Compounds targeting fibroblast activation protein and uses thereof

Abstract

This invention relates to conjugates targeting fibroblast activation protein α (FAP), comprising formula I: Y-L-X, wherein Y is a chelating agent or cytotoxic drug, L is a linker, and X comprises formula A-[Z-AA]. 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7 The molecule of the cyclic peptide ⁻Z⁻B. A is the N-terminal structure. B is the C-terminal structure, and each of Z, AA1, AA2, AA3, AA4, AA5, AA6, and AA7 is an amino acid residue.

Description

Background of the Invention

[0002] Fibroblast activating protein (FAP), also known as prolyl endopeptidase, is an enzyme encoded by the human FAP gene. FAP is a cell surface serine protease that acts on various hormones and extracellular matrix components. FAP is highly upregulated in a variety of cancers and is frequently used as a biomarker for pro-tumor matrix activity. It has also been proposed as a molecular target for cancer therapy, and in recent years, in particular, a significant amount of research has been devoted to the design and testing of various FAP-targeting therapies.

[0003] FAP is a type II transmembrane glycoprotein, 760 amino acids long. It contains a very short cytoplasmic N-terminal portion (6 amino acids), a transmembrane region (amino acids 7-26), and a larger extracellular portion containing an α / β-hydrolase domain and an octet β-helix domain. FAP is a non-classical serine protease, and its active site is located in the extracellular portion of the protein. In humans and mice, it contains Ser... 624 Asp 702 His 734 The catalytic triplet is composed of these components.

[0004] FAP expression is typically low to undetectable in most normal adult tissues, but is highly upregulated in a variety of cancers, including almost all carcinomas and sarcomas. FAP is present in cancer cells or cancer-associated fibroblasts in stromal tissues, including mesenchymal stem cells (MSCs), CAFs, sarcoma, and melanoma cells. FAP expression is also upregulated in non-cancerous diseases and tissue remodeling, including stromal and mesenchymal stem cells during embryogenesis, wound healing, fibrotic responses, arthritis, atherosclerotic plaques, and ischemic cardiac tissue after myocardial infarction.

[0005] Compared to adjacent normal tissue, FAP expression is higher in the cancer microenvironment, making FAP a potential therapeutic target for drug delivery. In this context, radiopharmaceuticals targeting FAP have specific potential. Radiopharmaceuticals are radiolabeled drugs used for disease imaging and treatment. These drugs are engineered in the form of CLTs, where C is a chelating agent that stably complexes (i.e., tightly binds) radionuclides (e.g., Pb-212, Ac-225, Lu-177, Cu-64, Cu-67, Ga-68, Pb-203) that decay through various forms of radioactive decay modes (e.g., beta-particle emission, alpha-particle emission, positron emission, gamma-ray emission, Auger electron emission); T is the structure of the targeting molecule (e.g., peptide, antibody, small molecule, aptamer) engineered to bind to diseased cells, typically by binding to cell surface receptors (e.g., G protein-coupled receptors, type II glycoproteins, or other antigens that may be expressed intracellularly); and L is a molecular linker connecting the chelating agent C and the binding moiety T.

[0006] Imaging can be achieved using certain radionuclides that emit gamma rays, commonly used for diagnosing and monitoring diseases. Other radionuclides that emit particles such as beta and alpha particles are used to treat diseases like cancer. In some cases, radionuclides used to treat cancer or other diseases undergo further decay, forming a series of secondary radionuclides (often called daughter radionuclides or "daughters"), which may or may not be complexed by chelating agents. The preparation of radiopharmaceuticals involves the reaction of CLT precursors with radionuclides. Examples of radionuclides used for this purpose that have a series of daughter radionuclides in their series include Pb-212 and Ac-225.

[0007] The industry needs effective methods to treat cancer. The inventors of this application have discovered a method to treat cancer by targeting FAP with a unique radiolabeled compound.

[0008] Detailed description of the attached diagram

[0009] Figure 1 This is a graph showing the %ID / g of VMT-FAP-2-26 according to Example 1.

[0010] Figure 2 It is a graph showing %ID / g of VMT-FAP-2-30 according to Example 1.

[0011] Figure 3 It is a graph showing %ID / g of VMT-FAP-2-42 according to Example 1.

[0012] Figure 4 It is a graph showing %ID / g of VMT-FAP-2-43 according to Example 1.

[0013] Figure 5 It is a graph showing %ID / g of VMT-FAP-2-33 according to Example 1.

[0014] Figure 6 It is a graph showing %ID / g of VMT-FAP-2-36 according to Example 1.

[0015] Figure 7 It is a graph showing %ID / g of VMT-FAP-2-39 according to Example 1.

[0016] Figure 8 It is a graph showing %ID / g of VMT-FAP-2-48 according to Example 1.

[0017] Figure 9 It is a graph showing %ID / g of VMT-FAP-2-51 according to Example 1.

[0018] Figure 10 This is a graph showing the %ID / g of VMT-FAP-2-53 according to Example 1.

[0019] Figure 11 This is a graph showing %ID / g of VMT-FAP-2-55 according to Example 1.

[0020] Figure 12 It is a graph showing %ID / g of VMT-FAP-2-57 according to Example 1.

[0021] Figure 13 It is a graph showing %ID / g of VMT-FAP-2-59 according to Example 1.

[0022] Figure 14 This is a graph showing %ID / g of VMT-FAP-2-60 according to Example 1.

[0023] Figure 15 This is a graph showing %ID / g of VMT-FAP-2-61 according to Example 1.

[0024] Figure 16 This is a graph showing the %ID / g of VMT-FAP-2-62 according to Example 1.

[0025] Figure 17 This is a graph showing the %ID / g of VMT-FAP-2-67 according to Example 1.

[0026] Figure 18 This is a graph showing %ID / g of VMT-FAP-2-68 according to Example 1.

[0027] Figure 19 This is a graph showing %ID / g of VMT-FAP-2-69 according to Example 1.

[0028] Figure 20 This is a graph showing %ID / g of VMT-FAP-2-70 according to Example 1.

[0029] Figure 21 This is a graph showing the %ID / g of VMT-FAP-2-72 according to Example 1.

[0030] Figure 22 This is a graph showing the %ID / g of VMT-FAP-2-74 according to Example 1.

[0031] Figure 23 This is a graph showing %ID / g of VMT-FAP-2-75 according to Example 1.

[0032] Figure 24 This is a graph showing the %ID / g of VMT-FAP-2-76 according to Example 1.

[0033] Figure 25 It is a structural instance of the VMT-FAP-2-26 sequence.

[0034] Figure 26 It is a structural instance of the VMT-FAP-2-30 sequence.

[0035] Figure 27 It is a structural instance of the VMT-FAP-2-42 sequence.

[0036] Figure 28 It is a structural instance of the VMT-FAP-2-43 sequence.

[0037] Figure 29 It is a structural instance of the VMT-FAP-2-33 sequence.

[0038] Figure 30 It is a structural instance of the VMT-FAP-2-36 sequence.

[0039] Figure 31 It is a structural instance of the VMT-FAP-2-39 sequence.

[0040] Figure 32 It is a structural instance of the VMT-FAP-2-48 sequence.

[0041] Figure 33 It is a structural instance of the VMT-FAP-2-51 sequence.

[0042] Figure 34 It is a structural instance of the VMT-FAP-2-53 sequence.

[0043] Figure 35 It is a structural instance of the VMT-FAP-2-55 sequence.

[0044] Figure 36 It is a structural instance of the VMT-FAP-2-57 sequence.

[0045] Figure 37 It is a structural instance of the VMT-FAP-2-59 sequence.

[0046] Figure 38 It is a structural instance of the VMT-FAP-2-60 sequence.

[0047] Figure 39 It is a structural instance of the VMT-FAP-2-61 sequence.

[0048] Figure 40 It is a structural instance of the VMT-FAP-2-62 sequence.

[0049] Figure 41 It is a structural instance of the VMT-FAP-2-67 sequence.

[0050] Figure 42 It is a structural instance of the VMT-FAP-2-68 sequence.

[0051] Figure 43 It is a structural instance of the VMT-FAP-2-69 sequence.

[0052] Figure 44 It is a structural instance of the VMT-FAP-2-70 sequence.

[0053] Figure 45 It is a structural instance of the VMT-FAP-2-72 sequence.

[0054] Figure 46 It is a structural instance of the VMT-FAP-2-74 sequence.

[0055] Figure 47 It is a structural instance of the VMT-FAP-2-75 sequence.

[0056] Figure 48 It is a structural instance of the VMT-FAP-2-76 sequence.

[0057] Figure 49 It is a structural instance of the VMT-FAP-2-77 sequence.

[0058] Figure 50 It is a structural instance of the VMT-FAP-2-78 sequence.

[0059] Figure 51 It is a structural instance of the VMT-FAP-2-82 sequence.

[0060] Figure 52 It is a structural instance of the VMT-FAP-2-83 sequence.

[0061] Figure 53 It is a structural instance of the VMT-FAP-2-84 sequence.

[0062] Figure 54 This demonstrates the binding affinity of VMT-FAP-2-59 in hFAP according to Example 3.

[0063] Figure 55 The GCI binding test of VMT-FAP-2-59 in hPREP and VMT-FAP-2-59 in hDPPIV according to Example 3 is described.

[0064] Figure 56 The following describes the [in Example 4] 203 Metabolic stability of Pb]VMT-FAP-2-59 after incubation in human serum for 96 hours.

[0065] Figure 57 This illustrates the human cancer cell and human cancer xenograft model according to Example 5. 203 Intake of Pb]VMT-FAP-2-59.

[0066] Figure 58 This is based on the hFAP-HT1080 xenograft in athymic nude mice according to Example 5. 203 Pb]VMT-FAP-2-59 and [ 212 Micro-SPECT imaging of Pb]VMT-FAP-2-59.

