Multivalent fibroblast activating protein ligands for targeted delivery applications

Abstract

The present invention relates to ligands of fibroblast activation protein (FAP) for the active delivery of various therapeutic payloads (e.g., cytotoxic drugs, therapeutic radionuclides, proteins, and immunomodulators) to disease sites. In particular, the present invention relates to the development of multivalent FAP ligands for therapeutic applications associated with diseases or disorders such as cancer, inflammation, or other diseases characterized by overexpression of FAP.

Description

[Technical Field]

[0001] Introduction The present invention relates to ligands of fibroblast activation protein (FAP) for the active delivery of various therapeutic payloads (e.g., cytotoxic drugs, therapeutic radionuclides, proteins, and immunomodulators) to disease sites. In particular, the present invention relates to the development of multivalent FAP ligands for therapeutic applications associated with diseases or disorders such as cancer, inflammation, or other diseases characterized by overexpression of FAP. [Background technology]

[0002] Chemotherapy remains widely used in the treatment of cancer patients and other diseases. Conventional anti-cancer chemotherapeutic agents act on the basic mechanisms of cell survival and cannot distinguish between healthy and malignant cells. Furthermore, these agents do not efficiently accumulate at disease sites upon systemic administration. The nonspecific mechanism of action and inefficient localization at tumor sites are responsible for the unsustainable side effects and poor therapeutic efficacy of conventional chemotherapy.

[0003] The development of therapeutic agents that can selectively localize to disease sites after systemic administration is highly desirable. Strategies for generating such therapeutic agents are typically achieved by chemically conjugating therapeutic payloads, such as cytotoxic drugs or therapeutic radionuclides, to ligands specific to disease markers. Disease-specific monoclonal antibodies, peptides, and small ligands have been considered as selective ligands for the development of targeted therapeutic products. The use of small ligands for therapeutic applications offers several advantages compared to large molecules such as peptides and antibodies, including faster and more efficient tumor penetration, lower immunogenicity, and lower production costs.

[0004] Small organic ligands specific for prostate-specific membrane antigen, folate receptor, and carbonic anhydrase IX have demonstrated excellent biodistribution profiles in preclinical cancer models and patients. These ligands have been conjugated with cytotoxic drugs and radionuclides to generate small molecule-drug conjugate and small molecule-radionuclide conjugate formulations (SMDCs and SMRCs) for cancer therapy. 177-Lutetium-PSMA-617 is an example of a late-stage SMRC and is currently being investigated in a Phase III trial (the VISION trial) for the treatment of patients with metastatic castration-resistant prostate cancer (mCRPC).

[0005] Fibroblast activation protein (FAP) is a membrane-bound gelatinase that promotes tumor growth and progression and is overexpressed in cancer-associated fibroblasts. FAP is an ideal target for the development of therapeutic SMDCs and SMRCs due to its low expression in normal organs.

[0006] WO 2019 / 154886 and WO 2019 / 154859 describe heterocyclic compounds as fibroblast activation protein-alpha inhibitors used to treat different types of cancer. WO 2019 / 118932 describes substituted N-containing cyclic compounds as fibroblast activation protein-alpha inhibitors used to treat various pathological conditions. WO 2019 / 083990 describes fibroblast activation protein-alpha (FAP-alpha) compounds targeted for imaging and radiotherapy as FAP-alpha inhibitors used in imaging of diseases associated with FAP-alpha and in treating proliferative diseases. It should be noted that the 4-isoquinolinoyl and 8-quinolinoyl derivatives described therein are characterized by very low FAP affinity. International Publication No. WO 2013 / 107820 describes substituted pyrrolidine derivatives for use in the treatment of proliferative diseases such as cancer, diseases characterized by tissue remodeling or chronic inflammation such as osteoarthritis. International Publication No. WO 2005 / 087235 describes pyrrolidine derivatives as dipeptidyl peptidase IV inhibitors for the treatment of type II diabetes. International Publication No. WO 2018 / 111989 describes conjugates comprising a fibroblast activation protein (FAP) inhibitor, a bivalent linker, and, for example, a near-infrared (NIR) dye, useful for the elimination of cancer-associated fibroblasts, imaging cell populations in vitro, and treating cancer. International Publication Nos. WO 2021 / 160825, WO 2022 / 171811, and WO 2021 / 016392 describe ligands of FAP for the active delivery of various payloads. Zboralski, D., Hoehne, A., Bredenbeck, A. et al., Eur J Nucl Med Mol Imaging 49, pp. 3651-3667 (2022) describes FAP-2286 (a FAP-binding peptide conjugated to a radionuclide chelator), and reports that radiolabeled FAP-2286 showed high tumor uptake and retention and demonstrated high efficacy in FAP-positive tumors.

[0007] Nevertheless, further improvements in FAP binders are needed for these and other applications.

[0008]

[0006] Accordingly, the present invention addresses the problem of providing improved therapeutic binding agents (ligands) for fibroblast activation proteins (FAPs) suitable for therapeutic use. The binding agents should be suitable for inhibiting FAPs and / or for targeted delivery of therapeutic payloads to sites affected by or at risk for diseases or disorders characterized by FAP overexpression, such as FAP-positive tumors. Preferably, the binding agents should exhibit improved binding parameters (e.g., FAP inhibitory activity, FAP binding affinity, and / or prolonged binding to FAP-positive cells) while exhibiting favorable tumor uptake or tumor-to-organ ratios (e.g., tumor-to-kidney ratios). Summary of the Invention

[0009] The present inventors have discovered a novel trivalent organic ligand ("Tri-ESV6") of fibroblast activation protein (FAP) that is suitable for therapeutic use. The compounds (also called ligands or binders) according to the present invention have the following structure: [ka] The nucleotide sequence comprises more than two (e.g., three) small binding moieties A having the following structure:

[0010] The compounds according to the invention have the following general formula I [ka] It can be represented by its individual diastereoisomer, its hydrate, its solvate, its crystalline form, its individual tautomer, or its pharmaceutically acceptable salt, where A is a linking moiety; B is a multifunctional moiety containing a branching point, covalently linking moiety A to C; and C is an atom, molecule, or particle and is a therapeutic agent. An exemplary structure is represented by general formula II, defined below:

[0011] Alternatively, moiety C contains a branch point such that three moieties A are attached to C and B is absent, i.e., moiety C can also function as a branch point, thereby replacing B, preferably when C is a chelator. An exemplary structure is represented by general formula III, defined below:

[0012] The present invention further provides a pharmaceutical composition comprising the compound and a pharmaceutically acceptable excipient.

[0013] The invention further provides said compound or pharmaceutical composition for use in a method of therapeutic treatment of the human or animal body; as well as a method of therapeutic treatment of the human or animal body comprising administering to a subject in need thereof a therapeutically effective amount of said compound or pharmaceutical composition.

[0014] The invention further provides said compound or pharmaceutical composition for use in a method of treating a subject suffering from or at risk of a disease or disorder; and a method of treating a disease or disorder comprising administering a therapeutically effective amount of said compound or pharmaceutical composition to a subject suffering from or at risk of said disease or disorder.

[0015] The invention further provides a method for targeted delivery of a therapeutic agent to a subject suffering from or at risk of a disease or disorder; and a compound or pharmaceutical composition for use in a method for targeted delivery of a therapeutically effective amount of said compound or pharmaceutical composition to a subject suffering from or at risk of a disease or disorder.

[0016] Preferably, said disease or disorder is characterized by overexpression of a FAP and is independently selected from cancer, inflammation, atherosclerosis, fibrosis, tissue remodeling and keloid disorders, and preferably the cancer is selected from the group consisting of breast cancer, pancreatic cancer, small intestine cancer, colon cancer, multidrug resistant colon cancer, rectal cancer, colorectal cancer, metastatic colorectal cancer, lung cancer, non-small cell lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocarcinoma, clear cell renal carcinoma, neuroendocrine tumors, oncogenetic osteomalacia, sarcoma, CUP (cancer of unknown primary), thymic carcinoma, desmoid tumor, glioma, astrocytoma, cervical cancer, skin cancer, kidney cancer and prostate cancer. [Brief explanation of the drawings]

[0017] [Figure 1-1] Figure 1 shows an inhibition assay with recombinant hFAP at a concentration of 66 pM. Tri-ESV6-DOTAGA exhibits a significantly lower IC50 compared to ESV6-DOTAGA, Bi-ESV6-DOTAGA, and FAP-2286. [Figure 1-2] Figure 1-1 continued [Figure 2-1] Fluorescence polarization assay with recombinant hFAP using fluorescent ligands at 1 nM concentration. Both ESV6-PEG2-FITC and Tri-ESV6-PEG2-FITC show significantly lower KD compared to FAP-2286-PEG2-FITC. [Figure 2-2] Figure 2-1 continued [Figure 3] Figure 1 shows an efflux assay of SK-RC-52.hFAP cells using 177Lu-labeled ligands. 177Lu-Tri-ESV6 exhibited a longer residence time in FAP-positive tumor cells, followed by 177Lu-Bi-ESV6, 177Lu-ESV6, and 177Lu-FAP2286. [Figure 4-1]Quantitative biodistribution analysis using 177Lu-ESV6, 177Lu-Bi-ESV6, 177Lu-Tri-ESV6, 177Lu-Tetra-ESV6, 177Lu-Hexa-ESV6, 177Lu-Octa-ESV6, and 177Lu-FAP-2286 (250 nmol / kg; 50 MBq / kg, respectively) in SK-RC-52.hFAP tumor-bearing mice. Tri-ESV6-DOTAGA exhibits significantly higher tumor uptake at 48, 72, and 96 hours post-injection. At the same dose, the Tri-ESV6 conjugate exhibits the best biodistribution profile, considering both tumor uptake over time and uptake in healthy organs. [Figure 4-2] Figure 4-1 continued [Figure 5] The therapeutic activity of 177Lu-ESV6-DOTAGA, 177Lu-Bi-ESV6-DOTAGA, and 177Lu-Tri-ESV6-DOTAGA, or saline as a single agent (single dose on day 8, 250 nmol / kg, 250 MBq / kg), in BALB / c nu / nu mice bearing SK-RC-52.hFAP tumors is shown. The efficacy of the different treatments was assessed by measuring tumor volume (mm3) daily during and after drug administration. Data points represent mean tumor volume ± SEM (n = 4 / group). ****P < 0.0001; *P < 0.1 (two-way ANOVA test followed by Bonferroni post-hoc test). Tri-ESV6-DOTAGA exhibited significant therapeutic activity. [Figure 6] The therapeutic activity of 177Lu-ESV6-DOTAGA, 177Lu-Bi-ESV6-DOTAGA, and 177Lu-Tri-ESV6-DOTAGA or saline as single agents (single dose on day 8, 250 nmol / kg, 250 MBq / kg) in BALB / c nu / nu mice bearing SK-RC-52.hFAP tumors is shown. The efficacy of the different treatments is assessed by daily measurement of tumor volume (mm3) during and after drug administration. The curves represent tumor growth values ​​for a single mouse. CR = complete remission. Tri-ESV6-DOTAGA shows significant therapeutic activity. [Figure 7]Figure 1 shows the tolerability of the different treatments with 177Lu-ESV6-DOTAGA, 177Lu-Bi-ESV6-DOTAGA, and 177Lu-Tri-ESV6-DOTAGA, as determined by assessing the % change in body weight during the experiment. All conjugates were very well tolerated in a comparable manner. [Figure 8-1] Inhibition assay using recombinant hFAP at a concentration of 66 pM shows that Tri-ESV6-DOTAGA exhibits an IC50 approximately 1000-fold lower than that of the derivative with a longer spacer between the ESV6 moiety and the branch (Tri-ESV6-PEG12-DOTAGA). [Figure 8-2] Figure 8-1 continued [Figure 9] Figure 9 shows the therapeutic activity in BALB / c nu / nu mice bearing SK-RC-52.hFAP tumors after intravenous administration of (i) L19-IL2 as a single agent at a dose of 0.05 mg / mouse on days 8, 10, and 12, (ii) Lu-Tri-ESV6 as a single agent at different doses of 5 MBq / mouse, 15 MBq / mouse, or 30 MBq / mouse on day 7, (iii) the combination of Lu-Tri-ESV6 and L19-IL2 at a dose of 5 MBq / mouse of Lu-Tri-ESV6, followed by three injections of L19-IL2 at a dose of 0.05 mg / mouse on days 8, 10, and 12, or (iv) saline. The efficacy of the different treatments was assessed by daily measurement of tumor volume (mm) during and after drug administration. Data points represent mean tumor volume ± SEM (n=4 / group). 177Lu-Tri-ESV6 exhibits dose-dependent therapeutic activity. [Figure 10-1] Inhibition assay with recombinant hFAP at a concentration of 66 pM is shown. Surprisingly, it was found that the compound with the shortest distance between the targeting moiety and the branch point (PEG units = 0) had the lowest IC50. [Figure 10-2] Figure 10-1 continued [Figure 10-3] Figure 10-2 continued [Figure 10-4] Figure 10-3 continued [Figure 10-5]Figure 10-4 continued [Figure 10-6] Figure 10-5 continued [Figure 11-1] Inhibition assay with recombinant hFAP at a concentration of 66 pM is shown. Inhibitory activity is directly proportional to valency up to Tetra-ESV6-DOTAGA (tetravalent compound). Surprisingly, despite their superior valency, Hexa-ESV6-DOTAGA (hexavalent compound) and Octa-ESV6-DOTAGA (octavalent compound) lose more than 10,000-fold activity compared to their tetravalent derivatives. Compounds with alternative linkers (ESV6-L-DOTAGA and Bi-ESV6-DOTAGA) show IC50 values ​​comparable to those of the parent derivatives (ESV6-DOTAGA and Bi-ESV6-DOTAGA in Figure 1). [Figure 11-2] Figure 11-1 continued [Figure 11-3] Figure 11-2 continued [Figure 11-4] Figure 11-3 continued [Figure 12] A preferred immunocytokine for use in the present invention is an L19-IL2 conjugate immunocytokine ("scFv2-format immunocytokine") in which, in each polypeptide chain, an IL2 polypeptide sequence is fused at its N-terminus via a linker to the C-terminus of the VL domain of a single-chain variable fragment (scFv) sequence comprising the VH and VL domains of L19, and the scFv unit of one L19-IL2 polypeptide chain forms a homodimer with the scFv unit of another L19-IL2 polypeptide chain. [Figure 13]FIG. 13 shows the results of the treatment of SK-RC-52.hFAP tumor-bearing BALB / c nu / nu mice with (i) L19-IL2 as a single agent at a dose of 0.05 mg / mouse on days 8, 10, and 12, (ii) 177Lu-Tri-ESV6 as a single agent at a dose of 5 MBq / mouse on day 7, and (iii) the combination of 177Lu-Tri-ESV6 and L19-IL2 (177Lu-Tri-ESV6 at a dose of 5 MBq / mouse on day 7, followed by L19-IL2 at a dose of 0.05 mg / mouse on days 8 and 10). The therapeutic activity after intravenous administration of L19-IL2 at a dose of 0.05 mg / mouse three times on days 12, 17, and 22 (Schedule 1), or L19-IL2 at a dose of 0.05 mg / mouse three times on days 12, 17, and 22 (Schedule 2), or L19-IL2 at a dose of 0.05 mg / mouse once on day 8 (Schedule 3), or L19-IL2 at a dose of 0.05 mg / mouse once on day 12 (Schedule 4), or (iv) saline is shown. The efficacy of the different treatments was evaluated by measuring tumor volume (mm3) daily during and after drug administration. Data points represent the mean tumor volume ± SEM (n = 4 / group). 177Lu-Tri-ESV6 exhibited dose-dependent therapeutic activity. [Figure 14] Figure 14 shows quantitative in vivo MMAE release in tumors and healthy organs by ESV6-GlyPro-MMAE (conjugate 58a of EP 3891138), Bi-ESV6-GlyPro-MMAE (conjugate 11 of WO 2022 / 171811), and Tri-ESV6-GlyPro-MMAE (conjugate 9 of the present invention). Each conjugate was injected at a dose of 250 nmol / kg, and MMAE release was recorded at different time points after administration. Values ​​are expressed as percentage of administered dose per gram of tissue (%ID / g). Quantification of MMAE released after administration of Tri-ESV6-GlyPro-MMAE demonstrated a distinct biodistribution profile, revealing higher and longer accumulation at tumor sites and lower release in healthy organs compared to ESV6-GlyPro-MMAE and Bi-ESV6-GlyPro-MMAE. [Figure 15]To identify the optimal dose for radiotherapy, 177Lu-Tri-ESV6-DOTAGA was injected into tumor-bearing mice at eight different doses ranging from 3 nmol / kg to 2250 nmol / kg. The results showed that the highest dose (>90 nmol / kg) resulted in a decrease in %ID / g in the tumor (A), while the lowest dose (<10 nmol / kg) resulted in partial uptake in healthy organs (B). The optimal dose range for mice was between 90 and 250 nmol / kg, which corresponds to 1–3 mg per human patient. [Figure 16] Figure 16 shows the evaluation of the therapeutic anticancer efficacy of ESV6-GlyPro-MMAE and Tri-ESV6-GlyPro-MMAE in HT-1080.hFAP tumor-bearing mice in terms of tumor volume (A) and survival rate (B) over time. Tolerability was assessed by evaluating body weight change (%) during the experiment, and all conjugates were shown to be well tolerated (C). [Figure 17] Figure 17 shows the data set underlying the results shown in Figure 16 (single plot; one curve for each animal): ESV6-GlyPro-MMAE at 50 nmol / kg (A) or 125 nmol / kg (B); Tri-ESV6-GlyPro-MMAE at 50 nmol / kg (D) or 125 nmol / kg (E); and saline (C). [Figure 18] FIG. 18 shows, inter alia, a comparative hFAP inhibition assay using trivalent binders 11, 12, 13 and 14 with different linker groups B and / or different payload groups C. [Figure 19] Figure 19 shows the biodistribution results of 177Lu at different radioisotope molar activities (MBq of 177Lu per nmol of ligand) using the binder compound 177Lu-Tri-ESV6-DOTAGA 24 hours after injection, suggesting that varying the molar activity does not significantly interfere with the favorable biodistribution of the binder. [Figure 20] FIG. 20 shows a comparative hFAP inhibition assay using tetravalent binders 10 and 29.

