embodiments, the detectable agent is a radioisotope. In some embodiments, the kit further includes instructions (e.g., in the form of a leaflet or instruction manual) on how to use the polypeptide. The kit may also include a means for administering the polypeptide or a pharmaceutical composition thereof, such as a syringe, pump, infuser, etc., a means for reconstituting the polypeptide, and / or a means for diluting the polypeptide.

[0086] In the context of the present invention, the term "kit" refers to two or more components (one of which corresponds to a polypeptide or pharmaceutical composition of the present invention) packaged together in a container, receptacle or other. A kit can thus be described as a set of products and / or materials sufficient to accomplish a certain purpose that can be sold as a single item.

[0087] All patents and other publications identified are expressly incorporated herein by reference, in their entirety or relevant portion, as will be apparent from the context of the citation, for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the information set forth herein.

[0088] The invention disclosed herein is further illustrated by the following examples. EXAMPLES

[0089] Example 1. Selection and screening of DLL3-binding peptides from a phage library Selections were performed using two Cys-restricted peptide phage libraries, 9-CYS and 12-C8C, displayed on the M13pIII major coat protein. Four different selection strategies were performed as shown in Table 2, and selection rounds were performed combining the use of DLL3 recombinant protein with three different stably transfected cell lines expressing different levels of human DLL3 on their surface.

[0090] [Table 2]

[0091] The first selection round was common to all four strategies and used recombinant human DLL3 (rDLL3). 10 μg of rDLL3-Fc was incubated with Protein A Dynabeads (Life Technologies) to immobilize the protein on the beads. The beads were then washed twice with PBS buffer before being incubated with the preblocked phages. Approximately 10 11 phages were used in this selection round. Briefly, phage particles were blocked in buffer at room temperature and then incubated with immobilized protein. At the end of the incubation, beads were collected with a magnet and washed five times in PBS buffer. Bound phages were eluted using 100 mM triethylamine (TEA) and then used to infect TG1 bacteria. After 1 h at 37°C, bacteria were plated on 2xTY / Amp / Glu plates and incubated overnight at 30°C.

[0092] The next day, colonies were harvested from the plates in 10 ml of culture medium and used to rescue the selection phage. The harvested bacteria were inoculated into 50 ml of 2xTY / Amp / Glu, grown at 37°C to OD 600nm=0.5, and then infected with K07 helper phage at an MOI of approximately 10 (10 phages per cell). The infected bacteria were spun down, resuspended in 2xTY / Amp / Kan, and grown overnight at 25°C. The next day, the supernatant was clarified by centrifugation, and the infected bacteria were then blocked in PBS with 4% milk.

[0093] Parental cell lines (CHO and HEK293) and DLL3-overexpressing cell lines were detached, counted, and cultured at 10 7Cells were aliquoted at 100 cells / tube. Cells were blocked in PBS with 3% milk and incubated with preblocked phage. DLL3-overexpressing cells and phage were incubated for 1 h and unbound phage were removed by washing in PBS. Phage were then eluted by resuspending cells in 500 μl of 100 mM HCl for 10 min. Eluted phage were used to infect a 10 ml mid-logarithmic culture of TG1. Bacteria were then plated on 2×TY / Amp / Glu plates and incubated overnight at 30° C. This process was repeated two more times for four selection rounds.

[0094] The pools of phages obtained after each selection round were tested for their binding to rDLL3-Fc by phage-ELISA and counterscreened against hIgG-Fc. Briefly, proteins were immobilized on NUNC-Maxisorp ELISA plates. Plates were then washed and blocked. 100 μl of preblocked phages were added to the blocked plates and incubated for 1 h at room temperature. Anti-M13 HRP-conjugated mAb was added and plates were read at A370 nm on a Multiskan Ascent (Thermo) Elisa reader after addition of TMB (Sigma).

[0095] Phage pools were also screened for their binding to cells overexpressing human DLL3 on their surface. Cells were detached, counted, and cultured at 1.8×10 in FACS buffer containing 2% FBS. 6 The cells were resuspended at 1000x cells / ml. The preblocked phage were then added to the cells and incubated for 1 h at room temperature. After washing, 1 μg of α-M13-FITC (clone MM05T, Sino Biologics) was added to the cells and incubated for an additional 1 h. The cells were then washed and resuspended in FACS buffer. Fluorescence was acquired using a flow cytometer (FACS Canto / Becton Dickinson).

[0096] Single phage clones were picked and grown after each selection round. Bacterial cultures were infected with helper phage at an MOI of approximately 10 and incubated at 37°C. Infected bacteria were spun down, resuspended, and grown overnight at 25°C. The next day, the supernatant was clarified by centrifugation. An aliquot of the supernatant was used for phage ELISA assays or FACS staining on DLL3 expressing cells using the same methods as above.

[0097] Phage clones with signals over 4-fold above background were further tested for their binding to DLL3 on the surface of DLL3-expressing cells by cell ELISA. Briefly, the day before the assay, parental cells and cells overexpressing DLL3 were plated on 96-well plates. The cells were then fixed and washed before adding phages preblocked as above. The cells were then washed and anti-M13-HRP was added. Signals were measured using a Multiskan Ascent (Thermo) ELISA reader at A370 nm. Phages that bound to DLL3-expressing cells were selected for sequencing of the displayed peptides.

[0098] Single phage clones that showed specific binding to rDLL3-Fc were then tested for binding to DLL3 stably expressed on the surface of cells to identify clones that recognized the native form of the protein. All phages that could recognize cells were subjected to DNA sequencing of the displayed peptides.

[0099] In total, 3400 single clones were analyzed, of which 1718 bind to recombinant DLL3 protein, and 387 of them also bind to DLL3 protein on the surface of cell lines stably expressing DLL3.