[0067] Figure 59 This describes the use of [According to Example 6] 212 Tumor volume and weight in athymic nude mice carrying fFAP-HT1080 tumors after Pb]VMT-FAP-2-59 treatment.

[0068] Figure 60 This describes the use of [According to Example 7] 212 Tumor volume and weight in mice carrying U87MG tumors after Pb]VMT-FAP-2-59 treatment.

[0069] Figure 61 The treatment outcomes for patients with metastatic lung adenocarcinoma according to Example 8 are described.

[0070] Figure 62 This describes the treatment outcome for a patient with metastatic ampullary neuroendocrine tumor according to Example 8.

[0071] Figure 63 The treatment outcomes of a patient with chondroblastic osteosarcoma according to Example 8 are described. Invention Details

[0073] This overview is provided to introduce a selection of concepts, which will be further described in the detailed description below. This overview is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid to limiting the scope of the claimed subject matter.

[0074] In some embodiments, the present invention provides conjugates that bind to human and mouse fibroblast activation protein α (FAP), thereby releasing radiation or cytotoxic drugs to target cancer cells and other cells (e.g., tumor stromal cells) in the tumor microenvironment. According to the invention, the chelating agent or cytotoxic drug is linked to a molecule containing a cyclic peptide. Therefore, the resulting conjugate can bind to FAP and release radiation or cytotoxic drugs at the tumor site.

[0075] In one embodiment of the invention, the coupling includes formula I:

[0076] YLX, where:

[0077] -Y is a chelating agent or cytotoxic drug.

[0078] -L is a connector, and

[0079] -X contains the cyclic peptide A-[Z-AA] 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7 The molecule of -Z]-B, where:

[0080]

[0081] -A is an N-terminal modification;

[0082] -B is a C-terminal modification;

[0083] -Z represents an amino acid residue, where X is cyclic;

[0084] -C is the cyclization part, where Z is coupled;

[0085] -Y and L are coupled to X on A, B, C, Z, or C.

[0086] AA 1 It is an L-, D-, or β-homotype of Thr or Ser.

[0087] AA 2 It is either an L- or D-amino acid of Pro; in addition, AA 2It can be any of the following amino acids in L-, D-, or β- form: 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), pyroglutamic acid (Pyr), homotype Pro, 4-aminopiperidin-4-carboxylic acid, 2-aminobenzoic acid (2-Abz), 3-aminocyclopentanecarboxylic acid, trans-3-hydroxyproline (Hyp), cis-3-hydroxyproline, trans-4-hydroxyproline, cis-4-hydroxyproline, α-methyl-proline, α-benzylproline, 3,4-dehydroproline, 4-oxaproline, 3-thiaproline, and 4-thiaproline.

[0088] AA 3 It is an L-, D-, or β-homotype of Trp, Phe, Tyr, or His; in addition, AA 3 It can be β-Phe or β-Tyr. Furthermore, AA 3 It can be any of the following amino acids in L-, D-, or β-form form: 1-naphthylalanine (1-Nal), 2-naphthylalanine (2-Nal), 3-(4-biphenylalanine (Bip), 2-pyridylalanine (2-Pal), 3-pyridylalanine (3-Pal), 4-pyridylalanine (4-Pal), 3-benzothiophene alanine (Bta), 2-cyanophenylalanine, 3-cyanophenylalanine, 4-cyanophenylalanine, 3-boronphenylalanine, 4-boronphenylalanine, 4-trifluoromethylphenylalanine, 2-chlorophenylalanine, 3-chlorophenylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-(4-) -Fluorophenylalanine, 4-fluorophenylalanine, 2-iodophenylalanine, 3-iodophenylalanine, 4-iodophenylalanine, 2-methylphenylalanine, 3-methylphenylalanine, 4-methylphenylalanine, 2-nitrophenylalanine, 3-nitrophenylalanine, 4-nitrophenylalanine, 4-pentafluorophenylalanine, phenylglycine (Phg), 4-aminophenylalanine, 4-methoxyphenylalanine, 5-hydroxytryptophan, 3,5-diiodotyrosine, 4-benzoylphenylalanine, 5-hydroxytryptophan, 3,5-diiodotyrosine, 4-benzoylphenylalanine (Bpa), cyclohexylglycine (Chg), or 2-thienylalanine (Thi).

[0089] AA 4 It is an L-, D-, or β-homotype of Glu, Asp, Gln, or Apn; in addition, AA 4 It can be β-Glu or β-Gln.

[0090] AA 5It is an L-, D-, or β-homoamino acid of Gln, Ala, Ile, Val, Trp, Tyr, Phe, His, Lys, Arg, Asp, Glu, Ser, Thr, Asn, or Gly; in addition, AA 5 It can be β-Glu, β-Gln, β-Phe, β-Tyr, or β-Lys. Additionally, AA... 5 It can be any of the following amino acids: L-, D-, or β-homotypes: leucine (Nle), citrulline (Cit), and valine. Additionally, AA... 5 It can be an L-, D-, or β-homotype of the following amino acids: 1-naphthylalanine (1-Nal), 2-naphthylalanine (2-Nal), 3-(4-biphenylalanine (Bip), 2-pyridylalanine (2-Pal), 3-pyridylalanine (3-Pal), 4-pyridylalanine (4-Pal), 3-benzothiophene alanine (Bta), 2-cyanophenylalanine, 3-cyanophenylalanine, 4-cyanophenylalanine, 3-boronphenylalanine, 4-boronphenylalanine, 4-trifluoromethylphenylalanine, 2-chlorophenylalanine, 3-chlorophenylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3- Fluorophenylalanine, 4-fluorophenylalanine, 2-iodophenylalanine, 3-iodophenylalanine, 4-iodophenylalanine, 2-methylphenylalanine, 3-methylphenylalanine, 4-methylphenylalanine, 2-nitrophenylalanine, 3-nitrophenylalanine, 4-nitrophenylalanine, 4-pentafluorophenylalanine, phenylglycine (Phg), 4-aminophenylalanine, 4-methoxyphenylalanine, 5-hydroxytryptophan, 3,5-diiodotyrosine, 4-benzoylphenylalanine, 5-hydroxytryptophan, 3,5-diiodotyrosine, 4-benzoylphenylalanine (Bpa), cyclohexylglycine (Chg), or 2-thienylalanine (Thi).

[0091] AA 6 It is an L-, D-, or β-homotype of Trp, Phe, Tyr, Ser, or Thr. Additionally, AA... 6 It can be β-Phe or β-Tyr. Furthermore, AA 6It can be any of the following amino acids in L-, D-, or β-form form: 1-naphthylalanine (1-Nal), 2-naphthylalanine (2-Nal), 3-(4-biphenylalanine (Bip), 2-pyridylalanine (2-Pal), 3-pyridylalanine (3-Pal), 4-pyridylalanine (4-Pal), 3-benzothiophene alanine (Bta), 2-cyanophenylalanine, 3-cyanophenylalanine, 4-cyanophenylalanine, 3-boronphenylalanine, 4-boronphenylalanine, 4-trifluoromethylphenylalanine, 2-chlorophenylalanine, 3-chlorophenylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-(4-) -Fluorophenylalanine, 4-fluorophenylalanine, 2-iodophenylalanine, 3-iodophenylalanine, 4-iodophenylalanine, 2-methylphenylalanine, 3-methylphenylalanine, 4-methylphenylalanine, 2-nitrophenylalanine, 3-nitrophenylalanine, 4-nitrophenylalanine, 4-pentafluorophenylalanine, phenylglycine (Phg), 4-aminophenylalanine, 4-methoxyphenylalanine, 5-hydroxytryptophan, 3,5-diiodotyrosine, 4-benzoylphenylalanine, 5-hydroxytryptophan, 3,5-diiodotyrosine, 4-benzoylphenylalanine (Bpa), cyclohexylglycine (Chg), or 2-thienylalanine (Thi).

[0092] AA 7 It is an L-, D-, or β-homotype of Trp, Met, Phe, or Tyr. Additionally, AA... 7 It can be β-Phe or β-Tyr. Furthermore, AA 7It can be any of the following amino acids in L-, D-, or β-form form: 1-naphthylalanine (1-Nal), 2-naphthylalanine (2-Nal), 3-(4-biphenylalanine (Bip), 2-pyridylalanine (2-Pal), 3-pyridylalanine (3-Pal), 4-pyridylalanine (4-Pal), 3-benzothiophene alanine (Bta), 2-cyanophenylalanine, 3-cyanophenylalanine, 4-cyanophenylalanine, 3-boronphenylalanine, 4-boronphenylalanine, 4-trifluoromethylphenylalanine, 2-chlorophenylalanine, 3-chlorophenylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-(4-) -Fluorophenylalanine, 4-fluorophenylalanine, 2-iodophenylalanine, 3-iodophenylalanine, 4-iodophenylalanine, 2-methylphenylalanine, 3-methylphenylalanine, 4-methylphenylalanine, 2-nitrophenylalanine, 3-nitrophenylalanine, 4-nitrophenylalanine, 4-pentafluorophenylalanine, phenylglycine (Phg), 4-aminophenylalanine, 4-methoxyphenylalanine, 5-hydroxytryptophan, 3,5-diiodotyrosine, 4-benzoylphenylalanine, 5-hydroxytryptophan, 3,5-diiodotyrosine, 4-benzoylphenylalanine (Bpa), cyclohexylglycine (Chg), or 2-thienylalanine (Thi).

[0093] A is an N-terminal structure of X and includes the following formula:

[0094]

[0095] Where R 1 It is -(CH2) n -, n=0-6; R2 is any side chain residue of Gly, Ala, Ser, Met, Lys, Arg, Asp, Glu, Asn.

[0096] B is the C-terminal structure of X, and can be selected from: -CO-NH2, -COOH, -OH, or -CO-NH-(CH2). n -CH3, where n = 0-6

[0097]

[0098] In some embodiments, the C-terminal structure B is selected from any of the following amino acids: lysine, ornithine, 2,4-diaminobutyric acid (Dab), 2,3-diaminopropionic acid (Dap), or 2,2-diaminoacetic acid, wherein the side chain residues are used for coupling with the linker and chelating agent.