[0018] Detailed Description of the Invention The present inventors have identified small molecule binders of fibroblast activation protein (FAP) that are suitable for therapeutic use.

[0019] As demonstrated by the in vitro data provided herein, therapeutic binding agents according to the invention can provide high FAP inhibitory activity, high FAP binding affinity, and prolonged binding to FAP-positive cells, and are therefore suitable candidates for targeted delivery of therapeutic payloads to sites affected by or at risk for diseases or disorders characterized by overexpression of FAP.

[0020] As demonstrated by the in vivo data provided herein, therapeutic binding agents according to the invention can provide high and prolonged tumor uptake and potent antitumor efficacy, while achieving significantly higher tumor-to-organ ratios (especially tumor-to-kidney).

[0021] Therapeutic binding agents according to the invention are surprisingly advantageous with respect to one or more of the above-mentioned effects and additional effects compared to related prior art FAP binding agents such as FAP-2286. As is evident from the results in Figures 1-5, binding agents according to the invention exhibit improved FAP inhibitory activity, improved FAP binding affinity, prolonged binding to FAP-positive cells, higher tumor uptake, and better tumor-to-organ ratios than FAP-2286.

[0022] The therapeutic binding agents according to the invention are surprisingly advantageous with respect to one or more of the above effects and additional effects compared to monovalent FAP binding agents, such as ESV6-DOTAGA, which has only one FAP-binding moiety A. As is evident from the results in Figure 1, a trivalent binding agent according to the invention (Tri-ESV6-DOTAGA) having three FAP-binding moieties A exhibits a significantly higher IC than the monovalent binding agent ESV6-DOTAGA. 50This is a surprising 43-fold improvement in IC, far exceeding the 3-fold improvement typically expected assuming additive behavior. Furthermore, the trivalent binder of the present invention (Tri-ESV6-DOTAGA) showed a significantly higher IC than the bivalent binder Bi-ESV6-DOTAGA. 50 This is surprising and far exceeds the 1.5-fold improvement typically expected assuming additive behavior. Thus, binding agents according to the invention can provide unexpected synergistic improvements in FAP inhibitory activity.

[0023] As can be seen from the results in Figure 3, the trivalent binding agents according to the invention show a surprisingly improved duration of efflux in FAP-positive tumor cells when compared to the bivalent binding agents, and even more so when compared to the monovalent binding agents. Thus, the binding agents according to the invention can provide an unexpected improvement in terms of prolonged duration of tumor uptake.

[0024] As is evident from the results in Figure 4, the trivalent binders according to the invention exhibit surprisingly improved in vivo tumor uptake in FAP-positive tumors compared to monovalent, bivalent, hexavalent, and octavalent binders (see also Tables 1-3 and 11-12), while maintaining surprisingly high tumor-to-organ ratios, particularly tumor-to-kidney ratios (see also Tables 5-7 and 13-15). This is surprising, since increasing the valency of a binder would typically be expected to result in higher organ accumulation and worse tumor-to-organ ratios. For example, at the same dose, the Tri-ESV6 binder exhibits much better (cleaner) biodistribution, i.e., more favorable tumor-to-organ ratios, compared to the other binders tested (e.g., hexavalent and octavalent).

[0025] Overall, 177Lu-TriESV6-DOTAGA showed the best biodistribution profile, considering both tumor uptake over time (i.e., approximately 49% ID / g, 6 h after injection) and uptake in healthy organs (e.g., approximately 0.3% ID / g in the spleen and approximately 0.7% ID / g in the liver at 6 h after injection), thereby highlighting trivalent TriESV6-based binders as best-in-class tumor-targeting ligands. Furthermore, trivalent compounds are smaller (low molecular weight), more atom-efficient, and easier and cheaper to manufacture than, for example, their tetravalent counterparts.

[0026] Thus, binding agents according to the present invention can provide unexpected improvements in tumor uptake while at the same time providing advantageous biodistribution and therefore have improved therapeutic potential.

[0027] As evidenced by the results in Figures 5 and 6, trivalent binding agents according to the invention have surprisingly improved in vivo therapeutic activity against FAP-positive tumors when compared to monovalent and bivalent binding agents, while maintaining tolerability as evidenced by Figure 7. Thus, binding agents according to the invention can provide unexpected improvements in therapeutic efficacy and / or target specificity.

[0028] As is clear from the results in Figures 8 and 10, the short spacer (L) between the trivalent ESV6 moiety and the branch point a gives superior inhibitory activity compared to longer spacers (see also Table 16). This is surprising considering that spacers in multivalent compounds long enough to reach multiple adjacent FAPs on target cells would be expected to be advantageous.

[0029] In view of the above, the present invention provides particularly advantageous therapeutic compounds which can be administered at lower doses (due to their improved binding properties) and which are expected to provide a favourable side effect profile (on the one hand due to the lower dose required and on the other hand due to improved biodistribution, in particular a high tumor:kidney ratio and a high tolerability profile).

[0030] In a preferred embodiment, the compounds of the present invention have the following formula II: [ka] (In the formula, L, B S and B L each independently represents alkylene, cycloalkylene, arylalkylene, heteroarylalkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, arylalkenylene, heteroarylalkenylene, heteroalkenylene, heterocycloalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, oxoalkylene, dioxoalkylene, aminoalkylene, a moiety comprising or consisting of structural units selected from: alkylenes, diaminoalkylenes, diacid esters, dialkylsiloxanes, amides, thioamides, thioethers, thioesters, esters, carbamates, hydrazones, thiazolidines, methylenealkoxycarbamates, disulfides, vinylenes, imines, imidamides, phosphoramides, saccharides, phosphate esters, phosphoramides, carbamates, dipeptides, tripeptides, and tetrapeptides, each of which is substituted or unsubstituted; J is alkylene, cycloalkylene, arylalkylene, heteroarylalkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, arylalkenylene, heteroarylalkenylene, heteroalkenylene, heterocycloalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, (oxo)alkylene, dioxoalkylene, aminoalkylene, diaminoalkylene a moiety containing a branch point comprising or consisting of structural units independently selected from the group consisting of: alkylene, diacid ester, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylenealkoxycarbamate, disulfide, vinylene, imine, imidamide, phosphoramide, saccharide, phosphate ester, phosphoramide, carbamate, dipeptide, tripeptide, tetrapeptide, each of which is substituted or unsubstituted; each x is an integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; each y is an integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; each z is an integer independently selected from 0, 1, 2, 3, 4, and 5; each a is an integer independently selected from 0, 1, 2, 3, 4, and 5).

[0031] In a further preferred embodiment, moiety C has a branch point, three moieties A are attached to C, and B is absent, i.e., moiety C serves as a branch point and thus replaces B, preferably when C is a chelator (e.g., a chelator group suitable for radiolabeling with a therapeutic nuclide). An exemplary structure is represented by the following general formula III: [ka] wherein all groups and variables are as defined in the claims, and preferably each a is 0.

[0032] In this embodiment, when C is a chelator having multiple -COOH groups, each A-(L) a The -arm is attached to a -C(O)- group in the chelator structure, originating from each -COOH group. For example, A-(L) a When the terminal atom of - is oxygen (i.e., the arm has the general structure ARO-), this arm can be attached to each -COOH group of a chelator, thereby forming an ester (-C(O)ORA); A-(L) a If the terminal atom of - is an amino nitrogen (i.e., the arm has the general structure AR-NH-), this arm can be attached to each -COOH group of a chelator, thereby forming an amide (-CONH-RA), where R is the number of atoms in each arm except for the terminal atom (L). a is a specific structure of

[0033] As used herein, the term "branch point," "RP," or ◯ (dotted line), unless otherwise specified, refers to a central atom bonded to at least three non-hydrogen atoms, at least two of which are located on a covalently bonded chain representing the shortest path between the A moiety and the central atom. If the path containing the minimum number of covalent bonds between two or more A moieties does not pass through the same atom but passes through the same ring system, the geometric center of such a ring system is considered a branch point. A molecule containing three A moieties, for example, can contain one or two branch points, and preferably contains only one branch point. If a molecule contains more than one branch point, the number of covalent bonds between each branch point and the nearest branch point is preferably seven or fewer, more preferably five or fewer, and most preferably three or fewer.

[0034] As used herein, unless otherwise specified, the term "distance" or "length" between moieties or groups refers to the longest interatomic distance (through space) between two non-hydrogen atoms belonging to the respective groups in their most extended conformation, where these atoms are connected via a path containing the fewest number of covalent bonds. For example, in compound G 1 -CH2-CH2-G 2 Group G in 1 (= 13 CH3) and G 2 The distance between the (=OH) atoms in the most extended (stretched) conformation is 13 This would be the longest interatomic distance (through space) between C and atom O.

[0035] Without wishing to be bound by theory, it is believed that the multiple FAP-binding moieties A, preferably up to four, more preferably three FAP-binding moieties A present in the molecule of the binding agent according to the present invention, are involved in binding and rebinding to the exact same FAP target molecule and / or establishing further synergistic interactions that are not possible with a single moiety. This is believed to be due to the interplay between the relatively large active pocket of the FAP protein, on the one hand, and the specific structural characteristics of the binding agent, on the other hand. Thus, one or more of the advantageous effects described herein are particularly pronounced when the intramolecular distance (length) between each moiety A and the nearest branch point is short, and the binding agent is preferably involved in binding to one FAP target molecule rather than multiple adjacent FAP molecules (e.g., on a target cell). That is, the moiety (L) a and J preferably will not provide a length for the arms of the multivalent compound to reach multiple adjacent FAPs on the target cell.

[0036] This is confirmed by the results shown in Figure 8, where Tri-ESV6-DOTAGA (with a distance of approximately 17 Å between each A moiety and the nearest branch point) exhibited much stronger FAP inhibition (approximately 1000-fold lower IC) compared to Tri-ESV6-PEG12-DOTAGA, which has a longer distance between each A moiety and the branch point (approximately 60 Å). 50In this regard, a particularly suitable distance (d1+d2) between each moiety A and the nearest branch point is believed to be 30 Å or less, preferably 24 Å or less, more preferably 17 Å or less.

[0037] Without wishing to be bound by theory, it is believed that reducing the distance d1+d2 contributes to improving the coupling properties.