[0100] Sequence analysis identified a total of 38 unique sequences (SEQ ID NOs: 39-76), which are listed in Table 1. Of these, 37 were derived from library 12-C8C and one was derived from library 9-CYS.

[0101] Example 2. Synthesis, modification and characterization of DLL3-binding peptides Materials and Methods Peptide synthesis: Peptide synthesis was performed by standard Fmoc stepwise solid phase synthesis (SPPS) using a Liberty Blue microwave synthesizer (CEM corp.). Synthesis was performed using Rink amide AM resin Novabiochem 0.29 mmol / g on a 150 μmol scale. Each amino acid (0.2 M in DMF) was acylated in 8-fold excess using equimolar amounts of DIC (0.5 M) and Oxyma (1 M) in DMF as activators. Amino acids were used with standard side chain protecting groups unless otherwise stated. Cysteines required for disulfide bridges were acylated as Fmoc-Cys(Trt)-OH on the solid phase. Aspartic acid was coupled as Fmoc-Asp(OMpe)-OH to minimize aspartimide formation, and Fmoc deprotection after incorporation of Asp-(OMpe)-OH was performed with 20% piperidine in DMF at room temperature. Single and double couplings were performed under microwave irradiation at 90°C for 2 min, with the exception of Fmoc-His(Trt)-OH (50°C). Double acylation reactions were performed for all Fmoc-Arg(Pbf)-OH and for the first three amino acids at the N-terminus (AET). Peptides were cleaved using a solution of 87.5% TFA, 5% HO, 2.5% TIS, 5% phenol for 1.5 h at room temperature and then precipitated with cold tert-butyl methyl ether. After centrifugation, the peptide pellet was washed with diethyl ether, dried, dissolved in 0.1% TFA in (1:1) HO / ACN, and lyophilized.

[0102] Analytical characterization: Crude and prepared peptides were analyzed by ultra-performance liquid chromatography with UV and mass spectrometry detection (UPLC-UV-MS). Analysis was performed on a Waters Acquity UPLC system equipped with an analytical Waters BEH130C4 (2.1×100 mm, 1.7 μm, 45° C.) column. Detection was performed by UV absorption at 214 nm. Mass spectrometry was performed on a Waters SQ detector with electrospray ionization in positive ion detection mode, with a mass-to-charge ratio scan range of 400-1800. Analysis was performed using a linear gradient of a binary mixture of H2O containing 0.1% TFA (A) and acetonitrile containing 0.1% TFA (B). The linear gradient of B used was 20% B to 20% B (1 min), 20% B to 60% B (4 min), 60% B to 80% B (0.2 min); flow rate: 0.4 mL / min; temperature: 45° C.

[0103] N-Terminal Acetylation: Upon completion of sequence assembly, the resin was acetylated using 10 molar equivalents of acetic anhydride in DMF. It is believed that blocking the N-terminal amino group reactivity of a polypeptide by acetylation may allow for a simple synthetic strategy for the attachment of detectable agents (e.g., fluorescent or radiolabeled moieties) to the polypeptide.

[0104] C-Terminal Amidation or Amination: C-terminal amidation or amination was performed during solid phase synthesis using methods known in the art.

[0105] Cyclization: Cyclization is believed to restrict the conformation of peptide binders for improved activity with targets. If necessary, peptides were cyclized via a disulfide bridge between two cysteine ​​groups to form a cyclic structure and purified using the methods described herein. Peptides were incubated overnight in 10% DMSO, 90% Tris 0.1M pH 8 (final peptide concentration of 1 mg / ml) for disulfide formation. The reaction was monitored by UPLC analysis on a BEH130 C4 Acquity Waters column (2.1 x 100 mm, 1.7 μm) with a gradient of 20% B to 20% B (1 min), 20% B to 60% B (4 min), 60% B to 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 complete dissolution of the peptide before purification.

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

[0107] Exemplary cyclized peptide sequences and structures are shown in Table 3 below.

[0108] [Table 3-1]

[0109] [Table 3-2]

[0110] [Table 3-3]

[0111] [Table 3-4]

[0112] [Table 3-5]

[0113] [Table 3-6]

[0114] Binding Kinetic Assays Using Biolayer Interferometry (BLI) and Surface Plasmon Resonance (SPR) Binding kinetic assays were performed using Biolayer Interferometry (BLI) technology. Recombinant DLL3 extracellular domain (rhDLL3-Fc) was biotinylated using the EZ-Link Sulfo-NHS-LC-LC-Biotin Kit according to the manufacturer's protocol (Pierce by Thermo Fisher Scientific). Recombinant protein was incubated with excess NHS-LC-LC biotin ester at a molar ratio of 1:3 (protein:biotin) for 90 min at 25°C. Unreacted biotin ester was removed by subsequent buffer exchange on a 0.5ml Zeba spin desalting column 7K MWCO (Pierce by Thermo Fisher Scientific).

[0115] Biomolecular interaction analysis was performed using an Octet Red 96e instrument (Forte Bio). Biotinylated hDLL3 was diluted to 12.5 μg / ml in 1× kinetic buffer (Forte Bio) and then captured on Streptavidin Dip and Read Biosensors (ForteBio) to 8–10 nm for 20 min, followed by sensor surface blocking with 10 μg / ml biocytin (Invitrogen by Thermo Fisher Scientific) for 60 s. To perform this assay, peptides were diluted to final concentrations of 20, 4 and 0.8 μM in binding assay buffer (1× kinetic buffer supplemented with 5% DMSO) and binding to DLL3 was assessed using a 90 s association followed by a 120 s dissociation. Peptides that showed positive binding were selected and tested over a dynamic range (10× dissociation constant (KD) to 0.1×KD), increasing the dissociation time up to 300 s to better evaluate affinity and binding kinetic parameters (e.g., k, k, and KD). Sensorgrams were analyzed by data analysis HT11.1 software (ForteBio): specific binding was obtained by subtracting binding from negative controls. Kinetic parameters were measured using a global fitting following a 1:1 Langmuir binding isotherm.