[0099] In some implementations, X contains the reverse sequence A-[Z-AA] 7 -AA 6 -AA5 -AA 4 -AA 3 -AA 2 -AA 1 -Z]-B, which loops at position Z through the looped portion C:

[0100]

[0101] X is cyclized at a side chain residue of amino acid Z, which comprises the following formula, where n = 0-4

[0102]

[0103] The cyclization part C is selected from the following structures, where n = 0-6.

[0104]

[0105] The R in these structures is selected from -H, -OH, -Br, -Cl, -I, and -(CH2). n -CH3 or -(CH2) n -SH-, where n = 0-6.

[0106] In some implementations, the cyclization portion C may also be selected from the following structures:

[0107]

[0108] In some implementations, Y and L are coupled to X via A or B, as shown in the following equation:

[0109]

[0110] In some implementations, Y and L are coupled to X via a cyclic portion C, as shown in the following equation:

[0111]

[0112] The cyclization part C is selected from the following structures, where n = 0-6:

[0113]

[0114] In some implementations, the cyclization portion C may also be selected from the following structures:

[0115]

[0116] In some implementations, X is metallized by technetium (Tc) or rhenium (Re).

[0117] In some embodiments, X is a homodimer or heterodimer that contains AA in at least one ring.1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7 sequence.

[0118] In some implementations, dimer X is cyclized via moiety C 2 Cycloning, where C 2 The following structures can be selected:

[0119]

[0120] L: Connector

[0121] In some embodiments, L is a chemical linker inserted between the peptide backbone that recognizes the FAP and the chelating agent for radiolabeling the composition using a radionuclide for diagnostic imaging and / or therapeutic purposes; and the linker improves the internalization of the composition into cells and improves the retention of the composition in tumors, thereby delivering radiation more precisely to cancerous tissue. In some embodiments, L is a hydrophobic linker composed of an aliphatic carbon chain linking the chelating agent and the peptide backbone.

[0122] In some embodiments, L is a hydrophilic linker comprising heteroatom substitutions in an aliphatic chain linking the chelating agent and the peptide backbone. In some embodiments, L is a mixture of hydrophilic and hydrophobic entities comprising piperidine or amino acid insertions to extend the chain and modulate the pharmacodynamic properties of the composition.

[0123] In some implementations, L is a polyethylene glycol linker PEGn, where n = 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0124] In some implementations, n is 2, 4, or 8 PEG subunits.

[0125] In some implementations, L is an aliphatic (ALP) linker with 2 or 4 carbon atoms.

[0126] In some implementations, L is a piperidine (PIP)-based linker with mixed properties.

[0127] In some embodiments, L is a mixture of hydrophilic and hydrophobic entities, including piperazine and amino acid inserts, to extend the chain and modulate the pharmacodynamic properties of the composition.

[0128] In some embodiments, L is a mixture of hydrophilic and hydrophobic entities, including benzene and amino acid insertions, to extend the chain and modulate the pharmacodynamic properties of the composition.

[0129] In some embodiments, L is a “cleavable” linker containing a mixture of natural amino acids Phe, Met, and Ile, which cleaves in the renal brush border membrane to facilitate clearance through the kidneys.

[0130] In some implementations, L may be coupled to an ε-amine of the lysine residue at the C-terminus of the peptide preceding Cys, where n may be equal to 0 to 4.

[0131]

[0132] In some implementations, L can be coupled to the N-terminus of A, or L can be coupled to the C portion.

[0133] In some embodiments, the C-terminus may be a carboxylic acid or an amide in the presence of B. In the absence of B, Cys may be a carboxylic acid or an amide.

[0134] In some embodiments, if the chelating agent and L are located on A, or if the chelating agent and L are located on C, the N-terminus may be modified with acetic acid, propionic acid, butyric acid, and incrementally added methyl groups, in the presence or absence of A, to increase hydrophobicity.

[0135] In some embodiments, peptide X is reacted with formula [chelating agent-L]. n Multiple chelating agents are coupled in -X, where the chelating agent-peptide ratio n = 2-4.

[0136] [ PubMed ] Bandari RP, Jiang Z, Reynolds TS, Bernskoetter NE, Szczodroski AF, Bassuner KJ, Kirkpatrick DL, Rold TL,Sieckman GL,Hoffman TJ,Connors JP,Smith C J.Synthesis and biological evaluation of radiolabeled copper-64[DUPA-6-Ahx-(NODAGA)-5-Ava-BBN(7-14)NH2],a novel bivalent targeting vector having affinity for two distinct biomarkers(GRPr / PSMA)of prostate cancer.Nucl Med Biol.2014:41(4):355-363.Doi:10.1016 / j.nucmedbio.2014.01.001.PubMed PMID:24508213;PMCID:PMC4041584:Dumont RA,Tamma M,Braun F,Borkowski S,Reubi JC,Maecke H,Weber WA,Mansi R.Targeted radiotherapy of prostate cancer with a gastrin-releasing peptide J NuclMed.2013:54(5):762-769.Doi:10.2967 / jnumed.112.112169.PubMed PMID:23492884;Gourni E,Mansi R,Jamous M,Waser B,Smerling C,Burian A,Buchegger F,Reubi JC,Maecke HR.N-terminal modifications improve the receptor affinity andpharmacokinetics of radiolabeled peptidic gastrin-releasing peptide receptorantagonists:examples of 68Ga-and64Cu-labeled peptides for PET imaging.J NuclMed.2014;55(10):1719-1725.D0i:10.2967 / jnumed.114.141242.PubMed PMID:25146125;Jamous M,Tamma M L,Gourni E,Waser B,Reubi J C,Maecke H R,Mansi R.PEG spacersof different length influence the biological profile of bombesin-basedradiolabeled antagonists.Nucl Med Biol.2014;41(6):464-470.Doi:10.1016 / j.nucmedbio.2014.03.014.PubMed PMID:24780298;Mansi R,Abiraj K,Wang X,Tamma ML,Gourni E,Cescato R,Berndt S,Reubi J C,Maecke H R.Evaluation of threedifferent families of bombesin receptor radioantagonists for targeted imagingand therapy of gastrin releasing peptide receptor(GRPR)positive tumors.J MedChem.2015;58(2):682-691.Doi:10.1021 / jm5012066.PubMed PMID:25474596;Pan D,XuYP,Yang R H,Wang L,Chen F,Luo S,Yang M,Yan Y.A new(68)Ga-labeled BBNpeptide with a hydrophilic linker for GRPR-targeted tumor imaging. AminoAcids. 2014; 46(6): 1481-1489. Doi: 10.1007 / s00726-014-1718-y. PubMed PMID: 24633452; Stott Reynolds TJ, Schehr R, Liu D, Xu J, Miao Y, Hoffman TJ, Rold TL, Lewis MR, Smith C J.Characterization and evaluation of DOTA-conjugatedBombesin / RGD-antagonists for prostate cancer tumor imaging and therapy. NuclMed Biol. 2015;42(2):99-108.Doi:10.1016 / j.nucmedbio.2014.10.002.PubMed PMID:25459113. .

[0137] Y: Payload of chelating agents, radionuclides, and cytotoxic drugs

[0138] In some embodiments, Y is a chelating substance (also referred to as a "chelating agent"). The chelating agent of the present invention is used to radiolabel the composition with a radioisotope to realize its dual potential in imaging diagnostics and therapeutic treatments.

[0139] In some implementations, X is used for radiolabeling of radionuclides for medical imaging and / or treatment of cancerous tumors.

[0140] In some embodiments, the chelating agent is radiometallized or radiolabeled with a radionuclide suitable for the treatment of FAP-positive malignancies and for radiological (or non-radiological) imaging.

[0141] In some implementations, peptides are used for radiolabeling of radionuclides in medical imaging and / or treatment of cancerous tumors.

[0142] In some embodiments, the radionuclide is selected from the following diagnostic radionuclides: Sc-43, Sc-44, Mn-51, Cu-64, Ga-67, Ga-68, Y-86, Zr-89, 99mTc, F-18, Br-76, Br-77, In-111, I-123, I-124, I-125, Tb-152, Pb-203.

[0143] In some embodiments, the radionuclide is selected from therapeutic radionuclides Sc-47, Cu-67, Sr-89, Y-90, I-131, Sm-153, Tb-149, Tb-161, Lu-177, Re-186, Re-188, At-211, Pb-212, Bi-212, Ra-223, Ra-224, Ac-225, Th-226, and Th-227.

[0144] In some implementations, the radionuclide is In-111, Pb-203; Cu-64, Ga-68, Zr-89, or other medical radionuclide used for imaging.

[0145] In some implementations, the radionuclide is Y-90, Pb-212, Bi-212, Bi-213, At-211, Ac-225, Lu-177, or other medical radionuclides used to treat cancerous tumors.

[0146] In some embodiments, the chelating agent Y is DOTA or other chelating agents for binding radionuclides used in diagnostic imaging or treatment of cancer or other diseases.

[0147] In some embodiments, the chelating agent Y is based on S-2-(4-nitrobenzyl)-1,4,7,10-tetraazacyclododecane or other variants of the cyclododecane.

[0148] In some embodiments, the chelating agent Y is based on 1,4,7,10-tetraazacyclododecane-1,4,7-tris(carbamoylmethyl)-10-acetic acid.

[0149] In some embodiments, the chelating agent Y is based on S-2-(4-nitrobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid.

[0150] In some embodiments, the chelating agent Y is based on S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid.

[0151] In some embodiments, the chelating agent Y is based on S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecanete-tert-butylacetate.

[0152] In some embodiments, the chelating agent is based on S-2-(4-benzyl isothiocyanate)-1,4,7,10-tetraazacyclododecanetetraacetic acid.