[0038] Preferably, the distance between each moiety A and J is 24 Å or less, preferably 18 Å or less, more preferably 11 Å or less. This distance represents the length of the spacer connecting the ligand A (which binds to fibroblast activation protein (FAP) on target cells) and the multi-point template J to which the arms of the compound are connected, and can also be denoted as d1, as illustrated in the following schematic diagram. In other words, d1 is the distance between the moiety (L) and the multi-point template J. a is the distance between the two atoms directly attached to each end of

[0039] Without wishing to be bound by theory, it is believed that reducing the distance d1 contributes to improving the coupling properties.

[0040] Preferably, the distance between each moiety L and the nearest branch point is 19 Å or less, preferably 12 Å or less, more preferably 6 Å or less. This distance represents the length from the branch point to the "first" atom belonging to moiety L along the chain representing the shortest path between the branch point and each moiety A, and encompasses the part of the distance between each moiety A and the nearest branch point that belongs to the multi-point template J. This distance can also be denoted as d2, as illustrated in the schematic diagram below. In other words, d2 is the distance between the group moiety (L) directly bonded to the moiety J. a (or A if a is 0) and the distance between the branch point.

[0041] Without wishing to be bound by theory, it is believed that reducing the distance d2 contributes to improving the coupling properties. [ka]

[0042] Part A Without wishing to be bound by any theory, it is believed that some of the surprising technical effects are related to the particular structure of the small binding moiety A, in which the quinoline ring is substituted at the 8-position with a nitrogen-containing group such as an amino or amide group: [ka]

[0043] It has previously been shown that a compound's higher target protein affinity may result in longer tumor retention in vivo (Wichert et al., Nature Chemistry 7, 241-249 (2015)). The therapeutic compounds of the present invention have increased affinity for FAPs, slower dissociation rates, and prolonged retention at disease sites at therapeutically relevant levels, preferably for more than 1 hour, more preferably for more than 6 hours, after injection, compared to prior art compounds. Preferably, peak enrichment is achieved after 5, 10, 20, 30, 45, 1, 2, 3, 4, 5, or 6 hours; and / or enrichment at disease sites is maintained at therapeutically relevant levels for 5, 10, 20, 30, 45, 1, 2, 3, 4, 5, or 6 hours, 24, 48, 72, or 96 hours after injection, or for at least that period, more preferably for more than 6 hours, and even more preferably for more than 24 hours.

[0044] Part B Moiety B is a covalent bond or a moiety comprising a chain of atoms that covalently bonds moiety A to payload C. Moiety B can be a cleavable or non-cleavable multifunctional moiety that can be used to link one or more payload and / or binder moieties to form the targeted conjugates of the invention.

[0045] Specifically, moiety B is a polyfunctional moiety that links one or more moieties C and / or A. The compound structure can include three moieties A per molecule. The compound structure can include more than one moiety C per molecule, preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 moieties C. Preferably, the compound structure includes three moieties A and one moiety C per molecule.

[0046] If a cleavable linker unit is present in moiety B, the release mechanism may be the same as that specific for the antibody linked to the cytotoxic payload. Indeed, the nature of the binding moiety is independent in that respect. Therefore, pH-dependent [Leamon, CP et al. (2006) Bioconjugate Chem., 17, 1226; Casi, G. et al. (2012) J. Am. Chem. Soc., 134, 5887], reducible [Bernardes, GJ et al. (2012) Angew. Chem. Int. Ed. Engl., 51. 941; Yang, J. et al. (2006) Proc. Natl. Acad. Sci. USA, 103, 13872] and enzymatic release [Doronina SO et al. (2008) Bioconjugate Chem, 19, 1960; Sutherland, MSK (2006) J. Biol. Chem, 281, 10540] are expected. In certain situations, when functional groups are present on either the binding moiety or the payload (e.g., thiol, alcohol), a linker-free connection can be established, thus releasing the intact payload and substantially simplifying pharmacokinetic analysis.

[0047] Moiety B may comprise or consist of the units shown in Table A below, where the substituents R and R nmay suitably be independently selected from H, halogen, substituted or unsubstituted (hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)aryl, (hetero)arylalkyl, (hetero)cycloalkyl, (hetero)cycloalkylaryl, heterocyclylalkyl, peptide, oligosaccharide or steroid group. Preferably, each of R, R, R2 and R3 is independently selected from H, OH, SH, NH2, halogen, cyano, carboxy, alkyl, cycloalkyl, aryl and heteroaryl, each of which is substituted or unsubstituted. Suitably, R and R n are independently selected from H, or C1-C7 alkyl or heteroalkyl. More suitably, R and R n is independently selected from H, methyl or ethyl. [Table 1]

[0048] Part B, Unit B L and / or Unit B S can suitably include disulfide bonds as cleavable bonds because these bonds are hydrolytically stable while at the same time providing suitable drug release kinetics to the target in vivo and traceless cleavage of the thiol-containing drug moiety.

[0049] Part B, Unit B L and / or Unit B SThe linker may be polar or charged to improve the water solubility of the conjugate. For example, the linker may contain about 1 to about 20, suitably about 2 to about 10, residues of one or more known water-soluble oligomers, such as peptides, oligosaccharides, glycosaminoglycans, polyacrylic acid or its salts, polyethylene glycol, polyhydroxyethyl (meth)acrylate, polysulfonates, etc. Suitably, the linker may contain a polar or charged peptide moiety containing, for example, 2 to 10 amino acid residues. The amino acid may refer to any natural or unnatural amino acid. The peptide linker suitably contains a free thiol group, preferably an N-terminal cysteine, for forming the cleavable disulfide bond with a thiol group on the drug moiety. Any peptide containing L- or D-amino acids may be suitable; particularly suitable peptide linkers of this type are Asp-Arg-Asp-Cys and / or Asp-Lys-Asp-Cys.

[0050] In these and other embodiments, moiety B, unit B L and / or Unit B S The peptide units may comprise cleavable or non-cleavable peptide units specifically tailored for selective enzymatic cleavage from the drug moiety by one or more proteases on the cell surface or extracellular region of the target tissue. The chain length of the amino acid residues of the peptide units suitably ranges from a single amino acid to about eight amino acid residues. Numerous specific cleavable peptide sequences suitable for use in the present invention can be designed and optimized for enzymatic cleavage by specific tumor-associated enzymes, e.g., proteases. Cleavable peptides for use in the present invention include those optimized for proteases MMP-1, 2, or 3, or cathepsin B, C, or D. Particularly preferred are peptides cleavable by cathepsin B. Cathepsin B is a ubiquitous cysteine ​​protease. It is an intracellular enzyme, except in pathological conditions such as metastatic tumors and rheumatoid arthritis. An example of a peptide cleavable by cathepsin B contains the sequence Val-Cit.

[0051] In any of the above embodiments, moiety B, in particular unit B L Preferably, the self-immolative linker further comprises a self-immolative moiety (which may or may not be present) following the linker. Self-immolative linkers are also known as electronic cascade linkers. These linkers undergo elimination and fragmentation upon enzymatic cleavage of the peptide, releasing the drug in an active, preferably free, form. The conjugate is stable extracellularly in the absence of an enzyme capable of cleaving the linker. However, upon exposure to an appropriate enzyme, the linker is cleaved, initiating a spontaneous self-immolative reaction that results in cleavage of the bond covalently linking the self-immolative moiety and the drug, thereby releasing the drug in its underivatized or pharmacologically active form. In these embodiments, the self-immolative linker is coupled to the binding moiety via an enzymatically cleavable peptide sequence that provides a substrate for the enzyme to cleave the amide bond and initiate the self-immolative reaction. Preferably, the drug moiety is linked to the self-immolative moiety of the linker via a chemically reactive functional group retained from the drug, such as a primary or secondary amine, hydroxyl, sulfhydryl, or carboxyl group.

[0052] Examples of self-immolative linkers include PABC or PAB (para-aminobenzyloxycarbonyl), which connects a drug moiety to a linking moiety in a conjugate (Carl et al. (1981) J. Med. Chem. 24:479-480; Chakravarty et al. (1983) J. Med. Chem. 26:638-644). The amide bond connecting the carboxy terminus of the peptide unit and the para-aminobenzyl of PAB is a substrate and can be cleaved by certain proteases. The aromatic amine becomes electron-donating and initiates an electronic cascade leading to the ejection of a leaving group, which releases the free drug after elimination of carbon dioxide (de Groot et al. (2001) Journal of Organic Chemistry 66(26):8815-8830). Additional self-immolative linkers are described in WO 2005 / 082023.

[0053] In yet another embodiment, the linker contains a glucuronyl group that can be cleaved by glucuronidase present on the cell surface or in the extracellular space of the target tissue. Lysosomal β-glucuronidase has been shown to be released extracellularly at high local concentrations in necrotic areas of human cancers, providing a route to targeted chemotherapy (Bosslet, K. et al., Cancer Res. 58, 1195-1201 (1998)).

[0054] In any of the above embodiments, moiety B suitably further comprises a spacer unit. The spacer unit is a unit B which may be linked to binding moiety A via, for example, an amide, amine or thioether bond. S The spacer unit may be, for example, of a length that allows the cleavable peptide sequence to be contacted by a cleaving enzyme (e.g., cathepsin B) and, suitably, also allows for hydrolysis of the amide bond coupling the cleavable peptide to the self-immolative moiety X. The spacer unit may comprise, for example, alkylene, arylene, heteroarylene, alkyloxy (e.g., polyethyleneoxy, PEG, polymethyleneoxy) and alkylamino (e.g., polyethyleneamino) repeating units, or divalent groups such as diacid esters and amides, including succinate, succinamide, diglycolate, malonate, and caproamide.

[0055] In any of the embodiments described herein, * is the attachment point to part A, or the shortest path to part A. ● represents an attachment point containing fewer atoms than in the ● is, in some cases, the attachment point to the treatment portion C or the shortest path to the treatment portion C. * represents the point of attachment to the therapeutic moiety C with fewer atoms than in the case of the reactive moiety L. The same applies when a reactive moiety L is present rather than a therapeutic payload moiety C. All of the following notations refer to the point of attachment of a particular group or atom (e.g., R) to a further moiety: [ka]

[0056] When the relevant structure is a peptide mono- or oligomer, each * is the shortest path to part A. ● represents an attachment point containing fewer atoms than in the case of ● is the shortest path to treatment part C. * represents attachment points containing fewer atoms than in the case of a , R b and R c When shown in any one of the structures, it may independently be present in one or more peptide monomer units, preferably in the one peptide monomer unit furthest from the other attachment points shown in the respective structures.

[0057] In any of the embodiments described herein, the terms "peptide," "dipeptide," "tripeptide," "tetrapeptide," etc. refer to peptide mono- or oligomers having a backbone formed by proteinogenic and / or non-proteinogenic amino acids. As used herein, the term "aminoacyl" or "amino acid" generally refers to any proteinogenic or non-proteinogenic amino acid. Preferably, in any of the embodiments disclosed herein, the side chain residue of a proteinogenic or non-proteinogenic amino acid is R a , R b and R c each of which is selected from the following list: [ka] (In the formula, R, R 1 , R 2 and R 3 each is independently selected from H, OH, SH, NH, halogen, cyano, carboxy, alkyl, cycloalkyl, aryl, and heteroaryl, each of which is substituted or unsubstituted; each X is independently selected from NH, NR, S, O, and CH, preferably NH; Each n and m is independently an integer preferably selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20).

[0058] Preferably, in any of the embodiments disclosed herein, the side chain residue of a proteinogenic or non-proteinogenic amino acid is R a , R b and R c and each of these groups may be part of a 3-, 4-, 5-, 6-, or 7-membered ring. For example, the α-, β-, and / or γ-positions of the side chain of the proteinogenic or non-proteinogenic amino acid may be the following amino acids (proline and hydroxyproline): [ka] or each of these may independently be part of an unsaturated structure (i.e., the respective group R a , R b and R c (there is no adjacent H atom), e.g., [ka] may be.

[0059] As used herein, the following peptide sequence notations refer to the sequence from N-terminus to C-terminus, and the attachment of a group via a horizontal bond (here: moiety C) refers to the covalent attachment to the peptide backbone via an amide bond to the respective terminal amino acid (here: AA3). [ka]

[0060] As used herein, the following peptide sequence notations refer to the sequence from N-terminus to C-terminus, and the attachment of groups via a vertical bond (here: moiety C) refers to a covalent bond via the side chain of the respective amino acid (here: AA3). [ka]

[0061] More preferred non-proteinogenic amino acids are listed below: [ka] may be selected from:

[0062] Particularly preferred embodiments of moiety B and of the compounds according to the invention are set out in the further paragraphs below and in the appended claims.

[0063] Preferably, B has the following structure: [ka] wherein each x is an integer independently selected from the range of 0 to 100, preferably 0 to 50, more preferably 0 to 30, and even more preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; each y is an integer independently selected from the range of 0 to 30, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; each z is an integer independently selected from the range of 0 to 5, preferably 0, 1, 2, 3, and 4; * represents the point of attachment to moiety A; and ● represents the point of attachment to the therapeutic moiety C).

[0064] L, B S and B Leach independently represents alkylene, cycloalkylene, arylalkylene, heteroarylalkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, arylalkenylene, heteroarylalkenylene, heteroalkenylene, heterocycloalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, oxoalkylene, dioxoalkylene, aminoalkylene , diaminoalkylene, diacid ester, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylenealkoxycarbamate, disulfide, vinylene, imine, imidamide, phosphoramide, saccharide, phosphate ester, phosphoramide, carbamate, dipeptide, tripeptide, and tetrapeptide, each of which is substituted or unsubstituted; L, B S and B L each of which independently [ka] [ka] [ka] [ka] wherein in each of the above structures: each n is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8; each m is independently 0, 1, 2, 3, or 4; Each R' is independently H or H, SH, NH, halogen, cyano, carboxy, C 1~6 Alkyl, O(C 1~6 alkyl), S(C 1~6 -alkyl), C 2~6 Alkenyl, C 2~6Alkynyl, C 1~6 Heteroalkenyl, C 1~6 Heteroalkynyl, C 3~10 Cycloalkenyl, C 1~10 Cycloheteroalkenyl, C 6~10 Aryl, and (C 6~10 Aryl)C 1~6 alkyl, each of which is optionally substituted with 1 to 3 substituents selected from -OH, oxo, and halo; Each R c , R d , and R e are independently H, optionally substituted C 1~6 Alkyl, (C3-C 10 Carbocyclyl)C 1~6 Alkyl, (C6-C 10 Aryl)C 1~6 Alkyl, (C1-C 10 Heterocyclyl)C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl and C6-C 10 aryl (in each of which optionally one or more of the carbon atoms may be replaced by a heteroatom); preferably selected from the side chain residues of proteinogenic or non-proteinogenic amino acids; each * is the shortest path to part A. ● represents an attachment point containing fewer atoms than in the case of ● is the shortest path to treatment part C. * represents attachment points containing fewer atoms than in the case of c , R d and R e When shown in any one of the structures, it can be independently present in one or more of the peptide monomer units, preferably in the one peptide monomer unit furthest from the other attachment points shown in the respective structures, wherein each of the above structures optionally includes an additional point of attachment to moiety A or C).