[0116] Peptide off-rate screening was performed using a Biacore T200 instrument (Cytiva, Sweden). Biotinylated rhDLL3-Fc was diluted to 12.5 μg / ml in HBS-P+1× buffer (Cytiva, Sweden) and then captured to 6000 RU on a series S SA sensor chip (Cytiva, Sweden). Peptides were diluted to 300 nM in PBS-P+1× buffer (Cytiva, Sweden) supplemented with 1% DMSO and 0.1% BSA, then injected at 30 μl / min over the ligand for 5 min and finally allowed to dissociate for 1 h. Sensorgrams were analyzed by Biacore T200 Evaluation Software 3.0 (Cytiva, Sweden): specific binding was obtained by subtracting non-specific binding to the bare biosensor and the zero analyte concentration made up from the binding assay buffer (double referencing). k was calculated using a global fitting following a 1:1 Langmuir binding isotherm. off The kinetic parameters were measured.

[0117] Flow cytometry HEK293-huDLL3, CHO-huDLL3 and the corresponding parental cell lines were detached with EDTA 2.5 mM and cultured at 1.8 × 10 6Cells were resuspended at 100 nM cells / ml, dispensed at 500 μl per tube, and pelleted in a Heraeus Multifuge X3R centrifuge at 1300 rpm for 5 min. To measure binding of fluorescently labeled peptides to cell surface hDLL3, cells were resuspended in 500 μl FACS buffer in the presence of 100 nM AF647-labeled peptide (e.g., PepSP1215 or PepSP1216). In competition experiments, cells were resuspended in 500 μl FACS buffer containing 30 nM peptide (e.g., PepSP1215) and increasing concentrations of unlabeled peptide (e.g., PepSP1146, PepSP1270, PepSP1271, or PepSP1272) were added from 500 μM to 4 nM. After 1 h incubation at room temperature, cells were washed once with FACS buffer, centrifuged, and resuspended in FACS buffer / 1% formaldehyde. Samples were acquired on a FACS ARIA flow cytometer (Becton Dickinson) and data were analyzed with FACS Express software.

[0118] result DLL3 binding characteristics: 35 polypeptides 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 and fit well according to a 1:1 Langmuir binding model. The remaining 12 showed poor (3) or heterogeneous (9) binding modes. In general, peptides with Kd in the micromolar range (e.g., >1 μM) were considered as poor binders, whereas peptides with Kd in the single- to double-digit nM range were considered as good binders. Peptides that did not fit well to the 1:1 Langmuir binding model were considered to have heterogeneous binding. 23 peptides were retested with broader dose-response curves ranging from approximately 10 × KD to 0.1 × KD, and kinetic parameters were determined using a global fitting procedure following the 1:1 Langmuir model. Affinity values ​​were obtained as the ratio of koff to kon. The characterized peptides showed total affinities below 1 μM, and seven peptides (PepSP1146, 1178, 1163, 1161, 1147, 1171 and 1182) showed affinities in the double-digit nM range with residence times ranging from 60 to 180 seconds. These seven peptides were retested in three independent experiments. The data for the 23 peptides as well as PepSP1213, 1214 and 1269 are summarized in Table 4 below.

[0119] [Table 4]

[0120] Peptides were functionalized at the N- or C-terminus to assess their effect on binding to DLL3. Acetylation of the N-terminus is a desirable feature for peptides because it blocks N-terminal amino group reactivity, which may allow for a simpler synthetic process for further modification of the peptide (e.g., conjugation to a radiolabeled moiety). Surface plasmon resonance (SPR) showed that neither acetylation of the N-terminal sequence nor C-terminal amidation or amination perturbed the binding properties of the peptide.

[0121] In addition, the N-terminal amino acids common to all unique sequences (AETVEF or AETVE) were deleted to assess the effect on binding. These amino acids were found to be responsible for binding of some of the peptides to DLL3, but are not required for all of the peptides to bind DLL3. See, for example, the binding data for PepSP1213 and PepSP1214 and for PepSP1161 and 1269.

[0122] Example 3. Labeling, dimerization and characterization of DLL3-binding peptides Materials and Methods Various exemplary reagents (e.g., linkers, click chemistry reagents, fluorescers) used in this series of experiments are listed below and are commercially available.

[0123] Linkers / Spacers: Trioxatridecane-succinamic acid (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.

[0124] Click chemistry reagents: homopropargylglycine (hPra), Fmoc-HPra-OH, Fmoc-Lys(N3)-OH

[0125] Fluorescent agents: AFDye™ 647 dibenzocyclooctyne (DBCO), Cy5 DBCO

[0126] PepSP1396 and PepSP1342: At the end of the PepSP1324 assembly, Fmoc-N-amide-PEG4-acid (CAS557756-85-1) was acylated on the resin in a 4-fold excess with equimolar amounts of DIC and HOAt in DMF as activators, followed by Fmoc deprotection (20% piperidine in DMF, 3 x 3 min) to give PepSP1396. Similarly, at the end of the PepSP1324 assembly, Fmoc-Ttds-OH (CAS172089-14-4) was acylated on the resin in a 4-fold excess with equimolar amounts of DIC and HOAt in DMF as activators, followed by Fmoc deprotection (20% piperidine in DMF, 3 x 3 min); the process was repeated for the second Fmoc-Ttds-OH present in the sequence to give PepSP1342.

[0127] Fluorescent labeling: PepSP1146 and 1171 were used for labeling and dimerization experiments. At the start of the synthesis, the unnatural amino acid Fmoc-Lys(N3)-OH (CAS159610-89-6) was acylated on the resin, and then the linker Fmoc-Ttds-OH was acylated using standard methods, and the peptides were synthesized using the same procedure as above. The peptides were then extended at the C-terminus with a linker (Ttds) and Lys(N3) group to generate the peptide precursors PepSP1273 and PepSP1274 (see Table 5 below). The peptide precursors were then labeled with a fluorescent agent, for example AlexaFluor647DBCO, using copper-free click chemistry to generate PepSP1215 and PepSP1216.