[0153] In some embodiments, the chelating agent Y is based on 1,4,7,10-tetraazacyclododecane-1,4,7-tritert-butylacetate-10-acetic acid.

[0154] In some embodiments, the chelating agent Y is based on 1,4,7,10-tetraazacyclododecane-1,4,7-tri-tert-butylacetate-10-succinimide acetate.

[0155] In some embodiments, the chelating agent Y is based on 1,4,7,10-tetraazacyclododecane-1,4,7-tri-tert-butylacetate-10-maleimide ethyl acetamide.

[0156] In some embodiments, the chelating agent Y is based on 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-10-maleimide ethyl acetamide.

[0157] In some embodiments, the chelating agent Y is based on 1,4,7,10-tetraazacyclododecane-1,4,7-tritert-butylacetate-10-(Na-Fmoc-Ne-acetamido-L-lysine).

[0158] In some embodiments, the chelating agent Y is based on 1,4,7,10-tetraazacyclododecane-1,4,7-tris(tert-butylacetate)-10-(3-butynylacetamide).

[0159] In some embodiments, the chelating agent Y is based on 1,4,7,10-tetraazacyclododecane-1,4,7-tris(tert-butylacetate)-10-(aminoethylacetamide).

[0160] In some embodiments, the chelating agent Y is based on 1,4,7,10-tetraazacyclododecane-1,4,7-tritert-butylacetate-10-(azidopropylethylacetamide).

[0161] In some embodiments, the chelating agent Y is based on 1,4,7,10-tetraazacyclododecane-1,4,7-tris(tert-butylacetate)-10-(4-aminobutyl)acetamide.

[0162] In some embodiments, the chelating agent Y is based on 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester.

[0163] In some embodiments, the chelating agent Y is based on 1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-(2-thioethyl)acetamide or other variants of DOTA.

[0164] In some embodiments, the chelating agent Y is based on S-2-(4-aminobenzyl)-diethylenetriaminepentaacetic acid or other variants of DTPA.

[0165] In some embodiments, the chelating agent Y is based on 3,6,9,15-tetraazabicyclo[9.3.1]pentadecano-1(15),11,13-triene-4-S-(4-aminobenzyl)-3,6,9-triacetic acid or other variants of the pentadecane macrocycle.

[0166] In some embodiments, the chelating agent Y is based on 1-oxa-4,7,10-tetraazacyclododecane-5-S-(4-aminobenzyl)-4,7,10-triacetic acid or other variants of an oxosubstituted macrocycle.

[0167] In some embodiments, the chelating agent Y is based on 2-S-(benzyl isothiocyanate)-1,4,7-triazacyclononane-1,4,7-triacetic acid or other variants of the cyclononane.

[0168] In some embodiments, the chelating agent Y is based on 1-(4-phenyl isothiocyanate)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxyamino)-6,11,17,22-tetraazaheptadecane]thiourea or other variants of deferoxamine.

[0169] In some embodiments, the chelating agent Y of the present invention is selected from PSC, DOTAM, DOTA, DO3A, DOTAGA, NOOTA, NODAGA, NODA-MPAA, HBED, TETA, CB-TE2A, DTPA, DFO, Macropa, HOPO, TRAP, THP, DATA, NOTP, sarcophagine, FSC, NETA, H4octapa, Pycup, NxS4-x (N4, N2S2, N3S), mTc(CO)3- chelating agent, DMSA, HIDA, PYP, HYNIC, and MDP. In some embodiments, the chelating agent is selected from PSC, DOTA, DOTAM, DO3A, and Macropa.

[0170] In some embodiments, F-18, I-123, I-124, I-125, I-131, and At-211 are directly coupled to X without a chelating agent, for example:

[0171]

[0172] In some embodiments, I-123, I-124, I-125, and I-131 are coupled to X via aryliodine bonds to form monoiodine or diiodine.

[0173] In some implementations, At-211 is coupled to X via an aryl-astatine bond.

[0174]

[0175] In some implementations, At-211 is coupled to X via a boratite bond.

[0176]

[0177] In some implementations, F-18, I-123, I-124, I-125, I-131, and At-211 are coupled to X via connectors.

[0178] In some implementations, F-18, I-123, I-124, I-125, I-131, and At-211 are coupled to X without a connector.

[0179] In some implementations, Y is a cytotoxic drug payload that is conjugated to X via A, B, or C to form a peptide drug conjugate.

[0180] In some implementations, the peptide drug conjugate delivers the cytotoxic drug payload in the form of a prodrug.

[0181] In some implementations, the cytotoxic drug payload is coupled to X via a cleavable linker.

[0182] In some embodiments, the cleavable linker can be an ester, amide, carbamate, dipeptide, tripeptide, hydrazone, or disulfide, with the following structure:

[0183]

[0184] In some implementations, the cytotoxic drug payload is released as free drug after linker lysis.

[0185] In some implementations, the cytotoxic drug payload is coupled to X via a non-cleavable linker.

[0186] In some embodiments, the cytotoxic drug payload may be paclitaxel, govitecan, auristatins, epothilone, maytansinoids, taxoids, tubulolysins, vinorelbine, mertansine (DM1), monomethyl auristatin E (MMAE), docetaxel, doxorubicin, thapsigargin, melphalan, 5-fluorouracil, calicheamicins, duocarmycin analogs, pyrrolobenzodiazepine, topotecan, or bleomycin A2. A2), dactinomycin D and / or mitomycin C.

[0187] In some implementations, the albumin-binding portion is coupled to X via A, B, or C to prolong the biological half-life in circulation and enhance accumulation in tumors.

[0188] In some embodiments, the albumin-binding portion may be a naphthalene-2-sulfonamide derivative, an Evans blue derivative, a 4-(p-iodophenyl)butyric acid derivative, a palmitic acid derivative, or a maleimide derivative.

[0189] Drug application, administration, and combined use with other anticancer drugs

[0190] In some embodiments, the present invention provides a method for treating cancer in patients in need, the method comprising administering the above-described conjugate. In some embodiments, the cancer is FAP-positive cancer or a malignant tumor. In some embodiments, the cancer is a solid tumor, including but not limited to sarcoma, salivary gland cancer, esophageal cancer, bile duct cancer, breast cancer, lung cancer, prostate cancer, pancreatic cancer, thymic cancer, head and neck cancer, ovarian cancer, desmoid tumor, chordoma, colorectal cancer, anal cancer, neuroendocrine tumors, small bowel cancer, medullary thyroid cancer, cervical cancer, endometrial cancer, hepatocellular carcinoma, gastric cancer, adenoid cystic cancer, pheochromocytoma, differentiated thyroid cancer, insulinoma, renal cancer, and skin cancer.

[0191] In some embodiments, the method also includes administering an anticancer composition.

[0192] In some embodiments, the anticancer composition includes, but is not limited to, phenylbutyric acid (PBA) or a pharmaceutically acceptable salt thereof, chloroquine, hydroxychloroquine (laquenil, Axemal (India), Dolquine and...

[0193] Quensyl), or as an antimalarial drug or a medicinal product used to overcome autophagy-related drug resistance by inhibiting the interaction between lysosomes and autophagosomes; triphenyl (TPP) derivatives, PBA, histone deacetylation inhibitors, MAPK pathway inhibitors (e.g., MEK inhibitors, RAS inhibitors, and / or RAF inhibitors).

[0194] In some embodiments, the invention further includes the administration of immunotherapy targeting immune system regulatory proteins. In some embodiments, the immunotherapy includes an anti-CTLA-4 monoclonal antibody, a Toll-like receptor (TLR) agonist, a CD40 agonist, and / or an anti-ganglioside monoclonal antibody. In some embodiments, the immunotherapy includes CTLA-4 and PD-1 inhibitors.

[0195] In some embodiments, the invention further includes the application of a radiosensitizer to enhance tumor-killing efficacy. In some embodiments, the radiosensitizer includes hyperbaric oxygen, carbogen, nicotinamide, metronidazole, mitomycin-C, tirapazamine, procaine, lidocaine, chlorpromazine, 5-fluorouracil, fludeoxyuridine, bromodeoxyuridine, iododeoxyuridine, hydroxyurea, gemcitabine, fludarabine, motexafin (gadolinium), N-ethylmaleimide, diamide, diethyl maleate, hyperthermia, paclitaxel, docetaxel, and irinotecan.

[0196] In some embodiments, the present invention further includes administering a DNA damage repair inhibitor to enhance tumor-killing efficacy. In some embodiments, the DNA damage repair inhibitor includes PARP inhibitors, ATR inhibitors, CHK inhibitors, WEE1 inhibitors, ATM inhibitors, and HDAC inhibitors.

[0197] In some implementations, the conjugate is administered intravenously or parenterally.

[0198] In some embodiments, the method also includes administering an anticancer composition.

[0199] In some implementations, the conjugate is administered in a single dose.

[0200] In some implementations, the conjugate is administered in multiple doses.

[0201] In some implementations, the conjugate is applied daily for several days.

[0202] In some implementations, the conjugate is applied once a week for one month.

[0203] In some implementations, the conjugate is applied once a week for up to 6 months.

[0204] In some implementations, the conjugate is administered at a dose of 1 mCi for medical imaging.

[0205] In some implementations, the conjugate is administered at a dose of up to 10 mCi for medical imaging.

[0206] In some implementations, the conjugate is administered at a dose of up to 50 mCi for medical imaging.

[0207] In some implementations, the conjugate is administered at a dose of 0.1 mCi for the medical treatment of cancerous tumors.

[0208] In some implementations, the conjugate is administered at a dose of up to 5 mCi for the medical treatment of cancerous tumors.

[0209] In some implementations, the conjugate is administered at a dose of up to 10 mCi for the medical treatment of cancerous tumors.

[0210] In some implementations, the conjugate is administered at a dose of up to 100 mCi for the medical treatment of cancerous tumors.