[0065] Part J Moiety J forms part of linking moiety B and is characterized in that it comprises a branch point, i.e. a central atom bonded to at least three non-hydrogen atoms, at least two of which are located on a covalent chain representing the shortest path between moiety A and the central atom. It can therefore be considered a multipoint template in which multiple arms bearing moieties A are bonded together.

[0066] As used herein, the moiety J is a group (L) a Together with these, they are collectively referred to as part K.

[0067] J independently represents alkylene, cycloalkylene, arylalkylene, heteroarylalkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, arylalkenylene, heteroarylalkenylene, heteroalkenylene, heterocycloalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, (oxo)alkylene, dioxoalkylene, aminoalkylene, diaminoalkenylene, The moiety may include a branch point that comprises or consists of structural units independently selected from the group consisting of: alkylene, diacid ester, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylenealkoxycarbamate, disulfide, vinylene, imine, imidamide, phosphoramide, saccharide, phosphate ester, phosphoramide, carbamate, dipeptide, tripeptide, tetrapeptide, each of which is substituted or unsubstituted.

[0068] Part C The moiety C in the present invention represents a therapeutic payload, which can generally be any atom (including H), molecule or particle. Preferably, the moiety C is not a hydrogen atom.

[0069] The payload may be a chelating agent for radiolabelling the therapeutic conjugate with a therapeutic nuclide. Suitably, the radionuclide is not released. Chelating agents are well known to those skilled in the art and include, for example, chelating agents such as sulfur colloid, diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane, N-(glutaric acid)-N',N'',N'''-triacetic acid (DOTAGA), 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (TETA), or any of the preferred chelating agent structures further listed in the following section or appended claims.

[0070] The therapeutic payload is 223 Ra, 89 Sr, 90 Y, 121 Sn, 177 Lu, 131 I, 211 At, 225 Ac, 188 Re, 149 Tb, 161 Tb and 227 Th, preferably 90 Y, 225 Ac or 177 Lu, more preferably 177 Radioactive groups may comprise or consist of therapeutic radioisotopes, including isotopes such as Lu; these may not be used for diagnostic applications.

[0071] The therapeutic payload may be a chelate of a therapeutic radioisotope, preferably an isotope listed above, with a chelator, preferably any of the chelators listed above or the preferred chelator structures listed further below; or a chelate with a group selected from the structures listed further below.

[0072] The payload may be a cytotoxic and / or cytostatic agent. Such agents may inhibit or prevent the function of a cell and / or cause destruction of the cell. Examples of cytotoxic agents include radiotherapeutic isotopes, chemotherapeutic agents, and toxins, such as small molecule or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including synthetic analogs and derivatives thereof. The cytotoxic agent may be selected from the group consisting of auristatins, DNA minor groove binders, DNA minor groove alkylating agents, enediynes, lexitropsins, duocarmycins, taxanes, puromycins, dolastatins, maytansinoids, and vinca alkaloids, or combinations of two or more thereof. Preferred cytotoxic and / or cytostatic payload moieties are further listed in section 8(e) below.

[0073] In one embodiment, the payload is selected from the group consisting of topoisomerase inhibitors, alkylating agents (e.g., nitrogen mustards; ethyleneimines; alkylsulfonates; triazenes; piperazines; and nitrosoureas), antimetabolites (e.g., mercaptopurine, thioguanine, 5-fluorouracil), antibiotics (e.g., anthracyclines, dactinomycin, bleomycin, adriamycin, mithramycin, dactinomycin), mitotic disruptors (e.g., plant alkaloids such as vincristine and / or microtubule antagonists such as paclitaxel), DNA methylating agents, DNA intercalators (e.g., The therapeutic chemotherapeutic agent is selected from the group consisting of carboplatin and / or cisplatin, daunomycin and / or doxorubicin and / or bleomycin and / or thalidomide), DNA synthesis inhibitors, DNA-RNA transcription regulators, enzyme inhibitors, gene regulators, hormone response modifiers, hypoxia-selective cytotoxins (e.g., tirapazamine), epidermal growth factor inhibitors, antivascular agents (e.g., xanthenone 5,6-dimethylxanthenone-4-acetic acid), radiation-activated prodrugs (e.g., nitroarylmethyl quaternary (NMQ) salts) or bioreductive drugs, or a combination of two or more thereof. In some embodiments, the payload (i.e., moiety C) is not derived from an anthracycline, and preferably is not derived from PNU 159682.

[0074] Therapeutic chemotherapeutic agents include erlotinib (TARCEVA®), bortezomib (VELCADE®), fulvestrant (FASLODEX®), Sutent (SU11248), letrozole (FEMARA®), imatinib mesylate (GLEEVEC®), PTK787 / ZK 222584, oxaliplatin (Eloxatin®), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, RAPAMUNE®), lapatinib (GSK572016), lonafarnib (SCH 66336), sorafenib (BAY43-9006), and gefitinib (IRESSA®), AG1478, AG1571 (SU 5271; Sugen), or a combination of two or more thereof.

[0075] Therapeutic chemotherapeutic agents include alkylating agents such as thiotepa, CYTOXAN®, and / or cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and / or piposulfan; alizyrines such as benzodopa, carboquone, meturedopa, and / or uredopa; ethyleneimines and / or methylamelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenediamine, methylamel ... ethylenethiophosphoramide and / or trimethylomelamine; acetogenins, such as bullatacin and / or bullatacinone; camptothecins; bryostatins; kallistatins; cryptophycins; dolastatins; duocarmycins; eleutherobin; pancrustatin; sarcodictyin; spongistatins; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide; mechlorethamine, mechlorethamine oxide hydrochloride oxide hydrochloride), melphalan, novembichin, phenesterine, prednimustine, trofosfamide and / or uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and / or ranimustine; dynemycin; bisphosphonates, such as clodronate; esperamycin; neocarzinostatin chromophore;Aclatinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®, doxorubicin, e.g., morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and / or deoxydoxol Bicine, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as dextromethorphan, benzodiazepines, benzodiazepines, benzophenone-3, benzodiazepines, benzodiazepines, benzophenone-4, benzodiazepines, benzodiazepines, benzophenone-5, benzodiazepines, benzodiazepines, benzophenone-6, benzodiazepines, benzophenone-8, benzophenone-9, benzophenone-10, benzophenone-11, benzophenone-12, benzophenone-13, benzophenone-14, benzophenone-15, benzophenone-16, benzophenone-17, benzophenone-18, benzophenone-19, benzophenone-20, benzophenone-21, benzophenone-22, benzophenone-23, benzophenone-24, benzophenone-25, benzophenone-26, benzophenone-27, benzophenone-28, benzophenone-29, benzophenone-30, benzophenone-31, benzophenone-32, benzophenone-33, benzophenone-34, benzophenone-35, benzophenone-36, benzophenone-37, benzophenone-38, benzophenone-39, benzophenone-40 purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, drostanolone propionate, epithiostanol, mepitiostane, tetanol Structone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; eflornithine; elliptinium acetate; epothilon; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; macrocyclic depsipeptides such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraelin; pentostatin; fenamet; pirarubicin;losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes, such as verracurin A, roridin A, and / or anguidine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside; cyclophosphamide; thiotepa; taxoids, such as taxol (TAXOL® )), paclitaxel, Abraxane, and / or TAXOTERE®, doxetaxel; chlorambucil; GEMZAR®, gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide; ifosfamide; mitoxantrone; vincristine; NAVELBINE®, vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronic acid; the topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids, such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, derivatives, or combinations of any two or more of the above.

[0076] Therapeutic payloads include taxanes such as paclitaxel and docetaxel, vinca alkaloids, discodermolide, epothilone A and B, desoxyepothilones, cryptophycin, curacin A, combretastatin A-4-phosphate, BMS 247550, BMS 184476, BMS 188791; LEP, RPR 109881A, EPO 906, TXD 258, ZD 6126, vinflunine, LU103793, dolastatin 10, E7010, T138067 and T900607, colchicine, phenstatin, chalcone, indanocine, T138067, oncocidin, vincristine, vinblastine, vinorelbine, vinflunine, halichondrin B, isohohohalichondrin B, ER-86526, pironetin, spongistatin 1, spike P, cryptophycin 1, LU103793 (cematodine or cemadotin), rhizoxin, sarcodictyin, eleutherobin, laurylamide, VP-16 and D-24851, as well as pharmaceutically acceptable salts, acids, derivatives or combinations of any two or more of the above.

[0077] The therapeutic payload may be a DNA intercalator, including, but not limited to, acridine, actinomycin, anthracycline, benzothiopyranoindazole, pixantrone, crisnatol, brostallicin, CI-958, doxorubicin (adriamycin), actinomycin D, daunorubicin (daunomycin), bleomycin, idarubicin, mitoxantrone, cyclophosphamide, melphalan, mitomycin C, bizelesin, etoposide, mitoxantrone, SN-38, carboplatin, cisplatin, actinomycin D, amsacrine, DACA, pyrazoloacridine, irinotecan, and topotecan, as well as pharmaceutically acceptable salts, acids, derivatives, or combinations of any two or more of the above.

[0078] The therapeutic payload may also be an antihormonal agent that acts to regulate or inhibit hormone action on tumors, such as antiestrogen agents and selective estrogen receptor modulators, including, but not limited to, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and / or fareston toremifene, as well as pharmaceutically acceptable salts, acids, derivatives, or combinations of two or more of any of the foregoing. The therapeutic payload may also be an aromatase inhibitor that inhibits aromatase, an enzyme that regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate, AROMASIN®, exemestane, formestany, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® and / or anastrozole, as well as pharmaceutically acceptable salts, acids, derivatives, or combinations of two or more of any of the foregoing.

[0079] The therapeutic payload may be an antiandrogen such as flutamide, nilutamide, bicalutamide, leuprorelin, goserelin and / or troxacitabine, as well as pharmaceutically acceptable salts, acids, derivatives or combinations of any two or more of the above.

[0080] The therapeutic payload may be a protein or an antibody. Preferably, the payload is a cytokine (e.g., an interleukin such as IL2, IL10, IL12, IL15; a member of the TNF superfamily; or an interferon such as interferon gamma).

[0081] Any therapeutic payload can be used in unmodified or modified form. A combination of some unmodified and some modified therapeutic payloads can also be used. For example, the therapeutic payload can be chemically modified. One form of chemical modification is derivatization of a carbonyl group, such as an aldehyde.

[0082] In a preferred embodiment, the payload moiety C is a topoisomerase inhibitor, preferably camptothecin (CPT) or a derivative thereof; more preferably derived from topotecan, irinotecan, ciratecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan, rubitecan, deruxtecan, DXd (e.g., by replacing a hydrogen atom); even more preferably exatecan; even more preferably [ka] wherein each n is 0, 1, 2, 3, 4, 5, or 6; most preferably [ka] is.

[0083] In a preferred embodiment, the therapeutic moiety C is an auristatin (i.e., having a structure derived from a member of the auristatin compound family) or an auristatin derivative. More preferably, the therapeutic moiety C has the following formula: [ka] (In the formula: R 1d are independently H or C1-C6 alkyl, preferably H or CH3; R 2d are independently C1-C6 alkyl; preferably CH3 or iPr; R 3d are independently H or C1-C6 alkyl, preferably H or CH3; R 4d are independently H, C1-C6 alkyl, COO(C1-C6 alkyl), CON(H or C1-C6 alkyl), C3-C 10 Aryl or C3-C 10 Heteroaryl; preferably H, CH3, COOH, COOCH3 or thiazolyl; R 5dare independently H, OH, or C1-C6 alkyl; preferably H or OH; R 6d are independently C3 to C 10 Aryl or C3-C 10 Heteroaryl; preferably optionally substituted phenyl or pyridyl).

[0084] More preferably, the therapeutic moiety C is derived from MMAE or MMAF.

[0085] In a preferred embodiment, the therapeutic moiety C has the following formula: [ka] (In the formula, n is 0, 1, 2, 3, 4 or 5; preferably 1; R 1e are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; R 2e are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; Each R 3e are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; R 4e are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; X is O, NH or S; preferably O).

[0086] In a preferred embodiment, the therapeutic moiety C has the following formula: [ka] (In the formula, n is 0, 1, 2, 3, 4 or 5; preferably 1; R 1fare independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; R 2f are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; R 3f are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; X is O, NH or S; preferably O).

[0087] Preferred embodiments of the therapeutic moiety C and the compounds according to the invention are further set out in the following paragraphs and appended claims.

[0088] As noted above, a preferred embodiment where C is a chelating agent is represented by formula III. An exemplary, particularly preferred structure of this type is represented by formula IIIa: [ka] wherein all variables and groups are as defined in the claims, and may exist in the form of a complex with any of the therapeutic radioisotopes disclosed herein, preferably 90-yttrium, 225-actinium or 177-lutetium, more preferably 177-lutetium.