[0128] Specifically, for labeling with AlexaFluor647, purified peptide precursors (final peptide concentration 30 mg / ml) were conjugated to AFDye™647DBCO (Click Chemistry Tools Catalog 1302) by incubation with 1.3 equivalents of Alexa-DBCO (dissolved in DMSO). The reaction was left overnight and monitored by UPLC analysis on a BEH300 C4 Acquity Waters column (2.1 × 100 mm, 1.7 μm) with gradients of 20% B to 20% B (1 min), 20% B to 90% B (4 min), 90% B to 90% B (0.2 min) and 25% B to 25% B (1 min) 25% B to 45% B (4 min), 45% B to 95% B (0.3 min) (eluent: A = HO + 0.1% TFA; B = CHCN + 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 on a reverse phase HPLC using a Delta Pak C4 200x25mm 300A 15um column and a linear gradient: 15%B to 15%B to 30%B in 20 minutes (eluent: A=H2O+0.1%TFA; B=CH3CN+0.1%TFA; flow rate: 50mL / min; λ: 214nm). Analytical characterization was performed on a BEH300 C4 Acquity Waters 2.1x100mm, 1.7μm column with a gradient of 25%B to 25%B (1min), 25%B to 45%B (4min), 45%B to 80%B (0.2min).

[0129] dimerization It is believed that peptide dimerization may improve the binding properties of peptides, for example due to improved avidity. For this purpose, peptide dimers were synthesized and tested. Three linkers, bis-propargyl-PEG6, bis-propargyl-PEG14 and bis-propargyl-PEG18, were used to homodimerize the precursor PepSP1273, since each linker has two terminal alkyne functional groups that react with the peptide precursor PepSP1273 by copper-catalyzed click chemistry to generate homodimers PepSP1270, 1271 and 1272. Specifically, one part of peptide azide precursor (PepSP1273) was incubated with 0.5 molar equivalents of bispropargyl-PEGx (x is 6, 14 or 16), 3 molar equivalents of CuSO4 and 5 molar equivalents of Na ascorbate. The reaction was carried out in DMSO with salts dissolved in water (water content <10%) and a final peptide concentration of 30 mg / ml. The reaction was complete after 5 min and the reaction was monitored by UPLC analysis on a BEH300 C4 Acquity Waters column (2.1 x 100 mm, 1.7 μm) with a gradient of 30% B to 30% B (1 min), 30% B to 50% B (4 min), 50% B to 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 on a preparative HPLC Waters system using the following conditions:

[0130] PepSP1270: Gradient: 20% B to 20% B (5 min) 20% B to 40% B in 25 min; Column: X Bridge C4 150×19 mm 300A 5um; Eluent: A=H2O+0.1% TFA; B=CH3CN+0.1% TFA; Flow rate: 20 mL / min; λ: 214 nm; PepSP1271: Gradient: 25% B to 25% B (5 min) 25% B to 45% B in 25 min; Column: Daisogel C4 200×20 mm 200A 5um; eluent: A=H2O+0.1%TFA; B=CH3CN+0.1%TFA; flow rate: 15mL / min; λ: 214nm; PepSP1272: gradient: 20%B to 20%B (5min) 20%B to 40%B in 25min; column: Delta Pak C4 200×25mm 300A 15um; eluent: A=H2O+0.1%TFA; B=CH3CN+0.1%TFA; flow rate: 50mL / min; λ: 214nm.

[0131] To improve the dimerization and labeling reactions, it is desirable to incorporate in the same linker molecule a) the best format and b) the ability to undergo simple conjugation reactions with radioactive moieties (e.g., 18F) for imaging and therapeutic purposes. To achieve this, a trifunctional linker was designed and synthesized using Scheme I shown below.

[0132] Scheme I. Synthesis of propargyl-trifunctionalized linkers for dimerization and labeling reactions [ka]

[0133] Starting from the linker PEG18, trifunctional linkers were synthesized according to Scheme I and functionalized with DBCO or BCN moieties. Radioisotopes (e.g., 18F) were then conjugated to peptides using copper-free click chemistry using linkers with DBCO or BCN moieties. PepSP1384: The peptide azide precursor PepSP1274 (1 eq.) was incubated with 0.5 eq. bis-propargyl-PEG18, 2 eq. CuSO4 and 2 eq. Na ascorbate. The reaction was carried out in DMSO with salts dissolved in water (water content <10%) and a final protein concentration of 20 mg / ml. The reaction was complete after 5 min and the reaction was monitored by UPLC analysis on a BEH300 C4 Acquity Waters column (2.1 × 100 mm, 1.7 μm) with a gradient of 30% B to 30% B (1 min), 30% B to 50% B (4 min), 50% B to 90% B (0.2 min) (eluent: A = HO + 0.1% TFA; B = CHCN + 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 on a reversed-phase HPLC using a Delta Pak C4 200x25mm 300A 15um column and a linear gradient: 25%B to 40%B in 20 minutes (eluent: A=H2O+0.1%TFA; B=CH3CN+0.1%TFA; flow rate: 50mL / min; λ: 214nm). Analytical characterization was performed on a BEH300 C4 Acquity Waters 2.1x100mm, 1.7μm column with gradient 30%B to 30%B (1min), 30%B to 50%B (4min), 50%B to 90%B (0.2min); flow rate: 0.4mL / min; λ: 214nm; temperature: 45°C; MS: Waters Acquity ESI+, single quadrupole. PepSP1321 and PepSP1324 were synthesized similarly.