[0211] In some implementations, the conjugate is administered at a dose of up to 200 mCi for the medical treatment of cancerous tumors.

[0212] In some implementations, the coupling agent is applied for more than a month.

[0213] In some implementations, the coupling agent is applied for more than one year.

[0214] In some implementations, the conjugate is administered at a dose of at least 1500 mg / day.

[0215] In some embodiments, the present invention provides a medicine box comprising the above-described conjugate, a container, and a package insert or label indicating the administration of the conjugate for the treatment of FAP-positive cancers.

[0216] In some embodiments, the present invention provides a product comprising the above-described conjugate for the treatment of FAP-positive cancers.

[0217] In some embodiments, the present invention provides the use of the above-described conjugate and one or more anticancer active agents for the treatment of melanoma.

[0218] In some embodiments, the present invention provides for the use of the above-described coupling, wherein:

[0219] a) The conjugate is administered simultaneously with one or more anticancer active agents; or

[0220] b) The conjugate and one or more anticancer active agents are administered sequentially; or

[0221] c) Administer the conjugate approximately 1 to 10 days after initiating treatment of one or more anticancer active agents; or

[0222] d) Administer one or more anticancer active agents approximately 1 to approximately 10 days after initiating the administration of its conjugate; or

[0223] e) On the same day, the administration of the conjugate and one or more anticancer active agents shall commence.

[0224] In some embodiments, the present invention provides a method for treating cells with upregulated FAP expression compared to comparable wild-type cells, the method comprising contacting the cells with the aforementioned peptide or conjugate.

[0225] In some implementations, the upregulation is a result of cancer.

[0226] In some implementations, the upregulation is a result of carcinoma.

[0227] In some implementation schemes, the upregulation is a result of sarcoma, salivary gland cancer, esophageal cancer, bile duct cancer, breast cancer, lung cancer, prostate cancer, pancreatic cancer, thymic cancer, head and neck cancer, ovarian cancer, desmoid tumor, chordoma, colorectal cancer, anal cancer, neuroendocrine tumors, small bowel cancer, medullary thyroid cancer, cervical cancer, endometrial cancer, hepatocellular carcinoma, gastric cancer, adenoid cystic carcinoma, pheochromocytoma, differentiated thyroid cancer, insulinoma, kidney cancer, and skin cancer.

[0228] In some implementations, the molecule is a radiolabeled peptide.

[0229] In some embodiments, the present invention provides a method for treating hyperplastic diseases in patients in need, the method comprising (a) administering an active agent that increases binding with FAP.

[0230] In some implementations, hyperplastic diseases include sarcoma, salivary gland cancer, esophageal cancer, bile duct cancer, breast cancer, lung cancer, prostate cancer, pancreatic cancer, thymic cancer, head and neck cancer, ovarian cancer, desmoid tumor, chordoma, colorectal cancer, anal cancer, neuroendocrine tumors, small bowel cancer, medullary thyroid cancer, cervical cancer, endometrial cancer, hepatocellular carcinoma, gastric cancer, adenoid cystic carcinoma, pheochromocytoma, differentiated thyroid cancer, insulinoma, kidney cancer, and skin cancer.

[0231] In some implementations, the molecule is a radiolabeled peptide.

[0232] Treatment using the complex of the present invention in combination with FAP can effectively detect and / or destroy cancer cells. In some embodiments, the cancer is resistant to the drug. As used herein, the term "drug resistance" refers to a reduced effectiveness of the drug in killing malignant cells; reducing the size and growth rate of cancerous tumors; and improving symptoms of the disease or condition. In some embodiments, the effectiveness of the drug is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% compared to the effect when first administered to a mammal.

[0233] In some embodiments, the present invention provides a method for treating cells with upregulated FAP expression compared to comparable wild-type cells, the method comprising contacting the cells with the aforementioned peptides and / or conjugates.

[0234] In some implementations, the radiolabeled conjugate is administered weekly for one month.

[0235] In some implementations, the radiolabeled conjugate is administered weekly for up to 6 months.

[0236] In some implementations, radiolabeled conjugates are administered at a dose of 1 mCi for medical imaging.

[0237] In some implementations, radiolabeled conjugates are applied at doses of up to 10 mCi for medical imaging.

[0238] In some implementations, radiolabeled conjugates are applied at doses of up to 50 mCi for medical imaging.

[0239] In some implementations, radiolabeled conjugates are administered at a dose of 0.1 mCi for the medical treatment of cancerous tumors.

[0240] In some implementations, radiolabeled conjugates are administered at doses of up to 1 mCi for the medical treatment of cancerous tumors.

[0241] In some implementations, radiolabeled conjugates are administered at doses of up to 10 mCi for the medical treatment of cancerous tumors.

[0242] In some implementations, radiolabeled conjugates are administered at doses of up to 100 mCi for the medical treatment of cancerous tumors.

[0243] In some implementations, the coupling agent is applied for more than a month.

[0244] In some implementations, the coupling agent is applied for more than one year.

[0245] In some implementations, the radiolabeled conjugate is administered at a dose of at least 1500 mg / day.

[0246] As used herein, “amino acid” or “amino acid sequence” includes oligopeptide, peptide, polypeptide, or protein sequences, or any fragments, portions, or subunits of these, as well as naturally occurring or synthetic molecules. The terms “polypeptide” and “protein” include amino acids interconnected by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain modified amino acids in addition to the 20 genetically encoded amino acids.

[0247] The term "polypeptide" also includes peptides and polypeptide fragments, motifs, etc. The uppercase abbreviation for amino acids refers to the natural L isomer. The lowercase abbreviation for amino acids represents the D isomer.

[0248] The terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to amino acid polymers of any length. Peptides and polypeptides can consist entirely of synthetic, non-natural amino acid analogs, or are chimeric molecules comprising a portion of natural peptide amino acids and a portion of non-natural amino acid analogs. In one aspect, polypeptides are used in the compositions, cell systems, or methods of the present invention (e.g., host cells having plasmids expressing at least one enzyme of the present invention). Furthermore, polypeptides can refer to compounds consisting of amino acid polymers covalently linked to another functional group (e.g., a solubilizing group, a targeting group, PEG, a non-amino acid group, or other therapeutic agents).

[0249] Amino acids and non-natural amino acids can be abbreviated using the names in parentheses below: proline (Pro)(P), valine (V), lysine (Lys)(K), ornithine (Orn)(O), leucine (Nle), glycine (Gly)(G), tryptophan (Trp)(W), alanine (Ala)(A), phenylalanine (Phe)(F), arginine (Arg)(R), histidine (His)(H), glutamic acid (Glu)(E), aspartic acid (Asp)(D), serine (S)(S), methionine (Met)(M), isoleucine (Ile)(I), tyrosine (Tyr)(Y), cyclohexylalanine (Cha), 4-fluoro-D-phenylglycine (4-fluoro-D-Phg), 2-thienyl-D-alanine (D-Thi), cysteine ​​(Cys)(C), threonine (Thr)(T), asparagine (Asn)(N), and glutamine (Gln)(Q).

[0250] The polypeptide compositions of the present invention may contain any combination of non-natural structural components. Individual peptide residues may be linked by Fmoc-based solid-phase synthesis. Linking groups that may replace conventional amide bonds (“peptide bonds”) include, for example, ketomethylene (e.g., -C(=O)-CH2-, for -C(=O)-NH-), aminomethylene (CH2-NH), ethylene, olefin (CH=CH), ether (CH2-O), thioether (CH2-S), tetrazolium, thiazole, reverse amide, thioamide, or ester (see, for example, Spatola (1983), Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp. 267-357, “Peptide Backbone Modifications,” Marcel Dekker, NY, incorporated herein by reference).

[0251] The polypeptides used to implement the methods of this invention can be modified by natural methods, such as post-translational processing (e.g., phosphorylation, acylation, etc.), or by chemical modification techniques to obtain modified polypeptides. Modifications can occur at any position on the polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl terminals. It should be understood that the same type of modification may be present at several sites on a given polypeptide to the same or different degrees. A given polypeptide may also have multiple types of modifications. Modifications include N-methylation, acetylation, acylation, ADP-ribosylation, amidation, covalent linkage of flavins, covalent linkage of heme moieties, covalent linkage of nucleotides or nucleotide derivatives, covalent linkage of lipids or lipid derivatives, covalent linkage of phosphatidylinositol, cross-linking cyclization, disulfide bond formation, demethylation, covalent cross-linking, cysteine ​​formation, pyroglutamic acid formation, formylation, γ-carboxylation, glycosylation, GPI anchoring, hydroxylation, iodination, methylation, myristylation, oxidation, PEGylation, proteolytic processing, phosphorylation, isopentenylation, selenization, sulfation, and transfer RNA-mediated addition of amino acids to proteins, such as arginylation. See, for example, Creighton, TE, Proteins-Structure and Molecular Properties, 2nd ed., WH Freeman and Company, New York (1993); Posttranslational Covalent Modification o f Proteins, edited by BC Johnson, Academic Press, New York, pp. 1-12 (1983), incorporated herein by reference.

[0252] The "bioactivity" section includes molecules or compounds that trigger or regulate physiological responses.

[0253] "Regulation" and "modulation" refer to the upregulation or downregulation of the activity of one or more proteins or protein subunits, such that their expression, level, or activity is greater or less than that observed in the absence of a regulator. For example, the term "regulation" can mean "inhibition" or "stimulation."

[0254] "N-terminal sequence" refers to the starting point of an amino acid chain that is typically, but not necessarily, terminated by a free amine (-NH2) group. The convention for writing peptide sequences is to place the N-terminus on the left and write the sequence from the N-terminus to the C-terminus. The N-terminal sequence can contain 1 to 100 amino acids, preferably 2 to 15 amino acids, and even more preferably 3 to 10 amino acids. The N-terminal sequence can be terminated with an amino group, or the end can be modified by methods known in the art to include functional members (e.g., targeting groups, retention signals, lipids, and anchors).