[0089] Further Aspects Fragment ((B S ) x (B L ) y ) z can be represented by one of the following structures: Single bond, (B S ) x , [ka] [ka] (wherein AA3, AA4, AA5, AA6, AA7, and AA8 each represent a proteinogenic or non-proteinogenic amino acid, or are absent; Here preferably: Each proteinogenic or non-proteinogenic amino acid preferably independently has the structure: [ka] represented by one of; and / or AA4 is an amino acid having a charged side chain and AA7 is an amino acid having an aliphatic side chain; Where more preferably: AA3 is selected from Asp, Glu, and Lys, or is absent; preferably Asp; AA4 is selected from Arg, HomoArg, Lys, Asp, and Glu, or is absent; preferably Lys or Arg; AA5 is selected from Asp, Glu, and Lys; preferably Asp; AA6 is selected from Cys, Lys, Gly and Val; preferably Cys or Lys; AA7 is selected from Gly, Ala, Val, Arg, Ile, Pro; and AA8 is selected from Pro and citrulline (Cit); Even more preferably, the sequences shown in the table below: [Table 2] or Fragment ((B S ) x (B L ) y ) z has the following structure: single bond, [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] can be represented by one of the following:

[0090] In some embodiments, the compound has the following structure: [ka] [ka] can be represented by one of the following:

[0091] Preferred compounds according to the present invention have the following formula: [ka] [ka] (In the formula, B S , B L , x, y, and n, and the remaining groups, are as defined elsewhere herein; Each of AA3, AA4, AA5, AA6, AA7 and AA8 represents a proteinogenic or non-proteinogenic amino acid, or is absent; Preferably, each (B S ) x and (B L ) y are independently a bond, -NHC(O)(CH2)n C(O)-, -NH(CH2) n C(O)-, -NHC(O)(CH2CH2O) m (CH2) n -, -C(O)(CH2CH2O) m (CH2) n -, -C(O)(CH2CH2O) m (CH2) n NH-, -(CH2CH2O) m (CH2) n -, -(CH2CH2O) m (CH2) n NH-, -(CH2CH2O) m (CH2) n NHC(O)-, -(CH2) n O(CH(CHCHO) m (CH2) n -, -(CH2) n O(CH2CH2O) m (CH2) n NH-, -(CH2CH2O) m (CH2) n NHC(O)-, -C(O)(CH2) n O(CH2CH2O) m (CH2) n -, -C(O)(CH2) n O(CH2CH2O) m (CH2) n NH-, -(CH2) n O(CH2CH2O) m (CH2) n NH-, -C(O)(CH2) n C(O)-, -C(O)(CH2) n -, -C(O)(CH2) n NH- or -(CH2) n C(O)-, more preferably -C(O)(CH2) n C(O)- or -(CH2) n C(O)—, where each n and m is independently an integer preferably selected from 0, 1, 2, 3, 4, 5, and 6; More preferably [ka] Most preferably by [ka] It can be expressed as:

[0092] Preferred compounds according to the invention are also those of the following formula: [ka] More preferably by: [ka] [ka] Most preferably by [ka] (In the formula, B S , B L , x, y, and n, and the remaining groups, are as defined elsewhere herein; Preferably, each (B S ) x and (B L ) y are independently a bond, -NHC(O)(CH2) n C(O)-, -NH(CH2) n C(O)-, -NHC(O)(CH2CH2O) m (CH2) n -, -C(O)(CH2CH2O) m (CH2) n -, -C(O)(CH2CH2O) m (CH2) n NH-, -(CH2CH2O) m (CH2) n -, -(CH2CH2O) m (CH2) n NH-, -(CH2CH2O) m (CH2) n NHC(O)-, -(CH2) n O(CH(CHCHO)m (CH2) n -, -(CH2) n O(CH2CH2O) m (CH2) n NH-, -(CH2CH2O) m (CH2) n NHC(O)-, -C(O)(CH2) n O(CH2CH2O) m (CH2) n -, -C(O)(CH2) n O(CH2CH2O) m (CH2) n NH-, -(CH2) n O(CH2CH2O) m (CH2) n NH-, -C(O)(CH2) n C(O)-, -C(O)(CH2) n -, -C(O)(CH2) n NH- or -(CH2) n represented by -C(O)-, more preferably -C(O)(CH2) n C(O)- or -(CH2) n C(O)—, where each n and m is independently an integer preferably selected from 0, 1, 2, 3, 4, 5, and 6.

[0093] Moiety C is a therapeutic agent which may be (a) a chelator group suitable for radiolabeling with a therapeutic nuclide; (b) a therapeutic radioactive group comprising a therapeutic radioisotope; (c) a chelate of a therapeutic radioisotope with a chelator; (d) a cytotoxic and / or cytostatic therapeutic agent, (f) an immunomodulatory agent, or (g) a protein, preferably: (a) Chelating groups suitable for radiolabeling with therapeutic nuclides include sulfur colloids, diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (TETA), iminodiacetic acid, bis(carboxymethylimidazole)glycine, 6-hydrazinopyridine-3-carboxylic acid (HYNIC), [ka] [ka] selected from; or The following formula: [ka] (In the formula: n is 0, 1, 2, 3, 4 or 5; preferably 1; R 1e are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; R 2e are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; Each R 3e are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; R 4e are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; and X is O, NH, or S; preferably O; or The following formula: [ka] (In the formula: n is 0, 1, 2, 3, 4 or 5; preferably 1; R 1f are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; R 2f are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; R 3f are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; and X is O, NH, or S; preferably O; (b) The therapeutic radioactive group is 223 Ra, 89 Sr, 90 Y, 121 Sn, 177 Lu, 131 I, 211 At, 225 Ac, 188 Re, 149 Tb, 161 Tb and 227 a radioisotope selected from Th, preferably 90 Y, 225 Ac or 177 Lu, more preferably 177 Contains Lu; these may not be used for diagnostic purposes; (c) The chelate of the therapeutic radioisotope is a chelate of an isotope listed in (b) above and / or a chelate with a chelating agent listed in (a) above; or moiety C has the following structure: [ka] [ka] (wherein M is preferably a therapeutic radioisotope selected from the list in (b) above); more preferably: [ka] or (d) The cytotoxic and / or cytostatic therapeutic agent is selected from a chemotherapeutic agent selected from the group consisting of topoisomerase inhibitors, alkylating agents, antimetabolites, antibiotics, mitotic disruptors, DNA intercalators, DNA synthesis inhibitors, DNA-RNA transcription regulators, enzyme inhibitors, gene regulators, hormone response regulators, hypoxia-selective cytotoxins, epidermal growth factor inhibitors, antivascular agents, and combinations of two or more thereof, preferably having the following structure: [ka] [ka] [ka] [ka] [ka] [ka] wherein each n is 0, 1, 2, 3, 4, 5, or 6; [ka] [ka] [ka] [ka] selected from; or The moiety C preferably has the following formula: [ka] (In the formula: R 1d are independently H or C1-C6 alkyl; preferably H or CH3; R 2d are independently C1-C6 alkyl; preferably CH3 or iPr; R 3d are independently H or C1-C6 alkyl; preferably H or CH3; R 4d are independently H, C1-C6 alkyl, COO(C1-C6 alkyl), CON(H or C1-C6 alkyl), C3-C 10 Aryl or C3-C 10 Heteroaryl; preferably H, CH3, COOH, COOCH3 or thiazolyl; R 5d are independently H, OH, C1-C6 alkyl; preferably H or OH; and R 6d are independently C3 to C 10 Aryl or C3-C 10 is heteroaryl; preferably, optionally substituted phenyl or pyridyl), Preferably, the moiety C is derived from MMAE or MMAF; (e) the Immunomodulatory Therapeutic Agent is selected from a molecule known to be capable of modulating the immune system, e.g., a ligand of CD3, CD25, TLR, STING, 4-1BBL, 4-1BB, PD-1, mTor, PDL-1, NKG-2D IMiD, which ligand may be an agonist and / or antagonist; or (f) The protein is a therapeutic protein selected from cytokines such as IL2, IL10, IL12, IL15, TNF, interferon gamma, etc., or is a therapeutic antibody.

[0094] treatment The compounds described herein can be used to treat diseases. Treatment can be a therapeutic treatment aimed at preventing, alleviating, or halting an undesirable physiological change or disorder. Treatment can prolong survival compared to the expected survival in the absence of treatment. The disease treated by the compound can be any disease that may benefit from treatment. This includes chronic and acute disorders or diseases, including pathological conditions that predispose to the disorder.

[0095] The terms "cancer" and "cancerous" are used in the broadest sense to refer to the physiological condition in mammals that is typically characterized by unregulated cell growth. A tumor contains one or more cancerous cells. When treating cancer, the therapeutic effect observed may be a reduction in the number of cancer cells; a reduction in tumor size; an inhibition or delay in cancer cell infiltration into peripheral organs; an inhibition of tumor growth; and / or an alleviation of one or more symptoms associated with the cancer.

[0096] In animal models, efficacy can be assessed by physical measurement of the tumor during treatment and / or by assessment of partial and complete remission of the cancer. For cancer treatment, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and / or by determining the response rate (RR).

[0097] Particularly preferred embodiments of the treatment methods related to the present invention are further set out in the following paragraphs and the appended claims.

[0098] Also disclosed herein are methods for the treatment of the human or animal body, e.g., by therapy administered to the human or animal body, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or pharmaceutical composition described herein. More specifically, disclosed herein are methods for therapy, e.g., by treating a subject suffering from or at risk of a disease or disorder; methods for targeted delivery of a therapeutic agent to a subject suffering from or at risk of a disease or disorder. In the above methods, the disease or disorder may be independently selected from cancer, inflammation, atherosclerosis, fibrosis, tissue remodeling, and keloid disorders, and preferably, the cancer is selected from breast cancer, pancreatic cancer, small intestine cancer, colon cancer, multidrug-resistant colon cancer, rectal cancer, colorectal cancer, metastatic colorectal cancer, lung cancer, non-small cell lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, bile duct cancer, renal clear cell carcinoma, neuroendocrine tumors, oncogenetic osteomalacia, sarcoma, CUP (cancer of unknown primary), thymic carcinoma, desmoid tumor, glioma, astrocytoma, cervical cancer, skin cancer, kidney cancer, and prostate cancer. When used in the methods disclosed herein, the compounds remain at the disease site at therapeutically relevant levels for an extended period of time, for example, for at least 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours after injection, preferably for more than 1 hour, more preferably for more than 6 hours, and even more preferably for more than 24 hours.

[0099] The disclosure of all compounds, compositions or combinations for use in any method for treatment of the human or animal body disclosed herein is also intended to extend to the corresponding method for treating a disease or disorder in a subject in need thereof, comprising administration to the subject of a therapeutically effective amount of the compound, composition or combination; and also to the corresponding use of the compound, composition or combination in the manufacture of a medicament for treating the respective disease or disorder.

[0100] dose The inventors have surprisingly found that the present compounds, when given at specific doses, can provide optimal tumor uptake and / or tumor-to-organ ratios and therefore exhibit minimal trapping in other organs (i.e., a particularly clean biodistribution profile).

[0101] The compounds, pharmaceutical compositions, or combinations described herein may be administered to a subject at a dose of 10-500 nmol / kg, preferably 30-250 nmol / kg, more preferably 90-250 nmol / kg, even more preferably 90-160 nmol / kg, and most preferably 90-125 nmol / kg, of a compound of general formula I, II, III, IV, V, VI, or VII, expressed as a mouse dose; or the corresponding human equivalent dose. The compounds, pharmaceutical compositions, or combinations described herein may be administered to a human subject at a dose of 0.8-40 nmol / kg, preferably 2-20 nmol / kg, more preferably 7-20 nmol / kg, even more preferably 7-13 nmol / kg, and most preferably 7-10 nmol / kg of the compound.

[0102] As shown in Figure 15, it was found that a particularly clean biodistribution profile and optimal tumor uptake could be achieved in mice, preferably at doses in the range of 10 to 500 nmol / kg, and particularly at doses of 90 nmol / kg or higher. Above 250 nmol / kg, saturation effects were observed. Therefore, a range of 90 nmol / kg or higher, preferably 90 to 250 nmol / kg (expressed as mouse dose), is particularly preferred. A range of 7 nmol / kg or higher, preferably 7 to 20 nmol / kg (expressed as human dose), is particularly preferred.

[0103] The mouse dose can be converted to a human equivalent dose (HED) by taking into account, for example, the recommendations reported by the FDA in the USFDA (United States Food and Drug Administration) Guidance for Industry: Estimating the Maximum Safe Starting Dose in Adult Healthy Volunteer (Rockville, MD): USFDA; 2005. To do this, the mouse dose (nmol / kg) must be divided by a factor of 12.3 to obtain the corresponding HED (nmol / kg). For conversion to a human equivalent dose expressed in nmol or mg, a human reference weight of 60 kg, 70 kg, or 80 kg, preferably 70 kg, can be used. This conversion takes into account the difference in surface area between the two different species (Mus musculus vs. Homo sapiens).

[0104] For example, for a compound with a molecular weight of 2246 g / mol, a range of 90 nmol / kg to 250 nmol / kg expressed as a mouse dose corresponds to an HED of 0.016 mg / kg to 0.046 mg / kg or 1.1 mg to 3.2 mg, taking into account an average body weight of 70 kg.

[0105] From the above viewpoint, it is particularly advantageous for the human dose to be 1 mg or more, preferably 1 to 3 mg per administration.

[0106] Pharmaceutical Composition The compounds described herein may be in the form of pharmaceutical compositions for human or animal therapeutic use in human and / or veterinary medicine, typically containing one or more pharmaceutically acceptable diluents, carriers, or excipients. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro, ed., 1985). The choice of pharmaceutical carrier, excipient, or diluent can be selected based on the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may contain any suitable binder, lubricant, suspending agent, coating agent, solubilizer as, or in addition to, the carrier, excipient, or diluent.

[0107] The pharmaceutical composition for treatment may contain preservatives, stabilizers, dyes, and flavoring agents. Examples of preservatives include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

[0108] Different delivery systems may have different composition / formulation requirements. As an example, a therapeutic pharmaceutical composition may be formulated to be administered using a minipump or via a mucosal route, e.g., as a nasal spray or aerosol for inhalation or an ingestible solution, or may be formulated to be administered parenterally (e.g., the composition is formulated in an injectable form for delivery via intravenous, intramuscular, or subcutaneous routes). Alternatively, a therapeutic formulation may be designed to be administered via a number of routes.

[0109] When a drug is administered mucosally through the gastrointestinal mucosa, the drug must remain stable during passage through the gastrointestinal tract; for example, the drug must be resistant to proteolysis, stable at acidic pH, and resistant to the cleansing effects of bile.