[0134] PepSP1343, PepSP1344 and PepSP1371 dimers were prepared using PepSP1318 as a precursor. Specifically, PepSP1318 was dimerized with a suitable PEG linker, propargyl-trifunctionalized linker (see Scheme I), to generate PepSp1343. PepSP1344 and PepSP1371 were generated from PepSP1343 and a suitable linker (PEG4-DBCO or PEG3-BCN) derivatized with either DBCO or BCN groups.

[0135] Specifically, for PepSP1343, PepSP1318 (precursor) (1 eq.) was incubated with 0.6 eq. of propargyl-trifunctionalized linker (synthesized according to Scheme III described herein below), and 1.9 eq. of CuSO4 and 1.9 eq. of Na-ascorbate were added to the solution. The reaction was carried out in DMSO with the salts dissolved in water (water content <10%) and with a final peptide concentration of 20 mg / ml. The reaction was complete after 5 min and the reaction was monitored by UPLC analysis on a BEH300 C4 Acquity Waters column (2.1 × 100 mm, 1.7 μm) with a gradient of 30% B to 30% B (1 min), 30% B to 50% B (4 min), 50% B to 90% B (0.2 min) (eluent: A = HO + 0.1% TFA; B = CHCN + 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 in DMSO and loaded on a reverse-phase HPLC using a Delta Pak C4 25×200 mm, 300A, 15 μm column and a linear gradient: 25% B to 25% B (5 min), 25% B to 40% B in 20 min (eluent: A=HO+0.1% TFA; B=CHCN+0.1% TFA; flow rate: 50 mL / min; λ: 214 nm). Analytical characterization was performed on a BEH300 C4 Acquity Waters 2.1 x 100 mm, 1.7 μm column with gradient 30% B to 30% B (1 min), 30% B to 50% B (5 min), 50% B to 90% B (0.2 min); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45 °C; MS: Waters Acquity ESI+, single quadrupole.

[0136] For PepSP1344, the dimeric precursor PepSP1343 was incubated with 1.1 equivalents of DBCO-PEG4-NHS ester (BroadPharm, MW: 649.7 Da) and 1% DIPEA was added. The reaction was carried out in DMSO with a final peptide concentration of 20 mg / ml. The reaction was completed after 1 h and the reaction was monitored by UPLC analysis on a BEH300 C4 Acquity Waters column (2.1 × 100 mm, 1.7 μm) with a gradient of 35% B to 35% B (1 min), 35% B to 55% B (4 min), 55% B to 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 on a reverse phase HPLC using a Waters Delta Pak C4 (200×25 mm 300A 15 um) column and a linear gradient: 25% B to 40% B in 20 min (eluent: A=HO+0.1% TFA; B=CHCN+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 a gradient of 35% B to 35% B (1 min), 35% B to 55% B (4 min), 55% B to 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. Similarly, PepSP1371 was synthesized using PepSP1343 as precursor and BCN-PEG3-NHS ester (BroadPharm, MW: 494.53 Da).

[0137] Further work was carried out to utilize a new synthetic scheme that can be used as an alternative route to Scheme I outlined above. The new scheme, Scheme II, is compatible with disulfide bridge formation and conjugation to radioligands. First, precursor compounds PepSP1321 and PepSP1324 were synthesized using Scheme II, both of which have a spacer Gly-Gly-Ttds at the C-terminus, which may benefit from greater flexibility for the final dimer. Other precursor compounds PepSP1396 and PepSP1342 were also synthesized, with linkers GG-Ttds-K(PEG4)-CONH2 and GG-Ttds-K(Ttds-Ttds)-CONH2, respectively.

[0138] The precursor compounds were used to synthesize compounds PepSP1462, PepSP1487, PepSP1488, and PepSP1489, which can be conjugated to radioisotopes, such as 18F. Specifically, PepSP1462 was prepared using PepSP1324 and the reagent PEG4-DBCO; PepSP1487 was prepared using PepSP1324 and an NHS-trifunctionalized linker (Scheme II); PepSP1488 was prepared using PepSP1396 and an NHS-trifunctionalized linker (Scheme II); and PepSP1489 was prepared using PepSP1342 and an NHS-trifunctionalized linker (Scheme II).

[0139] Specifically, for PepSP1462, precursor PepSP1324 (1 eq.) was dissolved in DMSO and 10 eq. DIPEA was added to the solution. Then, 1.1 eq. DBCO-PEG4-NHS (BroadPharm, MW: 649.7 Da) was dissolved in DMSO and slowly added dropwise to the solution containing the peptide precursor, resulting in a final concentration of 30 mg / mL peptide. The reaction was monitored by UPLC-MS analysis on a BEH300 C4 Acquity Waters column (2.1 × 100 mm, 1.7 μm) with a gradient of 30% B to 30% B (1 min), 30% B to 50% B (4 min), 50% B to 90% B (0.2 min) (eluent: A = HO + 0.1% TFA; B = CHCN + 0.1% TFA); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45 °C; MS: Waters Acquity ESI+, single quadrupole. The reaction was completed after 30 min and loaded onto a reverse-phase HPLC using a DeltaPak C4, 40×200 mm, 300 Å, 15 μm column and a linear gradient: 15% B to 15% B (5 min), 15% B to 35% B (20 min), 35% B to 40% B (5 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 x 100 mm, 1.7 μm column with gradient 30% B to 30% B (1 min), 30% B to 50% B (4 min), 50% B to 90% B (0.2 min); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45 °C; MS: Waters Acquity ESI+, single quadrupole.