[0255] "C-terminal sequence" refers to the end of an amino acid chain that is typically, but not necessarily, terminated by a carboxyl group. The convention for writing peptide sequences is to place the C-terminus on the right and write the sequence from the N-terminus to the C-terminus. The C-terminal sequence may contain 1 to 100 amino acids, preferably 2 to 15 amino acids, and even more preferably 3 to 10 amino acids. The C-terminal sequence may be terminated by a carboxyl group, or the end may be modified by methods known in the art to include functional members (e.g., targeting groups, retention signals, lipids, and anchors).

[0256] Anticancer active agents / cytotoxic active agents

[0257] As used herein, the terms “anticancer active agent” or “cytotoxic active agent” include therapeutic agents that kill cancer cells; slow tumor growth and cancer cell proliferation; and improve or prevent symptoms of one or more cancers. Anticancer active agents include pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the aforementioned compounds and includes pharmaceutically acceptable acid addition salts and base addition salts. Suitable pharmaceutically acceptable acid addition salts can be prepared from inorganic or organic acids. Examples of such inorganic acids are hydrochloric acid, sulfuric acid, and phosphoric acid. Suitable organic acids can be selected from aliphatic, cyclic aliphatic, aromatic, heterocyclic carboxylic acids, and sulfonic acids, examples of which are formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, fumaric acid, maleic acid, alkyl sulfonic acids, and aryl sulfonic acids. Further information on pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 19th edition, Mack Publishing Co., Easton, Pa. 1995. When the surfactant is a solid, those skilled in the art should understand that the compounds, surfactants, and salts of the present invention can exist in different crystalline or polymorphic forms, all of which should fall within the scope of the present invention and its specific formulations.

[0258] In some implementations, the anticancer / cytotoxic active agent is an MAPK pathway inhibitor, including but not limited to cobimetinib, dabrafenib, and / or trametinib.

[0259] In some implementations, the cytotoxic agent is conjugated to X via A, B, or C to form a peptide drug conjugate.

[0260] In some embodiments, the cytotoxic agent may be one or more of the following: paclitaxel, gavitenac, auristatin, epothilone, maytansine, taxanes, tubulolysin, vinorelbine, maytansine (DM1), monomethyl auristatin E (MMAE), docetaxel, doxorubicin, carotenoids, melphalan, 5-fluorouracil, cazithromycin, pyrrolobenzodiazepines, topotecan, bleomycin A2, actinomycin D, or mitomycin C.

[0261] In some implementations, the albumin-binding portion is coupled to X via A, B, or C to prolong the biological half-life in circulation and enhance accumulation in tumors.

[0262] In some embodiments, the albumin-binding portion may be one or more of a naphthalene-2-sulfonamide derivative, an Evans blue derivative, a 4-(p-iodophenyl)butyric acid derivative, a palmitic acid derivative, or a maleimide derivative.

[0263] Composition and method of application

[0264] The present invention provides a method for enhancing the anticancer effect of conventional cancer therapies (i.e., radiotherapy and / or chemotherapy) on mammalian cancer cells, the method comprising contacting the cancer cells with an effective amount of the conjugate as described above.

[0265] In some embodiments, the conjugate is administered in conjunction with other conventional cancer treatments. In some embodiments, the additional cancer treatment is chemotherapy and / or radiation therapy. In some embodiments, the conjugate and the anticancer active agent of the present invention are administered sequentially to a mammal, rather than as a single composition. In some embodiments, the mammal is a human.

[0266] The present invention provides a method for enhancing the anticancer effect of conventional cancer therapies (i.e., radiotherapy and / or chemotherapy) on mammalian cancer cells, the method comprising contacting cancer cells with an effective amount of an active agent that increases FAP expression.

[0267] The term "therapeutic effective amount" or "effective amount" is an amount sufficient to achieve a beneficial or desired clinical outcome. Effective amounts can be administered in the form of a single or multiple doses. Effective amounts are generally sufficient to alleviate, improve, stabilize, reverse, slow, or delay the progression of a disease state. This invention provides for "substantially pure compounds." The term "substantially pure compound" is used herein to describe a molecule, such as a polypeptide, which is substantially free of other proteins, lipids, carbohydrates, nucleic acids, and other biological materials naturally associated with it. For example, a substantially pure molecule (e.g., a polypeptide) may constitute at least 60% of the dry weight of the target molecule. The purity of a polypeptide can be determined using standard methods, including, for example, polyacrylamide gel electrophoresis (e.g., SDS-PAGE), column chromatography (e.g., high-performance liquid chromatography (HPLC)), and N-terminal amino acid sequence analysis.

[0268] As used in this article, “treatment,” “manipulation,” or “therapy” refers to the administration of an active agent to a mammal that can induce preventive, therapeutic, or other beneficial effects in an individual. Treatment may also alleviate or improve a subject’s disease or disease symptoms.

[0269] In some embodiments, the conjugate is administered in conjunction with other conventional cancer treatments. In some embodiments, the other cancer treatment is chemotherapy and / or radiation therapy. In some embodiments, the conjugate and the anticancer active agent are administered sequentially to a mammal, rather than as a single composition. In some embodiments, the mammal is a human.

[0270] In some embodiments of the above methods, the tumor volume is reduced by at least 10%. In some embodiments, the tumor is reduced by any amount between 1% and 100%. In some embodiments, the tumor uptake of a molecular imaging agent (e.g., fluoro-18deoxyglucose, fluoro-18thymidine, or other suitable molecular imaging agents) is reduced by any amount between 1% and 100%. In some embodiments, the imaging agent is fluoro-18deoxyglucose, fluoro-18thymidine, or other suitable molecular imaging agents. In some embodiments, mammalian symptoms (e.g., flushing, nausea, fever, or other symptoms associated with cancerous disease) are relieved.

[0271] It may be appropriate to administer the compound in the form of a pharmaceutically acceptable acid or base salt. Examples of pharmaceutically acceptable salts are addition salts of organic acids that form physiologically acceptable anions, such as toluenesulfonates, methanesulfonates, acetates, citrates, malonates, tartrates, succinates, benzoates, ascorbic acid salts, α-ketoglutarate, and α-glycerophosphates. Suitable inorganic salts may also be formed, including hydrochlorides, sulfates, nitrates, bicarbonates, and carbonates.

[0272] Pharmaceutically acceptable salts can be obtained using standard procedures known in the art, such as by reacting a sufficiently basic compound (e.g., an amine) with a suitable acid that provides a physiologically acceptable anion. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared.

[0273] The conjugates of the present invention can be formulated as pharmaceutical compositions and administered to mammalian hosts, such as human patients, via intravenous, intramuscular, local, or subcutaneous routes in various forms suitable for a chosen route of administration.

[0274] The active compound can also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, isotonic saline, phosphate-buffered saline, and optionally mixed with a non-toxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, triacetin, mixtures thereof, and in oils.

[0275] In some implementations, the final solution contains 0 to 20% v / v ethanol.

[0276] In some implementations, the final pH of the solution is 2-9.

[0277] In some embodiments, the radioactive compound is administered together with additional amino acids to reduce the renal uptake of the radioactive compound. In some embodiments, the amino acids are lysine and arginine.

[0278] Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.

[0279] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions, dispersions, or sterile powders containing the active ingredient, suitable for the ad hoc preparation of sterile solutions or dispersions for injection or infusion, optionally encapsulated in liposomes. In all cases, the final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or medium may be a solvent or liquid dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glycerides, and suitable mixtures thereof. For example, appropriate flowability can be maintained by forming liposomes, maintaining the desired particle size in the case of dispersions, or using surfactants. Microbial action can be prevented by various antimicrobial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, isotonic agents, such as sugars, buffers, or sodium chloride, are preferred. The absorption of the injectable composition can be prolonged by using substances that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0280] A sterile injectable solution is prepared by mixing the desired amount of the active compound with various other ingredients listed above (as needed) in a suitable solvent, followed by sterilization through filtration. In the case of sterile powders used to prepare the sterile injectable solution, a preferred preparation method is vacuum drying and freeze-drying techniques to obtain the active ingredient powder while retaining any other desired components present in the previously sterile filtered solution.

[0281] For topical application, the compounds of the present invention can be applied in their pure form, i.e., when they are liquids. However, it may be desirable to apply the compounds of the present invention in combination with a dermatologically acceptable carrier (which may be solid or liquid) to the skin, either as a composition or a formulation.

[0282] Useful solid carriers include finely crushed solids such as talc, clay, microcrystalline cellulose, silica, alumina, etc. Useful liquid carriers include water, alcohol, or ethylene glycol or water-alcohol / ethylene glycol mixtures, wherein the compounds of the present invention can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Fragrances and other adjuvants such as antimicrobial agents can be added to optimize performance for a given application. The resulting liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using a pump or aerosol sprayer.

[0283] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified cellulose, or modified mineral materials can also be used with liquid carriers to form spreadable pastes, gels, ointments, soaps, etc., for direct application to the user's skin.

[0284] Examples of useful skin compositions that can be used to deliver the compounds of the present invention to the skin are known in the art; see, for example, Jacquet et al. (US Patent No. 4,608,392), Geria (US Patent No. 4,992,478), Smith et al. (US Patent No. 4,559,157) and Wortzman (US Patent No. 4,820,508).

[0285] The dosage of the conjugate of this invention varies depending on the subject's age, weight, and condition. Treatment may begin with a low dose below the optimal dose, then the dose may be increased until the desired effect is achieved, or even the optimal effect in that case. Generally, the dose for patients weighing less than 20 kg is approximately 450-600 mg / kg / day, and the dose for heavier patients is approximately 9.9-13.0 g / m². 2 / day. However, higher or lower doses may also be considered, and thus within the scope of this invention. Healthcare professionals may prescribe small doses and observe the effect on the patient's symptoms. Subsequently, they may increase the dose if appropriate. Generally, the active agents of this invention can be administered at concentrations that provide effective results without causing any excessively harmful or toxic side effects, and can be administered in the form of a single unit dose, or, if desired, in convenient subunits administered at appropriate times.