[0110] Where appropriate, therapeutic pharmaceutical compositions can be administered by inhalation, in the form of suppositories or pessaries, topically in the form of lotions, solutions, creams, ointments, or powders, by the use of skin patches, orally in the form of tablets containing excipients such as starch or lactose, or in the form of capsules or ovules alone or mixed with excipients, or in the form of elixirs, solutions, or suspensions containing flavorings or coloring agents, or the pharmaceutical compositions can be injected parenterally, for example, intravenously, intramuscularly, or subcutaneously. For parenteral administration, therapeutic compositions are best used in the form of a sterile aqueous solution, which may contain other substances, for example, sufficient salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration, therapeutic compositions can be administered in the form of tablets or lozenges, which can be formulated in conventional manner.

[0111] The therapeutic compounds of the present invention can be administered in the form of pharmaceutically acceptable salts or active salts. Pharmaceutically acceptable salts are well known to those skilled in the art and include, for example, those mentioned by Berge et al. in J. Pharm. Sci., 66, 1-19 (1977). Salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoic acid)) salts.

[0112] Routes of administration (delivery) include, but are not limited to, one or more of oral (e.g., as a tablet, capsule, or ingestible solution), topical, mucosal (e.g., as a nasal spray or aerosol for inhalation), nasal, parenteral (e.g., via an injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intraventricular, intracerebral, subcutaneous, ocular (including intravitreal or intracameral), transdermal, rectal, buccal, vaginal, epidural, and sublingual.

[0113] Typically, a physician will determine the actual therapeutic dose that will be most suitable for an individual subject. The specific dose level and frequency of administration for any particular patient can vary and will depend on a variety of factors, including the activity of the particular compound used, the metabolic stability and duration of action of that compound, age, body weight, general health, sex, diet, method and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual being treated.

[0114] Therapeutic formulations may be packaged in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example, water, for administration. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules, and tablets of the kind described above. Exemplary unit-dose formulations contain a daily dose or daily sub-dose, or an appropriate fraction thereof, of the active ingredient.

[0115] Pharmaceutical combinations As used herein, the term "pharmaceutical combination" means that the individual components contained therein can be administered simultaneously or sequentially, for example, in the same dosage form or as separate dosage forms; preferably, a subject is exposed at a given time to a therapeutically or prophylactically effective amount of each of the individual components of the combination. For example, a pharmaceutical combination can include two or more individual components within the same formulation or as separate formulations intended (e.g., as part of a therapeutic administration regimen / scheme or by instructions for use) or arranged (e.g., as a kit of parts) for simultaneous or sequential administration.

[0116] The compounds described herein may be administered to a patient in combination with a therapeutic agent, such as an immunoconjugate, preferably an immunocytokine, most preferably an immunocytokine comprising a human IL2 polypeptide conjugated to an scFv polypeptide sequence capable of binding to the epitope set forth in SEQ ID NO: 40 (and even more preferably an L19-IL2 immunocytokine further comprising linked VL and VH domains derived from the antibody L19, optionally capable of homodimerization in an scFv2 format), as described herein below.

[0117] Combination with immune cytokines Pharmaceutical combinations comprising a compound or pharmaceutical composition disclosed herein and an immunocytokine are provided.

[0118] As used herein, the term "immunocytokine" refers to a conjugated or fused protein comprising a cytokine and an antibody, antibody fragment, or antibody derivative. A fusion protein is a polypeptide that is the translation product resulting from the fusion of two or more genes or nucleic acid coding sequences into one open reading frame (ORF). The fusion expression product of two genes or ORFs may be conjugated by a linker. As used herein, the terms conjugated protein and fusion protein are generally used interchangeably. A fusion protein may further comprise a signal peptide sequence, usually located upstream (5') of the specific binding member and subunit.

[0119] Immune cytokines, including IL2 Preferably, the immunocytokine comprises a sequence having IL2 activity, i.e., an IL2 polypeptide, i.e., the cytokine IL2 or a functional fragment thereof. Preferably, the immunocytokine comprises one (i.e., a single) IL2 polypeptide per polypeptide chain. As used herein, the terms "IL2" and "IL2 polypeptide" are used interchangeably.

[0120] IL2 can be derived from any animal, for example, human, rodent (e.g., rat, mouse), horse, cow, pig, sheep, dog, etc. Human IL2 is preferred in conjugates for administration to humans. The amino acid sequence of human IL2 is set forth in SEQ ID NO: 21. The immunocytokine conjugate preferably comprises a single IL2 polypeptide. The IL2 polypeptide in the immunocytokine of the present invention retains the biological activity of IL2, for example, the ability to promote the proliferation and / or differentiation of activated T and B lymphocytes and natural killer (NK) cells, and / or the ability to induce cytotoxic T cell (CTL) activity and / or the ability to induce anti-tumor cytotoxicity of NK / lymphokine-activated killer (LAK) cells.

[0121] Antibodies, fragments or derivatives thereof contained in immunocytokines Immunocytokines include, in addition to cytokines (e.g., IL2), antibodies, antibody fragments, or antibody derivatives. Preferably, the immunocytokines herein include antibody derivatives. Antibody derivatives may include, for example, single-chain variable fragments (scFv), diabodies, or single-chain diabodies (scDb), or Fab, or Fab2, or nanobodies, or "SIP" (WO 2003 / 076469), or "Crab" (Neri et al., (1995) J Mol Biol, 246, 367-73). Immunocytokines may include IgG antibodies or IgG derivatives.

[0122] Preferably, the antibody derivative comprises an scFv. As known in the art, an scFv comprises a VH domain and a VL domain, where the domains are linked by a linker that allows association of the VH and VL domains to form an antigen-binding site. An scFv can be stabilized by the incorporation of a disulfide bridge linking the VH and VL domains

[48] .

[0123] Single-chain Fv (scFv) antibody polypeptide sequences are particularly preferred for incorporation into immunocytokines (e.g., with an additional polypeptide sequence having IL2 activity) due to the small size of the scFv format, which offers physiological and therapeutic advantages for in vivo use of the immunocytokine conjugate. Furthermore, the absence of an Fc region in scFvs may reduce anti-idiotypic responses and minimize undesirable properties related to complement activation and interactions with Fc receptors, which can hinder tumor targeting and lead to nonspecific cell activation.

[0124] The linker connecting the VH and VL domains within an scFv chain may be a peptide linker sequence that is not long enough to allow pairing of the VH and VL domains within the same scFv polypeptide chain. Thus, scFv homodimers can alternatively be formed in which the VH of one scFv chain pairs with the VL of the other scFv chain (or vice versa). This general format is referred to as the "scFv2" format, or sometimes referred to as a "diabody." Examples of suitable short linker sequences are GSSGG (SEQ ID NO: 26) and GGSGG (SEQ ID NO: 27). Preferably, the linker is a 12-residue linker such as SEQ ID NO: 22.

[0125] In so-called single-chain diabodies ("scDb"), two sets of VH and VL domains (i.e., the two polypeptides that pair to form a dimer in a diabody or scFv2) are linked as a single chain by a peptide linker ("scDb linker") as follows: (VH-VL)-linker-(VH-VL) (where "(VH-VL)" represents an scFv unit consisting of a set of VH and VL domains connected by a short linker as described above).

[0126] The scDb linker sequence is of sufficient length and / or flexibility to allow pairing of the VH domain of one scFv unit (i.e., a first VH- and VL-containing polypeptide) with the VL domain of another complementary scFv unit (i.e., a second VH- and VL-containing polypeptide), and vice versa, within a single polypeptide chain. Generally, a long and / or flexible linker that allows two complementary VH- and VL-containing polypeptides to dimerize in this manner within a single polypeptide chain is 10-20 amino acids in length.

[0127] Antigen-binding specificity of immune cytokines The antibody, antibody fragment, or antibody derivative suitably binds specifically to an extracellular matrix (ECM) component associated with tumor growth and / or angiogenesis. The antibody, antibody fragment, or derivative thereof (e.g., scFv, diabody, or single-chain diabody) comprises an antigen-binding site having complementarity-determining regions (CDRs) or VH and / or VL domains of an antibody capable of specifically binding to an antigen of interest. In particular, it may comprise one or more CDRs or VH and / or VL domains of an antibody capable of specifically binding to an antigen of ECM.

[0128] The antigen-binding sites of an antibody, antibody fragment, or antibody derivative (e.g., scFv, diabody, or scFv2, or single-chain diabody) may be identical or different, but are preferably identical (e.g., the L19 antigen-binding site of antibody L19, see below). Each of the antigen-binding sites may bind to the same antigen or epitope. This can be achieved by providing two identical antigen-binding sites, such as two identical VH-VL domain pairs, or by providing two different antigen-binding sites, for example, comprising different VH and VL domains, but which nevertheless both bind to the same antigen or epitope. Alternatively, the antibody, antibody fragment, or antibody derivative may be bispecific. "Bispecific" means that each of the antigen-binding sites binds to a different antigen. Optionally, the two antigen-binding sites may bind to two different antigens referred to herein, such as two different antigens of the extracellular matrix, or two different domains of a particular antigen (e.g., fibronectin or tenascin-C).

[0129] The antigen may be an antigen that is preferentially expressed by cells of the tumor or tumor neovasculature, or that is associated with the ECM. Such antigens include fibronectin and tenascin C, as discussed above.

[0130] As used herein, the term "specific binding" means that one member of a specific binding pair does not exhibit any significant binding to molecules other than its specific binding partner. This term is also applicable, for example, to cases where an antigen-binding site is specific for a particular epitope present on many different antigens, in which case an antibody, antibody fragment, or antibody derivative bearing the antigen-binding site can bind to a variety of antigens bearing the epitope.

[0131] The antibody, fragment, or derivative thereof can specifically bind to fibronectin. Fibronectin is an antigen that undergoes alternative splicing, and many alternative isoforms of fibronectin are known, including alternatively spliced ​​isoforms A-FN and B-FN, which contain domains ED-A and ED-B, respectively, known markers of angiogenesis. Preferably, the antibody, fragment, or derivative thereof binds to fibronectin isoform B-FN, and most preferably to the ED-B domain (extra domain B) of fibronectin isoform B-FN. The amino acid sequence of the ED-B domain of B-FN is provided by residues 1266 to 1356 of UniProt database entry P02751 (human fibronectin) and is represented herein as SEQ ID NO: 39.

[0132] Thus, preferably, the antibody, fragment or derivative portion of the immunocytokine binds to the epitope of SEQ ID NO: 39. Preferably, the antibody, fragment or derivative (e.g., scFv polypeptide sequence) binds to the epitope represented by SEQ ID NO: 40.

[0133] The fibronectin isoform B-FN is one of the best-known markers of angiogenesis (WO 1997 / 045544). The 91-amino acid extra domain "ED-B" is found in the B-FN isoform and is identical in mice, rats, rabbits, dogs, and humans. B-FN accumulates around the neovasculature of invasive tumors and other tissues undergoing angiogenesis, such as the proliferative endometrium and some pathological ocular structures, but is not detected in other normal adult tissues.

[0134] Preferably, the antibody derivative is a human monoclonal scFv sequence that specifically binds to the alternatively spliced ​​ED-B domain of fibronectin isoform B-FN, and preferably the scFv sequence binds to the epitope of SEQ ID NO: 39, e.g., the epitope represented by SEQ ID NO: 40.

[0135] Also preferably, the antibody, antibody fragment, or antibody derivative (e.g., scFv sequence) that specifically binds to the alternatively spliced ​​ED-B domain of fibronectin isoform B-FN comprises an antigen-binding site derived from antibody L19. Antibody L19 specifically binds to the alternatively spliced ​​ED-B domain of fibronectin isoform B-FN, and the epitope is represented by SEQ ID NO: 40. The sequence of antibody L19 is disclosed in Pini et al. (1998) J. Biol. Chem. 273:21769-21776 or U.S. Pat. No. 8,097,254. Thus, an example of a most preferred antibody derivative is the L19 scFv described previously (WO 1999 / 058570; WO 2006 / 119897 or WO 2003 / 076469); see also SEQ ID NO: 15 of WO 2020 / 070150 and the immunocytokine comprising the L19 scFv in Figure 1B).

[0136] Most preferably, the immunocytokine comprises (as an antibody derivative) a human monoclonal scFv polypeptide sequence specific for the alternatively spliced ​​ED-B domain of fibronectin isoform B-FN as described above (e.g., the epitope represented by SEQ ID NO: 40) and (as a cytokine) IL2 (see, e.g., SEQ ID NO: 15 and FIG. 1B of WO 2020 / 070150, and SEQ ID NO: 12 herein).

[0137] Antigen-binding sites and sequences The antigen-binding site may comprise, for example, one, two, three, four, five, or six CDRs of antibody L19. The amino acid sequences of the CDRs of L19 are as follows: SEQ ID NO: 13 (CDR1 VH); SEQ ID NO: 14 (CDR2 VH); SEQ ID NO: 15 (CDR3 VH); SEQ ID NO: 16 (CDR1 VL); SEQ ID NO: 17 (CDR2 VL), and / or SEQ ID NO: 18 (CDR3 VL).

[0138] SEQ ID NOs: 13 to 15 are the amino acid sequences of the VH CDR region (1 to 3 respectively) of human monoclonal antibody L19. SEQ ID NOs: 16 to 18 are the amino acid sequences of the VL CDR region (1 to 3 respectively) of human monoclonal antibody L19.

[0139] The antigen-binding site may be flanked by one, two, three, four, five, six, seven, or eight framework regions of antibody L19. The amino acid sequences of the framework regions of L19 are as follows: SEQ ID NO:30 (framework region 1 VH); SEQ ID NO:31 (framework region 1 VH); SEQ ID NO:32 (framework region 1 VH); SEQ ID NO:33 (framework region 1 VH); SEQ ID NO:34 (framework region 1 VL); SEQ ID NO:35 (framework region 1 VL); SEQ ID NO:36 (framework region 1 VL); and / or SEQ ID NO:37 (framework region 1 VL).

[0140] The amino acid sequences of the VH and VL domains of antibody L19 correspond to SEQ ID NOs: 19 and 20, respectively.

[0141] Preferably, the antibody derivative comprises a VH domain having an amino acid sequence comprising the VH CDR1, VH CDR2, and / or VH CDR3 of L19, and a VL domain having an amino acid sequence comprising the VL CDR1, VL CDR2, and / or VL CDR3 of L19.

[0142] The antibody derivatives described above may comprise a VH domain having an amino acid sequence that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (preferably 80%) sequence identity to the amino acid sequence of the L19 VH domain set forth in SEQ ID NO: 22, and / or may comprise a VL domain having an amino acid sequence that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (preferably 80%) sequence identity to the amino acid sequence of the L19 VL domain set forth in SEQ ID NO: 23.