[0140] For PepSP1487, 1488 and 1489, the precursors PepSP1324, 1396 and 1342, respectively (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) was dissolved in DMSO and slowly added dropwise to the solution containing the peptide precursor, resulting in a final concentration of peptide of 1 mg / mL. The reaction was monitored by UPLC-MS analysis on a BEH300 C4 Acquity Waters column (2.1 × 100 mm, 1.7 μm) with a gradient of 30% B to 30% B (1 min), 30% B to 50% B (4 min), 50% B to 90% B (0.2 min) (eluent: A = HO + 0.1% TFA; B = CHCN + 0.1% TFA); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45 °C; MS: Waters Acquity ESI+, single quadrupole. After overnight the reaction was complete and the reaction was diluted 1:2 with HO+0.05% NH and loaded onto a reversed-phase HPLC using an XBridge Protein BEH C4, 30×150 mm, 300 Å, 5 μm column and the following linear gradient: 5% B to 5% B (5 min) - 5% B to 25% B (20 min), 25% B to 30% B (5 min) (eluent: A=HO+0.05% NH; B=CHCN; flow rate: 80 mL / min; λ: 214 nm). Analytical characterization was performed on a BEH300 C4 Acquity Waters 2.1 x 100 mm, 1.7 μm column with gradient -30% B to 30% B (1 min), -30% B to 50% B (4 min), -50% B to 90% B (0.2 min); flow rate: 0.4 mL / min; λ: 214 nm; temperature: 45 °C; MS: Waters Acquity ESI+, single quadrupole.

[0141] The DBCO derivatives PepSP1462, PepSP1487, PepSP1488 and PepSP1489 were finally conjugated with fluoroethyl azide (FEA) to give the final fluoro-labeled compounds PepSP1538, PepSP1581, PepSP1582 and PepSP1583, respectively, as follows:

[0142] Synthesis of monomeric fluoroethyl azide (FEA) derivative PepSP1538: 1 equivalent of 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 analysis on a BEH300 C4 Acquity Waters column (2.1 × 100 mm, 1.7 μm) with a gradient of 30% B to 30% B (1 min), 30% B to 60% B (4 min), 60% B to 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, the reaction was diluted 1:2 with HO+0.05% NH and loaded on a reverse phase HPLC using a C4, 25×200 mm, 15 um, 300A Waters Deltapak column and a linear gradient: 20% B to 20% B (5 min) to 35% B (20 min) (eluent: A=HO+0.05% NH; B=CHCN; 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 gradient 30% B to 30% B (1 min), 30% B to 60% B (4 min), 60% B to 90% B (0.2 min); eluents: A: HO + 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.

[0143] Synthesis of dimeric FEA derivatives PepSP1581, PepSP1582 and PepSP1583: 1 equivalent of dimeric 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 was added to the reaction mixture. The reaction was monitored by UPLC-MS analysis on a BEH300 C4 Acquity Waters column (2.1 × 100 mm, 1.7 μm) with a gradient of 30% B to 30% B (1 min), 30% B to 60% B (4 min), 60% B to 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, the reaction was diluted 1:2 with HO+0.05% NH and loaded on a reverse phase HPLC using a C4, 20×150 mm, 5 um, 300A Waters XBridge column and a linear gradient: 10% B to 10% B (5 min) to 25% B (20 min) (eluent: A=HO+0.05% NH; B=CHCN; 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 gradient 30% B to 30% B (1 min), 30% B to 60% B (4 min), 60% B to 90% B (0.2 min); eluents: A: HO + 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.

[0144] Scheme II. Synthesis of trifunctional linkers for dimerization and labeling reactions [ka]

[0145] Scheme III below shows a synthetic scheme for a propargyl-trifunctionalized linker. [ka]

[0146] The following steps were carried out to synthesize the propargyl-trifunctionalized linker (Scheme III). Step 1: 1.3 eq. Boc-Gly-OH (4530-20-5) was dissolved in DMF with 1.3 eq. HATU, after 5 min the mixture was added to 1 eq. NH-bis(PEG4-acid) and DIPEA (3 eq.) dissolved in DMF. The coupling reaction was complete after 15 min. Reaction monitoring was performed by UPLC-MS. Step 2: HATU and DIPEA (2.3 eq.) were added to the reaction mixture and stirred at room temperature, after 5 min 2 eq. propargyl-PEGx-amine dissolved in DMF was 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, stirred at room temperature for 10 min, then concentrated to dryness. The crude material was purified by RP flash chromatography using a Luknova C18 column (Gradient: (%B) 0% for 4 CV, 0%-35% in 10 CV, 35% for 3 CV. A: HO+0.1% TFA; B: ACN+0.1% TFA; λ: 214 nm; collected fractions were lyophilized. UPLC-MS: Acquity BEH C18, 2.1×100 mm, 1.7 um, 130A Flow rate: 0.4 mL / min; Gradient (%B): 20% in 1 min, 20%-70% in 4 min; A: HO+0.1% TFA; B: ACN+0.1% TFA; λ: 214 nm).

[0147] Scheme 4 below shows the synthesis of an NHS-trifunctionalized linker. [ka]

[0148] The following steps were carried out to synthesize the NHS-trifunctionalized linker (Scheme 4): Step 1: 1 equivalent of NH-bis(PEG3-acid) HCl salt (BroadPharm, 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 was complete after 15 min. The reaction was purified by flash chromatography using a Luknova SuperSep HP 25 g column (Gradient: (%B) 0% for 2 CV, 0% to 15% in 8 CV, 15% for 2 CV, A: DCM+0.2% acetic acid; B: MeOH+0.2% acetic acid; λ: 206 nm; collected fractions were lyophilized. UPLC-MS: Acquity BEH C18, 2.1×100 mm, 1.7 um, 130A Flow rate: 0.4 mL / min; Gradient (%B): 5% B to 5% B (1 min), 5% B to 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, then concentrated to dryness. The product was dissolved in DCM and 3 equivalents of DIPEA was added, followed by 1 equivalent of DBCO-PEG4-NHS ester (BroadPharm, MW: 649.7 Da), stirred for 15 minutes and monitored by UPLC-MS. Step 3: 3 equivalents of TEA was added to 1 equivalent of product dissolved in DCM; 3 equivalents of N,N'-disuccinimidyl carbonate (BroadPharm, 256.17 Da) dissolved in DMF were added to have a reaction in DCM:DMF 1:1; the reaction was stirred for 1 hour and monitored by UPLC-MS. This was loaded on a reverse phase HPLC and purified using a Waters XBridge C18 (50×150 mm, 130A, 5 mm) column and a linear gradient: 30% B to 30% B (5 min), 30% B to 50% B (20 min); eluent: A=H2O+0.1% TFA; B=CH3CN+0.1% TFA; flow rate: 80 mL / min; λ: 214 nm; collected fractions were lyophilized.Analytical characterization was performed on an Acquity BEH C18, 2.1×100 mm, 1.7 um, 130A Flow rate: 0.4 mL / min; Gradient (%B): 5% B to 5% B (1 min), 5% B to 95% B (4 min); A: HO+0.1% TFA; B: CH3CN+0.1% TFA; λ: 214 nm; Temperature: 45° C.; MS: Waters Acquity ESI+, single quadrupole.