[0286] The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. For example, the therapeutic agent can be directly introduced into the target cancer by direct injection. Other examples of routes of administration include oral, parenteral, such as intravenous, slow infusion, intradermal, subcutaneous, oral (e.g., ingestion or inhalation), transdermal (topical), transmucosal, and rectal administration. Such compositions typically contain a pharmaceutically active agent and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration and diet-based food forms. The use of such media and agents for pharmaceutically active substances is well known in the art, and food as a medium of administration is also well known in the art.

[0287] Solutions or suspensions may include the following components: sterile diluents, such as water for injection, saline solutions (e.g., phosphate-buffered saline (PBS)), non-volatile oils, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), glycerol, or other synthetic solvents; antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.; antioxidants, such as ascorbic acid, gentianic acid, sodium bisulfite; chelating agents, such as EDTA, DTPA, DMSA, DMPS; buffers, such as acetates, citrates, or phosphates; and substances for regulating tension, such as sodium chloride or glucose. For example, appropriate flowability can be maintained by using a coating layer such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. In many cases, isotonic agents, such as sugars, polyols such as mannitol or sorbitol, and sodium chloride are preferably included in the composition. Extended administration of the injectable composition can be achieved by including substances that delay absorption. For example, such active agents include, for example, aluminum monostearate and gelatin. Parenteral preparations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0288] Formulating the composition into dosage units facilitates ease of administration and dosage uniformity. The dosage unit form used herein refers to a physically discrete unit suitable as a unit dose for an individual to be treated; each unit contains a predetermined amount of the active compound, calculated to produce the desired therapeutic effect when used with the desired drug carrier. The dosage unit form of the present invention depends on the amount of compound required to produce the desired effect. The required amount of compound can be formulated as a single dose or as multiple dosage units. Treatment may require a single dose or repeated doses.

[0289] As used herein, "systemic delivery" refers to the delivery of an active agent or composition resulting in its widespread biodistribution within a living organism. Some administration techniques can lead to systemic delivery of certain active agents, but not others. Systemic delivery means exposing a useful, preferably therapeutic, amount of active agent to most parts of the body. Achieving widespread biodistribution typically requires a certain blood lifetime, ensuring that the active agent is not rapidly degraded or cleared (e.g., via first-pass organs (liver, lungs, etc.) or via rapid, nonspecific cell binding) before reaching the disease site distal to the application site. Systemic delivery of lipid particles can be performed by any method known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal delivery. In a preferred embodiment, systemic delivery of lipid particles is performed via intravenous delivery.

[0290] As used in this article, "local delivery" refers to the direct delivery of an active agent to a target site within a living organism. For example, an active agent can be delivered locally by direct injection into a disease site, other target sites, or target organs (such as the skin).

[0291] The term "mammal" refers to any mammal species, such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, livestock, etc.

[0292] The term "treatment" refers to therapeutic procedures and preventative or preventative measures aimed at preventing or reducing undesirable physiological changes or impairments, such as the progression or spread of cancer. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction of disease severity, stabilization (i.e., non-deterioration) of the disease state, delay or slowing of disease progression, improvement or reduction of the disease state, and remission, whether detectable or undetectable (partial or complete). "Treatment" can also refer to extended survival compared to expected survival without treatment. Those requiring treatment include those already suffering from the condition or disease, those susceptible to the condition or disease, or those requiring prevention of the condition or disease.

[0293] The following examples are intended to further illustrate the invention. They are not intended to limit the invention in any way.

[0294] Example 1

[0295] Binding affinity for human fibroblast activation protein (FAP)

[0296] Given the K of the FAP substrate Z-GP-AMC m When the value is within the range, an enzyme inhibition assay is performed to determine the IC50 value. 50 The value was then calculated using the Cheng-Prusoff equation to determine the Ki value. For this determination, FAP (5 ng / well) and different concentrations (i.e., 10 ng / well) were used. -6 Up to 10 -12 The test peptide was incubated in a black 96-well plate at room temperature on a shaking plate for 10 minutes. Then, substrate Z-GP-AMC was added to a final concentration of 50 μM. The total reaction volume per well was 100 μL. The reaction mixture was incubated on a shaking plate at room temperature in the dark for 1 hour, and fluorescence values ​​were read at ex / em (360 / 40 and 460 / 40) using a BioTek Synergy LX plate reader. Experiments were performed in at least duplicate, with controls including buffer only, buffer and FAP enzyme, and buffer containing only substrate and FAP enzyme. The assay buffer consisted of 50 mM Tris-HCl, 1 M NaCl, and 1 mg / mL BSA, pH 7.5. Data were plotted in GraphPad Prism, and IC50 was calculated using nonlinear regression. 50 Value. The compounds tested and their K values. i The values ​​are as follows:

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311] Example 2

[0312] In vivo biodistribution of Pb-203-labeled FAP-targeting radiopeptides in tumor models

[0313] To determine the biodistribution of the example conjugate, athymic nude mice were inoculated with hFAP-HT1080 cells overexpressing FAP containing 50% Cultrex matrix gel, and tumors were allowed to grow for 2–3 weeks. Then... 203 Pb-conjugates were radiolabeled, and 74 kBq was injected intravenously into mice carrying hFAP-HT1080 tumors. Molar activity ranged from 5 to 50 MBq / nmol. Mice were euthanized at 2 and 24 hours according to animal protocol, and blood, lungs, spleen, muscle, tumor tissue, kidneys, and livers were collected. Samples were then analyzed using a Packard COBRA II automated gamma counter and compared with a reference dose to determine %ID / g (n=2). Results are as follows. Figure 1-24 As shown.

[0314] Example 3

[0315] VMT-FAP-2-59 binding specificity

[0316] Enzyme inhibition assays were performed to determine the inhibitory constants (i.e., Ki) against human FAP, mouse FAP, human dipeptidyl peptidase 4 (DPPIV), and human prolyl endopeptidase (PREP). Z-GP-AMC was used as a substrate for human FAP, mouse FAP, and human PREP. GP-AMC was used as a substrate for human DPPIV. For this assay, different concentrations (i.e., 10...) were... -6 Up to 10 -12VMT-FAP-2-59 was placed in a black 96-well plate with 5 ng hFAP, 5 ng mFAP, 2.5 ng DPPIV, or 5 ng PREP and incubated on a shaking plate at room temperature for 10 minutes. Substrate was added to bring the final concentrations of Z-GP-AMC and GP-AMC to 50 μM. The total reaction volume per well was 100 μL. The reaction was incubated on a shaking plate at room temperature in the dark for 1 hour, and then fluorescence was read at ex / em (360 / 40 and 460 / 40) using a BioTek Synergy LX plate reader. Experiments were performed in at least duplicate. Buffer-only, buffer / enzyme buffer / substrate, and enzyme-only were included as controls. Data were plotted in GraphPad Prism, and IC50 was calculated using nonlinear regression. 50 The binding kinetics (Kd) of VMT-FAP-2-59 with human FAP, human DPPIV, and human PREP were also determined by grating-coupled interferometry (GCI). hFAP, hDPPIV, and hPREP proteins were immobilized on PCH (N=3) or PCP (N=2) sheets, respectively. On PCH sheets, the binding affinity of VMT-FAP-2-29 with hFAP was determined at concentrations ranging from 61 pM to 125 nM. The binding affinity of VMT-FAP-2-29 with hDPPIV and hPREP was determined at concentrations ranging from 61 pM to 1 μM. On PCP sheets, the binding affinity of VMT-FAP-2-29 with hFAP was determined at concentrations ranging from 61 pM to 1 μM. The binding affinity of VMT-FAP-2-29 with hDPPIV and hPREP was determined at concentrations ranging from 61 pM to 1 μM. The binding affinity of VMT-FAP-2-29 to hFAP at concentrations ranging from 39 pM to 20 nM was determined on PCP tablets. The binding affinity with hDPPIV and hPREP at concentrations ranging from 39 pM to 50 nM was also determined. The results are summarized in the table below. Representative GCI kinetic curves are shown below. Figures 54-55 As shown.

[0317] FAP 2nM 0.4nM mouse FAP N / A 4.2nM Human DPPIV No combination No combination People PREP No combination 152nM

[0318] Example 4

[0319] [ 203 Radiochemical stability of Pb]VMT-FAP-2-59 in human serum

[0320] Fresh human serum was collected from blood obtained from the DeGowin Blood Center at the University of Iowa. The blood was allowed to clot at room temperature for 1 hour, then centrifuged at 1500g for 10 minutes, and the supernatant serum was collected. The human serum was then mixed with 3.3 MBq... 203Pb]VMT-FAP-2-59 was incubated at 37°C for 96 hours. After 96 hours, serum aliquots containing the radiotracer were obtained, and proteins were precipitated by methanol. 1.5 v / v ice-cold methanol was added and incubated on ice for 15 minutes. The methanol-containing aliquots were then centrifuged at 10,000 g for 10 minutes to precipitate proteins, and the supernatant was collected. To determine stability in serum, the supernatant was analyzed by HPLC using a radiometric detector. Gradient elution was performed using an Agilent 1200 series HPLC system: gradient elution in 0.1% trifluoroacetate acetonitrile aqueous solution at concentrations from 5% to 60% in water over 10 minutes. After 96 hours of incubation in fresh human serum at 37°C, stable [[] was found in the human serum. 203 Pb]VMT-FAP-2-59, such as Figure 56 As shown.

[0321] Example 5

[0322] [ 203 Biodistribution and imaging of Pb]VMT-FAP-2-59 in human cancer xenograft models

[0323] After injecting 74kBq[ 203 Following Pb]PSV-359 (10⁻¹² MBq / nmole), the levels of Pb were measured in female athymic nude mice carrying hFAP-HT1080 human fibrosarcoma xenografts (n=4, at each time point) and in U87MG human glioma xenografts (n=3, at each time point).