[0143] Preferably, the antibody derivative is an L19 scFv (i.e., an scFv derived from and comprising one or more antigen-specific portions of the antibody L19; interchangeably referred to as "scFv(L19)"). Thus, the L19 scFv may comprise one, two, three, four, five, or six CDRs (preferably, all six CDRs) of the antibody L19. Optionally, the L19 scFv may comprise the L19 VH domain (SEQ ID NO: 22) and / or the L19 VL domain (SEQ ID NO: 23), e.g., both the L19 VH and L19 VL.

[0144] In the scFv unit, the VH and VL domains are connected by a linker. The linker may be a peptide linker sequence that is not long enough to allow pairing of the VH and VL domains. Thus, scFv homodimers can alternatively be formed in which the VH of one scFv chain pairs with the VL of the other scFv chain (or vice versa). This general format is referred to as the "scFv2" format, or sometimes as a "diabody." Examples of suitable short linker sequences are GSSGG (SEQ ID NO: 26) and GGSGG (SEQ ID NO: 27). Preferably, the linker is a 12-residue linker such as SEQ ID NO: 22.

[0145] The antibody derivative may be an L19 diabody having an amino acid sequence that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (preferably 80%) sequence identity to the amino acid sequence set forth in SEQ ID NO:28, or an L19 scDb having an amino acid sequence that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (preferably 80%) sequence identity to the amino acid sequence set forth in SEQ ID NO:29.

[0146] In a preferred embodiment, the antibody derivative is an scFv(L19) having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (preferably 80%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 38.

[0147] Linking segments within immunocytokines The immunocytokine comprises a cytokine (e.g., an IL2 polypeptide) conjugated to an antibody, antibody fragment, or antibody derivative. This conjugation can be via any suitable covalent bond or linker moiety, such as a disulfide bond or a peptide bond, most preferably a peptide linker sequence. The peptide linker sequence can be a short (2-30, preferably 10-20) stretch of amino acids. Suitable examples of peptide linker sequences are known in the art. Examples of suitable linker sequences are (G4S)3 (SEQ ID NO: 24) or GSLDGAGGSAGADGG (SEQ ID NO: 25). One or more different linkers may be used. Preferably, the linker can be the 17-residue linker of SEQ ID NO: 23.

[0148] Thus, the antibody, antibody fragment, or antibody derivative and IL2 may be produced and / or secreted as a single polypeptide chain.

[0149] IL2 is preferably linked to the C-terminus of the antibody, antibody fragment, or antibody derivative. Preferably, the linkage may be via a peptide linker sequence, as disclosed herein. When IL2 is conjugated to the C-terminus, the N-terminus of the antibody, antibody fragment, or antibody derivative is preferably free. "Free" in this context refers to the N-terminus not being linked to another moiety, such as IL2, or conjugated in any other way.

[0150] Preferred L19-IL2 immunoconjugates. Preferably, the immunocytokine comprises an L19-derived scFv unit linked to an IL2 polypeptide in a conjugated polypeptide chain, forming a homodimer by complementary pairing between the VH and VL domains of the scFv units in the two polypeptide chains (according to the scFv2 or diabody format). Thus, preferably, the immunocytokine comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (preferably 80%) sequence identity with the amino acid sequence of the "L19-IL2" immunocytokine represented by SEQ ID NO: 12, which comprises an L19-derived scFv unit linked to IL2, has such functionality, and thus forms a homodimer via the scFv unit. More preferably, the immunocytokine comprises the sequence of SEQ ID NO: 12 (i.e., the amino acid sequence of the scFv format L19-IL2 polypeptide).

[0151] Production and purification of the L19-IL2 construct may be performed as described in WO 01 / 062298. A schematic diagram of the scFv-format L19-IL2 conjugate is shown in FIG.

[0152] Further definitions of immune cytokines Sequence identity is generally defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego, USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximizing the number of matches and minimizing the number of gaps. Generally, default parameters are used, with a gap creation penalty of 12 and a gap extension penalty of 4. Although GAP is preferred, other algorithms may also be used, such as BLAST (using the method of Altschul et al. (1990) J. Mol. Biol. 215:405-410), FASTA (using the method of Pearson and Lipman (1988) PNAS USA 85:2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol. Biol. 147:195-197), or the TBLASTN program of Altschul et al. (1990) supra, which generally utilize default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may also be used.

[0153] Variants of these VH and VL domains and CDRs may be used in antibody molecules for use in conjugates as described herein. Suitable variants can be obtained by methods of sequence modification or mutation and screening.

[0154] Particular variants for use as described herein may contain one or more amino acid sequence modifications (additions, deletions, substitutions and / or insertions of amino acid residues) (perhaps less than about 20 modifications, less than about 15 modifications, less than about 10 modifications, or less than about 5 modifications, 4, 3, 2 or 1 modification).

[0155] precursor compound In one aspect of the present invention, a compound, its individual diastereoisomers, its hydrates, its solvates, its crystalline forms, its individual tautomers, or its salts is disclosed herein, wherein the compound (precursor compound) comprises three moieties A and a reactive moiety G that can react with a conjugation partner to form a covalent bond. Upon conjugation (i.e., reacting to form a covalent bond), the former precursor compound binds to the former conjugation partner, which in turn binds to a therapeutic payload moiety C. The conjugation partner can be an atom, molecule, particle, or therapeutic agent. Preferably, the conjugate is a therapeutic agent, which can correspond to the payload moieties already described in detail above for the therapeutic conjugates according to the present invention.

[0156] Each moiety A preferably has the structure A defined above. 1 or A 2 It has.

[0157] Preferably, the precursor compound has the following formula: [ka] where B is a covalent bond or a multifunctional moiety that covalently attaches moiety A to G.

[0158] Preferably, G is an amide, ester, carbamate, hydrazone, thiazolidine, methylenealkoxycarbamate, disulfide, alkylene, cycloalkylene, arylalkylene, heteroarylalkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, arylalkenylene, heteroarylalkenylene, heteroalkenylene, heterocycloalkenylene, alkynylene, heteroalkynyl, heteroalkynyl, heterocycloalkenylene, ... capable of forming an nylene, arylene, heteroarylene, aminoacyl, oxyalkylene, aminoalkylene, diacid ester, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylenealkoxycarbamate, disulfide, vinylene, imine, imidamide, phosphoramide, saccharide, phosphate ester, phosphoramide, carbamate, dipeptide, tripeptide, or tetrapeptide linking group; and / or Moiety B preferably has a structure as detailed above for the conjugates according to the invention.

[0159] The moiety G preferably undergoes reaction to form an amide, ester, carbamate, hydrazone, thiazolidine, methylenealkoxycarbamate, disulfide, alkylene, cycloalkylene, arylalkylene, heteroarylalkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, arylalkenylene, heteroarylalkenylene, heteroalkenylene, heterocycloalkenylene, alkynylene, hetero The linking group can be formed as an alkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, aminoalkylene, diacid ester, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylenealkoxycarbamate, disulfide, vinylene, imine, imidamide, phosphoramide, saccharide, phosphate ester, phosphoramide, carbamate, dipeptide, tripeptide or tetrapeptide. As will be appreciated by those skilled in the art, there are multiple possibilities for providing a reactive group that can react with a conjugation partner to form a linking group according to the foregoing list, all of which are encompassed by the present disclosure.

[0160] Moiety B may be a cleavable or non-cleavable multifunctional moiety and can be used to link one or more reactive moieties and / or binder moieties to form the conjugate precursor of the invention. In some embodiments, the structure of the compound independently comprises more than one moiety A per molecule, preferably 2, 3, 4, 5, 6, 7, 8, 9, or 10 moieties A; and / or more than one moiety G per molecule, preferably 2, 3, 4, 5, 6, 7, 8, 9, or 10 moieties G. Preferably, the structure of the compound comprises three moieties A and one moiety G; or three moieties A and two moieties G per molecule.

[0161] The moiety G is preferably H, NH2, OH, N3, COOH, SH, Hal, [ka] [ka] wherein each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each m is independently 0, 1, 2, 3, 4, or 5; each Hal is F, Cl, Br, or I; and each R 4 is independently selected from carboxy, alkyl, cycloalkyl, aryl, and heteroaryl, wherein each of the foregoing is substituted or unsubstituted halogen and cyano.

[0162] In all structures, unless otherwise specified, all groups and variables are as further defined above throughout this disclosure.

[0163] Further compounds Also disclosed herein is a novel tetravalent organic ligand of fibroblast activation protein (FAP) suitable for therapeutic use ("Tetra-ESV6"), which comprises four small binding moieties A as defined above.

[0164] The compounds according to the present invention have the following general formula IV [ka] and an individual diastereoisomer thereof, a hydrate thereof, a solvate thereof, a crystalline form thereof, an individual tautomer thereof, or a pharmaceutically acceptable salt thereof, wherein A is a linking moiety; B is a multifunctional moiety containing a branching point, covalently linking moiety A to C; and C is an atom, molecule, or particle and is a therapeutic agent.

[0165] Alternatively, moiety C contains a branch point such that four moieties A are attached to C and B is absent, i.e., moiety C can also function as a branch point, replacing B, preferably when C is a chelator. An exemplary structure is represented by the general formula V: [ka] is expressed by

[0166] All exemplary structures, aspects, embodiments and definitions disclosed for the trivalent binders of general formulas I and III disclosed herein also apply mutatis mutandis to general formulas IV and V.

[0167] For example, the tetravalent linking agent may be represented by formula VI or VII: [ka] wherein all groups and variables are otherwise the same as defined for Formula II or III.

[0168] Exemplary structures of the moiety J' include: [ka] wherein all groups and variables are otherwise as defined herein.

[0169] Specific structures of the portion J' include: [ka] [ka] (wherein each i and j is an integer independently selected from 0, 1, 2, 3, and 4; preferably, each i is 1 or 2, and each j is 1, 2, or 3).

[0170] Preferred structures of J' include: [ka] Examples include:

[0171] Fragment ((L) a Particularly preferred structures of 4J' include: [ka] Examples include:

[0172] Particularly preferred tetravalent compounds are shown in Table 17.

[0173] Methods for preparing therapeutic conjugates In one aspect of the invention, disclosed herein is a method for preparing a therapeutic conjugate, comprising conjugating a precursor compound as described above with a conjugation partner. Preferably, the precursor compound is conjugated to the conjugation partner by reacting therewith to form a covalent bond. Preferably, the conjugate thus obtained is a therapeutic conjugate compound as described elsewhere herein.

[0174] The conjugation partner may be an atom, molecule or particle that is a therapeutic agent and may correspond to the therapeutic payload moieties already described in detail above for the conjugates according to the invention.

[0175] Preferably, the method further comprises formulating the conjugate as a therapeutic pharmaceutical composition. The pharmaceutical composition may be for human or animal treatment in human and veterinary medicine and typically comprises any one or more pharmaceutically acceptable diluents, carriers, or excipients. Therapeutically acceptable carriers or diluents are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro, ed., 1985). The choice of carrier, excipient, or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may comprise any suitable binder, lubricant, suspending agent, coating agent, solubilizer as, or in addition to, the carrier, excipient, or diluent. All formulation details and aspects disclosed in the "Pharmaceutical Compositions" section above apply fully here.

[0176] General Technology The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology and pharmacology, which are known to those skilled in the art, and such techniques are explained fully in the literature. See, for example, Gennaro, AR (ed.) (1990) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Hardman, JG, Limbird, LE, and Gilman, AG (eds.) (2001) The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill Co.; Colowick, S. et al. (eds.), Methods in Enzymology, Academic Press, Inc.; Weir, DM and Blackwell, CC (eds.) (1986) Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications; Maniatis, T. et al. (eds.) (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel, FM et al. (eds.) (1999) Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons; Ream et al. (eds.) (1998) See Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press; Newton, CR, and Graham, A. (eds.) (1997) PCR (Introduction to Biotechniques Series), 2nd ed., Springer Verlag.

[0177] chemical synthesis The compounds described herein can be prepared by chemical synthesis techniques. It will be apparent to those skilled in the art that sensitive functional groups may need to be protected and deprotected during compound synthesis. This can be accomplished by conventional techniques, such as those described in "Protective Groups in Organic Synthesis" by TW Greene and PGM Wuts, John Wiley and Sons Inc. (1991), and "Protecting Groups" by PJ Kocienski, Georg Thieme Verlag (1994). During some reactions, for example, when a base is used in a reaction with a substrate having an optical center containing a base-sensitive group, it is possible that any stereocenters present may be epimerized under certain conditions. As is well known in the art, such potential problems can be avoided by the selection of reaction sequences, conditions, reagents, protection / deprotection regimes, and the like.

[0178] definition Antibody. The term "antibody" is used in its broadest sense and encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), veneered antibodies, antibody fragments, and small immune proteins (SIPs) (see Int. J. Cancer (2002) 102, 75-85). Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. A target antigen generally has multiple binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen can have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to a desired target antigen or a portion thereof. The antibody can be of any type, such as IgG, IgE, IgM, IgD, and IgA, or any class, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, or subclass thereof. The antibody can be from a mouse, human, rabbit, or other species.

[0179] Antibody Fragments. The term "antibody fragment" refers to a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; dAbs, camelid V HH These include, but are not limited to, antibodies and single domain antibodies, including cartilaginous fish IgNAR antibodies. Antibodies and their fragments may be replaced by alternative non-immunoglobulin scaffolds, peptide aptamers, nucleic acid aptamers, structured polypeptides containing polypeptide loops embedded in non-peptide backbones, binding molecules based on natural receptors or domains thereof.

[0180] Derivatives. Derivatives include chemical modifications of compounds. Examples of such modifications include replacement of hydrogen with halo, alkyl, acyl, or amino groups, etc. The modifications may increase or decrease one or more hydrogen bonding interactions, charge interactions, hydrophobic interactions, van der Waals interactions, and / or dipole-dipole interactions.

[0181] Analogs. This term encompasses all enantiomers, racemates and stereoisomers, and all pharmaceutically acceptable salts and hydrates of such compounds.

[0182] Unless otherwise stated, the following definitions apply to chemical terms used in connection with the compounds of the present invention and compositions containing such compounds.