[0149] The sequences and structures of the labeling and / or dimerization peptides are shown in Table 5 below.

[0150] [Table 5-1]

[0151] [Table 5-2]

[0152] [Table 5-3]

[0153] [Table 5-4]

[0154] [Table 5-5]

[0155] [Table 5-6]

[0156] [Table 5-7]

[0157] Peptides containing various linkers and / or labeled with fluorescent agents were tested for binding to DLL3 by BLI, SPR, or flow cytometry using cells expressing human DLL3. 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 does not adversely affect binding to DLL3. The fluorescently labeled peptides were also found to specifically bind to DLL3 protein expressed on the surface of cells (CHO cells); see Figure 1.

[0158] [Table 6]

[0159] Peptide dimers were tested for binding to DLL3. As shown in Table 7 below, dimers significantly increased complex stability, resulting in 30-100 fold more stable off-rates. Dimers made using the bis-PEG18 linker showed the best 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 monomeric PepSP1146, as shown by their higher efficiency in competing for binding of AF647-labeled PepSP1146. The peptide dimerized via the bis-PEG18 linker (PepSP1172) was shown to be the most efficient binder. The data in Table 7 below are kinetic parameters measured from curve fitting of the peptide SPR sensorgrams.

[0160] [Table 7]

[0161] The binding data for dimeric peptides PepSP1344, 1371, 1384, 1462, 1487, 1488 and 1489 are shown in Table 8 below. The data in the table was determined from curve fitting of the SPR sensorgrams of the peptides. As can be seen from the data, dimerization significantly improved DLL3.

[0162] [Table 8]

[0163] Four fluoro-labeled conjugates (PepSP1538, PepSP1581, PepSP1582 and PepSP1583) were tested in a single point SPR binding assay against rhDLL3-Fc to measure the complex stability (koff) of each molecule. The results show the peptides as tight rhDLL3-Fc binders with stable complex half-lives (Table 9).

[0164] [Table 9]

[0165] Example 4 Binding and Cross-Reactivity of DLL3-Binding Peptides Materials and Methods: DLL3 expressing cells used in the experiments included CHO cells expressing human (hu), cynomolgus monkey (cyno), mouse (mu) and rat DLL3. CHO DHFR cells and CHO cells expressing human FLT3 were used as negative controls. DLL3 target expression was analyzed by flow cytometry. DLL3 expressing or DLL3 negative CHO cells listed above were suspended in FACS buffer (1x PBS + 1% fetal bovine serum) and incubated with 10μg / ml AMG757 for 40 minutes at 4°C. The cells were then washed twice in FACS buffer and incubated with allophycocyanin-labeled anti-human IgG Fcγ antibody for 20 minutes at 4°C. DLL3 cell surface expression was analyzed using an LSR Fortessa flow cytometer (Becton Dickinson) and FACSDiva software (Becton Dickinson). The percentage of CHO cells expressing DLL3 from different species is summarized in the table below.

[0166] [Table 10]

[0167] The peptide PepSP1462 identified herein was biotinylated. Anti-streptavidin antibody conjugated to AF488 was used to detect the biotinylated peptide. AF488-labeled peptide was detected directly.

[0168] The binding of the labeled PepSP1462 peptide to DLL3-expressing cells was tested by flow cytometry. A DLL3-negative cell line was used to evaluate the binding specificity of the labeled peptide for DLL3. DLL3-expressing and DLL3-negative cells were incubated for 30 minutes at 4°C in the presence or absence of 100 μM PepSP1462-biotin peptide. The cells were then washed twice with FACS buffer and incubated with anti-streptavidin antibody conjugated to AF488 (Thermo Fisher). As a control, the cells were incubated only with anti-streptavidin-Alexa Fluor488 antibody. The cells were then washed twice with FACS buffer and resuspended in FACS buffer containing 0.5 μg / ml propidium iodide. The cells were analyzed by flow cytometry using an LSR Fortessa and FACSDiva software (Becton Dickinson) or Flow Jo (Flow Jo LLC).

[0169] Figure 3 shows that PepSP1462, labeled with biotin and detected with anti-streptavidin-AF488 antibody, binds to CHO cells expressing human, mouse, cynomolgus monkey or rat DLL3, but not to CHO cells not transfected with DLL3 (CHO huFLT3). 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 show that PepSP1462 cross-reacts with DLL3 from other species.

[0170] This specification is best understood in light of the teachings of the references cited herein. The embodiments herein provide an explanation of the embodiments of the invention and should not be construed as limiting the scope of the invention. Those skilled in the art will readily recognize that many other embodiments are encompassed by the invention. All publications, patents, and sequences cited in this disclosure are incorporated by reference in their entirety. To the extent that the material incorporated by reference contradicts or is inconsistent with this specification, this specification takes precedence over any such material. The citation of any reference herein is not an admission that such reference is prior art to the present invention.