[0324] [ 203 In vivo biodistribution of Pb]VMT-FAP-2-59. Tumors and target organs were harvested at each specified time point and measured on automated gamma rays. 203 The radioactivity of Pb]PSV-359 was determined and normalized to a percentage of the injected dose per gram (%ID / g). Notably, HT1080 human fibrosarcoma cells were genetically engineered to express human FAP on the cancer cell membrane, while the expression of human FAP in U87MG human glioma cells was limited, as shown in in vitro cell binding assays, indicating that FAP in U87MG xenograft tumors in vivo was expressed in the stroma in the form of mouse FAP. Results are as follows. Figure 57 As shown.

[0325] After injecting 0.8 MBq [ 203 Pb]PSV-359 or 1.2MBq[ 212Micro-SPECT imaging of hFAP-HT1080 xenografts harvested from athymic nude mice 2 hours and 24 hours after Pb]PSV-359 administration. Rapid accumulation, rapid clearance, and low retention in normal tissue were observed. Results are as follows: Figure 58 As shown.

[0326] Example 6

[0327] [ 212 Radiotherapy efficacy of Pb-VMT-FAP-2-59 in a human fibrosarcoma xenograft model

[0328] In vivo efficacy was assessed in a sarcoma model where hFAP was expressed on the membrane of cancer cells. HT1080 cells were genetically modified to express hFAP. Eight days prior to treatment, athymic nude mice were inoculated with approximately 2 million cells. Mice were then randomly assigned to control and treatment groups based on tumor size. The treatment group received 4.5–5.4 MBq of hFAP. 212 Pb-Pb-VMT-FAP-2-59 was administered in three doses, approximately two weeks apart. Two mice received a fourth treatment with an additional 1.9–2.1 MBq on days 48 and 53. Tumor volume was measured with calipers, and weight was recorded over 70 days. No treatment-related toxicities were observed. The efficacy of VMT-FAP-2-59 in thymic-agnostic nude mice carrying HT1080-FAP tumors was assessed. Tumor volume and weight were monitored over 70 days. The treatment component was administered in three doses of 4.5–5.4 MBq. 212 Pb-VMT-FAP-2-59. Two mice with tumor recurrence were given an additional 1.9–2.1 MBq, as shown by the dashed arrows. Results are as follows. Figure 59 As shown.

[0329] Example 7

[0330] [ 212 Radiotherapy efficacy of Pb-VMT-FAP-2-59 in a human glioma xenograft model

[0331] In vivo efficacy was determined in a U87MG human glioma xenograft model. In this U87MG xenograft model, FAP was expressed in the matrix as mouse FAP, while human FAP expression was limited on the surface of U87MG cells. Two million U87MG cells were subcutaneously seeded into athymic nude mice. Mice were then randomly assigned to control and treatment groups according to tumor size. 4.6 MBq [ 212 The Pb-Pb-VMT-FAP-2-59 treatment group received three doses approximately two weeks apart. Tumor volume was measured using calipers, and weight was recorded over 40 days. [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 212Following treatment with Pb]VMT-FAP-2-59, a significant inhibition of the growth of U87MG xenograft tumors was observed, indicating that [ 212 Pb]VMT-FAP-2-59 was able to inhibit the growth of tumors that expressed high levels of FAP in the matrix but low levels of FAP on cancer cells. Results are as follows... Figure 60 As shown.

[0332] Example 8

[0333] Patients with different solid tumors 203 Pb]PSV359SPECT / CT Imaging

[0334] Diagnostic SPECT imaging isotopes 203 Pb (half-life 52 hours) was used in patients with different types of solid tumors at the Fortis Memorial Institute (Gurgaon, India). 203 First human SPECT / CT imaging of Pb]PSV359, the solid tumor including metastatic lung adenocarcinoma (51-year-old male, Figure 61 Metastatic ampullary neuroendocrine tumor (71-year-old male) Figure 62 ) and chondroblastic osteosarcoma (16-year-old male, Figure 63 According to the conventional preparation method, [ 203 Pb]PSV359 was radiolabeled and purified. The final product had a radiochemical purity of over 96%. The mean injection activity was 262.7 MBq (7.1 mCi). Serial SPECT / CT imaging was performed at 1, 4, and 18 hours post-injection. The patient also received... 18 F-FDG and / or [ 68 PET / CT imaging was performed using Ga]FAP-2286. Rapid tumor uptake was observed 1 hour after injection. 203 Pb]PSV359 was rapidly cleared by the kidneys via the bladder in all three patients. Minimal accumulation was observed in normal organs (brain, heart, liver, kidneys), indicating highly specific tumor targeting and favorable pharmacokinetics. In the same patient, [ 203 Pb]PSV359 tumor uptake and [ 68 Ga]FAP-2286 (a clinically validated FAP-targeted PET imaging agent) showed good concordance. Importantly, [Ga]FAP-2286 was observed in the tumors of all three patients. 203 Pb]PSV359 within 18 hours (approximately therapeutic isotope) 212 Excellent retention of Pb half-life (twice that of Pb) indicates that... 212The favorable pharmacokinetics of Pb]PSV359 delivers optimal alpha radiation to tumors. These first-in-human trial data demonstrate the superior pharmacodynamic and pharmacokinetic performance of PSV359 in patients with various solid tumors.

[0335] It should be understood that minor dosage and formulation modifications may be made to the compositions and ranges expressed herein, while still remaining within the scope and spirit of the invention.

[0336] The invention has been described with reference to specific compositions, efficacy theories, etc. Those skilled in the art will understand that the invention is not limited to these illustrative embodiments or mechanisms, and modifications can be made without departing from the scope or spirit of the invention as defined in the appended claims. All such obvious modifications and variations should be included within the scope of the invention as defined in the appended claims. The claims are intended to cover the claimed components and steps in any order that effectively achieves their intended purpose, unless the context clearly indicates otherwise.

[0337] The foregoing description is for illustrative and descriptive purposes only. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. It is conceivable that other alternative methods and approaches, obvious to those skilled in the art, should also be considered to be included in this invention. This specification is merely an example of embodiments. It should be understood that any other modifications, substitutions, and / or additions may be made within the intended spirit and scope of this disclosure. As can be seen from the foregoing, the exemplary aspects of this disclosure achieve at least all the intended purposes.

Claims

1. Fibroblast activating protein (FAP) targeting conjugates, which have one of the following structures: VMT-FAP-2-59 VMT-FAP-2-42 VMT-FAP-2-82 VMT-FAP-2-83 VMT-FAP-2-85 VMT-FAP-2-48 VMT-FAP-2-51 VMT-FAP-2-55 VMT-FAP-2-57 VMT-FAP-2-67 VMT-FAP-2-68 VMT-FAP-2-69 VMT-FAP-2-70 VMT-FAP-2-72 VMT-FAP-2-74 VMT-FAP-2-75 VMT-FAP-2-76 VMT-FAP-2-77 and VMT-FAP-2-78.

2. The FAP-targeting conjugate according to claim 1, having the following structure: VMT-FAP-2-59.

3. The FAP targeting conjugate according to claim 1 or claim 2, wherein the structure is complexed with a radionuclide.

4. The FAP-targeting conjugate according to claim 3, wherein the radionuclide is used for medical imaging of FAP-positive malignant tumors, optionally wherein the radionuclide is selected from diagnostic radionuclides Sc-43, Sc-44, Mn-51, Cu-64, Ga-67, Ga-68, Y-86, Zr-89, Tc-99m, F-18, Br-76, Br-77, In-111, I-123, I-124, I-125, Tb-152, and Pb-203.

5. The FAP-targeting conjugate according to claim 3, wherein the radionuclide is used for the medical treatment of FAP-positive malignant tumors, optionally wherein the radionuclide is selected from therapeutic radionuclides Sc-47, Cu-67, Sr-89, Y-90, I-131, Sm-153, Tb-149, Tb-161, Lu-177, Re-186, Re-188, At-211, Pb-212, Bi-212, Bi-213, Ra-223, Ra-224, Ac-225, Th-226, and Th-227.

6. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the FAP-targeting conjugate according to any one of claims 1 to 5.

7. The pharmaceutical composition of claim 6, wherein the composition may be administered intravenously or intraperitoneally by infusion or injection.

8. Use of the FAP-targeting conjugate according to any one of claims 1 to 5 or the pharmaceutical composition according to any one of claims 6 to 7 for the preparation of a medical imaging agent for FAP-positive malignant tumors.

9. The use according to claim 8, wherein the coupling has the following structure: VMT-FAP-2-59 It also complexes with the diagnostic radionuclide Pb-203.

10. Use of the FAP-targeting conjugate according to any one of claims 1 to 5 or the pharmaceutical composition according to any one of claims 6 to 7 for the preparation of a medicament for treating FAP-positive malignant tumors.

11. The use according to claim 10, wherein the coupling has the following structure: VMT-FAP-2-59 It also complexes with the therapeutic radionuclide Pb-212.

12. The use according to claim 10 or claim 11, wherein the use further comprises administering the anticancer composition.

13. The use according to claim 8 or claim 10, wherein the FAP-positive malignant tumor is a solid tumor, optionally wherein the solid tumor is a sarcoma, salivary gland cancer, esophageal cancer, bile duct cancer, breast cancer, lung cancer, prostate cancer, pancreatic cancer, thymic cancer, head and neck cancer, ovarian cancer, desmoid tumor, chordoma, colorectal cancer, anal cancer, neuroendocrine tumor, small bowel cancer, cervical cancer, endometrial cancer, hepatocellular carcinoma, gastric cancer, adenoid cystic carcinoma, differentiated thyroid cancer, renal cancer, or skin cancer.

14. A kit comprising the FAP-targeting conjugate of any one of claims 1 to 5, a container, and a package insert or label instructing the administration of the conjugate for the treatment of FAP-positive cancers.

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