[0171] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the embodiments described above.

Claims

1. a) C-X1-X2-X3-X4-X5-X6-X7-X8-C (SEQ ID NO: 78) (In the formula, 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 A polypeptide comprising an amino acid sequence selected from:

2. 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 The polypeptide of claim 1, wherein X8 is T.

3. The polypeptide of claim 1, comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 23.

4. The polypeptide of claim 1, comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 7.

5. The polypeptide of claim 1 , wherein the amino acid sequence further comprises amino acid residues AETVEF or AETVE at the N-terminus of the amino acid sequence.

6. The polypeptide of claim 5, comprising the amino acid sequence of any one of SEQ ID NOs: 39 to 61.

7. The polypeptide of claim 5, comprising the amino acid sequence of any one of SEQ ID NOs: 39 to 45.

8. A polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 38.

9. 9. The polypeptide of claim 8, wherein the amino acid sequence further comprises amino acid residues AETVEF or AETVE at the N-terminus of the amino acid sequence.

10. 10. The polypeptide according to claim 9, comprising the amino acid sequence of any one of SEQ ID NOs: 39 to 76, preferably SEQ ID NOs: 39 to 61, more preferably SEQ ID NOs: 39 to 45.

11. The polypeptide of claim 1, which is modified at the N-terminus, C-terminus, or both.

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

13. The polypeptide of claim 11 , wherein the C-terminus of the polypeptide is amidated or aminated.

14. The polypeptide of claim 1, comprising a dimer of said amino acid sequence.

15. The polypeptide of claim 14, wherein the dimer is a homodimer.

16. The polypeptide of claim 14 , wherein the dimer comprises a first linker connecting the two amino acid sequences.

17. 17. The polypeptide of claim 16, wherein the first linker is independently a peptide linker or a non-peptide linker.

18. 18. The polypeptide of claim 17, wherein the first linker comprises an unnatural amino acid.

19. 18. The polypeptide of claim 17, wherein the first linker comprises a poly(ethylene glycol) (PEG) linker.

20. 20. The polypeptide of claim 19, 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 a combination thereof.

21. 17. The polypeptide of claim 16, wherein the first linker further comprises hPra, Lys(N)3, trioxatridecane-succinamic acid (Ttds), Gly-Gly, or a combination thereof.

22. A polypeptide described in any one of claims 1 to 21, further comprising a detectable agent.

23. The polypeptide described in claim 22, wherein the detectable agent is attached to the polypeptide via a second linker, a chelating agent, or a combination thereof.

24. The polypeptide described in claim 23, wherein the second linker is independently a peptide linker or a non-peptide linker.

25. The polypeptide described in claim 24, wherein the second linker comprises an unnatural amino acid.

26. The polypeptide described in claim 24, wherein the second linker comprises a poly(ethylene glycol) (PEG) linker.

27. The polypeptide of claim 26, 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 a combination thereof.

28. 24. The polypeptide of claim 23, wherein the second linker comprises a bicyclo[6.1.0]nonyne (BCN) group or a dibenzocyclooctyne (DBCO) group.

29. 23. The polypeptide of claim 22, wherein the detectable agent comprises a fluorescent agent or a radioisotope.

30. 30. The polypeptide of claim 29, wherein the fluorescent agent is Cy3, Cy5, fluorescein isothiocyanate (FITC), anthranilyl, 2-aminobenzoyl (Abz), 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), carboxytetramethylrhodamine (TAMRA), 5-(dimethylamino)naphthalene-1-sulfonyl (dansyl), 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (EDANS), or 7-methoxycoumarinyl-4-acetyl (Mca).

31. 30. The polypeptide of claim 29, wherein the radioactive isotope is 67Ga, 99mTc, 111In, 68Ga, 64Cu, 44Sc, 86Y, 89Zr, 18F, 125I, 123I, 124I or 203Pb.

32. 32. The polypeptide of claim 31, wherein the radioactive isotope is 18F.

33. 30. The polypeptide of claim 29, wherein the radioactive isotope is 47Sc, 114mIn, 177Lu, 90Y, 212 / 213Bi, 212Pb, 225Ac, 186 / 188Re, 67Cu, 131I, 227Th, 211At or 90Y.

34. The polypeptide of claim 1 that binds to DLL3.

35. 35. The polypeptide of claim 34, which binds to human DLL3 expressed on the surface of a cell.

36. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 21.

37. 37. The pharmaceutical composition of claim 36, further comprising N-tert-butyl-α-phenylnitrone (PBN), ethanol, sodium ascorbate, gentisic acid, or a combination thereof.

38. 37. The pharmaceutical composition of claim 36, having a pH of 4.5 to 8.

0.

39. 23. A composition for use in a method for detecting DLL3 in a sample, the composition comprising the polypeptide of claim 22, the method comprising contacting the composition with the sample and detecting DLL3 in the sample.

40. 40. The composition of claim 39, wherein the sample comprises cells that express DLL3.

41. 40. The composition of claim 39, wherein the DLL3 is human DLL3.

42. 42. The composition of claim 41 , wherein the cell is in a subject's body, and the method comprises administering the composition to the subject and detecting DLL3 in the subject using an imaging technique.

43. 43. The composition of claim 42, wherein the subject is a human with a DLL3-expressing tumor or cancer.

44. 43. The composition of claim 42, 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 lung 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.

45. 43. The composition of claim 42, wherein the imaging technique is a positron emission tomography (PET) scan.

46. 34. A composition for treating a DLL3-expressing tumor or cancer disease, comprising the polypeptide of claim 33, wherein the composition is administered to a subject in need of treatment for a DLL3-expressing tumor or cancer disease.

47. 47. The composition of claim 46, wherein the subject is a human.

48. 42. The composition of claim 41, wherein the administration is intravenous administration.

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