[0183] Alkyl refers to a branched or unbranched saturated hydrocarbyl group. Suitably, the alkyl group contains 1 to 100, preferably 3 to 30, more preferably 5 to 25 carbon atoms. Preferably, alkyl refers to methyl, ethyl, propyl, butyl, pentyl, or hexyl.

[0184] Alkenyl refers to a branched or unbranched hydrocarbyl group containing one or more carbon-carbon double bonds. Suitably, an alkenyl group contains from 2 to 30 carbon atoms, preferably from 5 to about 25 carbon atoms.

[0185] Alkynyl refers to a branched or unbranched hydrocarbyl group containing one or more carbon-carbon triple bonds. Suitably, the alkynyl group contains from about 3 to about 30 carbon atoms, for example, from about 5 to about 25 carbon atoms.

[0186] Halogen refers to fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.

[0187] Cycloalkyl refers to an alicyclic moiety, preferably one having 3, 4, 5, 6, 7, or 8 carbon atoms. This group may be a bridged or polycyclic ring system. More often, the cycloalkyl group is monocyclic. This term includes reference to groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and bicyclo[2.2.2]octyl.

[0188] Aryl refers to an aromatic carbocyclic ring system, preferably containing 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring carbon atoms. Aryl may also be a polycyclic ring system having two or more rings, at least one of which is aromatic. The term includes reference to groups such as phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthryl, and the like.

[0189] The prefix (hetero) herein means that one or more of the carbon atoms of the group may be replaced with nitrogen, oxygen, phosphorus, silicon, or sulfur. Heteroalkyl groups include, for example, alkyloxy and alkylthio groups. Heterocycloalkyl or heteroaryl groups herein may have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring atoms, at least one of which is selected from nitrogen, oxygen, phosphorus, silicon, and sulfur. Particularly preferred are 3- to 10-membered rings or ring systems, more particularly 5- or 6-membered rings, which may be saturated or unsaturated. For example, oxiranyl, azinyl, 1,2-oxathiolanyl, imidazolyl, thienyl, furyl, tetrahydrofuryl, pyranyl, thiopyranyl, thianthrenyl, isobenzofuranyl, benzofuranyl, chromenyl, 2H-pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidyl, piperazinyl, pyridazinyl, morpholinyl, thiomorpholinyl, in particular thiomorpholino, indolizinyl, 1,3-dioxo-1,3-dihydro-isoindolyl, 3H-indolyl, indolyl, benzimidazolyl, coumaryl, indolyl, It is selected from dazolyl, triazolyl, tetrazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolyl, benzofuranyl, dibenzofuranyl, benzothiophenyl, dibenzothiophenyl, phthalazinyl, naphthyridinyl, quinoxalyl, quinazolinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, [β]-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, furazanyl, phenazinyl, phenothiazinyl, phenoxazinyl, chromenyl, isochromanyl, chromanyl, 3,4-dihydro-2H-isoquinolin-1-one, 3,4-dihydro-2H-isoquinolinyl, and the like.

[0190] "Substituted" means that one or more, particularly up to five, more particularly one, two, or three hydrogen atoms in the moiety are independently replaced by a corresponding number of substituents. As used herein, the term "optionally substituted" includes substituted or unsubstituted. Of course, substituents are present only in chemically possible positions, and it will be understood that one skilled in the art can determine (experimentally or theoretically) whether a particular substitution is possible without undue effort. For example, an amino or hydroxy group having a free hydrogen atom may be unstable if it is attached to a carbon atom through an unsaturated (e.g., olefinic) bond. Preferably, the term "substituted" means that one or more, particularly up to five, more particularly one, two, or three hydrogen atoms in the moiety are independently replaced by a corresponding number of substituents selected from OH, SH, NH, halogen, cyano, carboxy, alkyl, cycloalkyl, aryl, and heteroaryl. Furthermore, the substituents described herein may themselves be substituted by any substituent, subject to the aforementioned restrictions on suitable substitution recognized by those skilled in the art. Preferably, any of the aforementioned substituents may be further substituted with any of the aforementioned substituents, each of which may be further substituted with any of the aforementioned substituents.

[0191] Suitable substituents include halogen atoms and halomethyl groups, such as CF3 and CCl3; oxygen-containing groups, such as oxo, hydroxy, carboxy, carboxyalkyl, alkoxy, alkoyl, alkoyloxy, aryloxy, aryloyl, and aryloyloxy; nitrogen-containing groups, such as amino, alkylamino, dialkylamino, cyano, azido, and nitro; sulfur-containing groups, such as thiol, alkylthiol, sulfonyl, and sulfoxide; heterocyclic groups, which may themselves be substituted; alkyl groups, which may themselves be substituted; and aryl groups, such as phenyl and substituted phenyl, which may themselves be substituted. Alkyl includes substituted and unsubstituted benzyl.

[0192] When two or more moieties are described as being "independently" selected from a list of atoms or groups, this means that the moieties can be the same or different, and thus the identity of each moiety is independent of the identity of one or more other moieties.

[0193] Example 1. General Description and Procedures Yields refer to chromatographically purified compounds unless otherwise specified.

[0194] Mass spectrometry (LC-ESI-MS) spectra were recorded on an Agilent 6100 Series single quadrupole MS system coupled to an Agilent 1200 Series LC system using a linear gradient of solvents A and B (A = Millipore water containing 0.1% formic acid [FA], B = MeCN containing 0.1% formic acid [FA]) at a flow rate of 2 mL / min on an InfinityLab Poroshell 120 EC-C18 column, 4.6 mm × 56 mm; or on an InfinityLab Poroshell 120 EC-C18 column, 2.7 μm, 4.6 × 50 mm, at a flow rate of 0.8 mL / min, from 10% ACN in 0.1% HCOOH in water to 100% ACN in 3 or 10 min.

[0195] High-resolution mass spectrometry (HRMS) spectra and analytical reversed-phase ultra-performance liquid chromatography (UPLC) were recorded on a Waters Xevo G2-XS QTOF coupled to a Waters Acquity UPLC H-Class system equipped with a PDA UV detector using an ACQUITY UPLC BEH C18 column, 130 Å, 1.7 μm, 2.1 mm × 50 mm, with a linear gradient of solvents A and B (A = Millipore water with 0.1% FA, B = MeCN with 0.1% FA) at a flow rate of 0.6 mL / min.

[0196] Preparative reversed-phase high-pressure liquid chromatography (RP-HPLC) was performed on an Agilent 1200 Series system using a Phenomenex Gemini® 5 μm NX-C18 semi-preparative column, 110 Å, 150 mm × 10 mm, at a flow rate of 5 mL / min with a linear gradient of solvents A and B (A = Millipore water with 0.1% trifluoroacetic acid [TFA], B = MeCN with 0.1% trifluoroacetic acid [TFA]); or on an Agilent 1200 Series RP-HPLC equipped with a PDA UV detector using a Synergi 10 μm, MAX-RP 80 Å, 10 × 250 mm C18 column with a linear gradient of solvents A and B (A = Millipore water with 0.1% TFA, B = ACN with 0.1% TFA) at a flow rate of 5 mL / min.

[0197] 2. Synthesis of precursors and reference compounds [ka]

[0198] Synthesis of ESV6-succinic acid-COOH (P4) for therapeutic applications Step 1: (S)-8-Amino-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)quinoline-4-carboxamide (P3). Commercially available 8-amino-quinoline-4-carboxylic acid (19.0 mg, 100 μmol, 1.0 equiv.), DIPEA (70.0 μL, 400 μmol, 4.0 equiv.), and HATU (38.0 mg, 100 μmol, 1.0 equiv.) were dissolved in a 1:1 DCM / DMF mixture (2 mL). After 15 min, a DCM solution of (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile trifluoroacetate (30.3 mg, 100 μmol, 1.0 equiv.) was added. The reaction mixture was stirred at room temperature for 1 h, washed with water, dried over NaSO, filtered, and concentrated to give the crude brown product as a sticky oil. The residue was purified by flash chromatography (DCM / MeOH 91:1 to 90:10) to give the pure product as a brownish oil (24.8 mg, 68.9 μmol, 69% yield). MS (ES)+ ) m / z 360(M+H) + .

[0199] Step 2: (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)4-oxobutanoic acid (P4). Triethylamine (20.8 μL, 150 μmol, 2.0 equiv) and 4-dimethylaminopyridine (0.91 mg, 10.0 μmol, 0.1 equiv) were added to a cooled (0° C.) solution of P3 (26.8 mg, 70.0 μmol, 1.0 equiv) in DCM, followed by the dropwise addition of succinic anhydride (15.0 mg, 150 μmol, 2.0 equiv). The reaction mixture was allowed to warm to room temperature. The reaction mixture was placed in a preheated 40° C. oil bath until complete conversion was observed. The solvent was evaporated and the residue was purified by RP-HPLC to give the pure product as a white powder (9.42 mg, 20.0 μmol, 28% yield). MS (ES + ) m / z 460(M+H) + . [ka]

[0200] Alternative synthesis of ESV6-succinate-COOH (P4) for therapeutic applications Step 1: 8-(4-(tert-butoxy)-4-oxobutanamido)quinoline-4-carboxylic acid To a solution of 4-(tert-butoxy)-4-oxobutanoic acid (57 mg, 0.33 mmol, 1.2 equiv.) in dry THF (1.5 mL) was slowly added SOCl (24 μL, 0.33 mmol, 1.2 equiv.) at 0 °C. Then, a solution of 8-aminoquinoline-4-carboxylic acid (50 mg, 0.25 mmol, 1 equiv.) in DMF (500 μL) was added, and the mixture was stirred at 0 °C for 30 min. The crude product was concentrated under reduced pressure and purified by reverse-phase MPLC (98:2 to 0:100 AcCN / water + 0.1% HCOOH, 45 min). The fractions were collected and lyophilized to give a white solid (50 mg, 0.15 mmol, 57%).

[0201] Step 2: tert-Butyl (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4-oxobutanoate. 8-(4-(tert-Butoxy)-4-oxobutanamido)quinoline-4-carboxylic acid (50 mg, 0.15 mmol, 1 equiv.), (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride (33 mg, 0.15 mmol, 1 equiv.), and HATU (55 g, 0.15 mmol, 1 equiv.) were suspended in 2.0 mL of DMF. DIPEA (100 μL, 0.6 mmol, 4 equiv.) was added dropwise, and the reaction was stirred for 15 minutes. The crude product was diluted with DCM, washed with water, dried over anhydrous NaSO, filtered, and the solvent was evaporated in vacuo. The crude product was concentrated under reduced pressure and purified by reverse-phase MPLC (98:2 to 0:100 AcCN / water + 0.1% HCOOH, 45 min). The fractions were collected and lyophilized to give a white solid (40 mg, 0.077 mmol, 52%).

[0202] Step 3: (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4-oxobutanoic acid (P4). (S)-tert-Butyl 4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4-oxobutanoate (40 mg, 0.077 mmol, 1 equiv) was dissolved in DCM (600 μL) and TFA (285 μL, 3.85 mmol, 50 equiv) was added dropwise at 0° C. The mixture was stirred for 2 hours and then concentrated under reduced pressure. The crude product was purified by reverse-phase MPLC (98:2 to 0:100 AcCN / water + 0.1% HCOOH, 45 min). The fractions were collected and lyophilized to give a white solid (25 mg, 0.054 mmol, 71%). [ka]

[0203] Synthesis of Bi-ESV6-COOH (P16) for therapeutic applications. 2-Chlorotrityl resin (300 mg) was added to a solid-phase synthesis syringe and then swollen with dry DCM for 15 min. Fmoc-1-Lys(Fmoc)-OH (89 mg, 0.15 mmol, 1 equiv.) and 4-methylmorpholine (45 μL, 0.40 mmol, 2.7 equiv.) were added sequentially to the resin, and the mixture was allowed to react for 3 h. A capping step with methanol / 4-methylmorpholine / DCM (1:2:7 ratio, 5 mL, 30 min) was then performed, followed by washing with DMF and removal of the Fmoc residue with 20% piperidine in DMF (10 mL). The resin was then treated with a solution of ESV6-succinic acid-COOH (P4, 137 mg, 0.300 mmol, 2.0 equiv.), HATU (86 mg, 0.22 mmol, 1.5 equiv.), and DIPEA (97 μL, 0.75 mmol, 5.0 equiv.) in DMF (5 mL) for 1 h. After multiple washes with DMF, the resin was cleaved with 30% TFA in DCM (10 mL) for 1 h. The cleaved solution was collected, concentrated in vacuo, and purified by RP-chromatography (gradient: water / acetonitrile + 0.1% FA 98:2 to 0:100, 45 min). Fractions were collected and lyophilized to afford Bi-ESV6-COOH (P16) as a white solid (30 mg, 0.029 mmol, 19% yield). MS (ES+) m / z 1029.3 (M+H). + . [ka]

[0204] Synthesis of Bi-ESV6-DOTAGA for therapeutic applications. To a DMF solution (500 μL) of Bi-ESV6-COOH (P16, 12 mg, 0.012 mmol), N-hydroxysuccinimide (2.0 mg, 0.017 mmol, 1.5 equiv.), HATU (6.7 mg, 0.017 mmol, 1.5 equiv.), and DIPEA (8 μL, 0.05 mmol, 4.0 equiv.) were added. After 30 min, a solution of DOTA-GA-NH2 (12 mg, 0.023 mmol, 2.0 equiv.) in water (500 μL) was added dropwise. The reaction mixture was stirred at room temperature for an additional 30 min and then purified by RP-HPLC (Agilent 1200 Series system equipped with a Synergi 4 μm Polar-RP 80 Å 10 × 150 mm C18 column using a gradient of 90:10 to 0:100 water / acetonitrile + 0.1% TFA in 12 min). Fractions were collected and lyophilized to give a white solid (10 mg, 0.007 mmol, 56% yield). MS (ES+) m / z 1530.5 (M+H). + . [ka]

[0205] Bi-ESV6-DOTAGA- for therapeutic use 175 Synthesis of Lu. Bi-ESV6-DOTAGA (4.0 mg, 2.6 μmol, 1 equiv.) was dissolved in pH 8 acetate buffer (300 μL). Subsequently, a solution of LuCl3 hexahydrate (2.0 mg, 5.2 μmol, 2 equiv.) dissolved in 0.05 N HCl (1.50 mL) was added. The reaction was stirred at 90 °C for 20 min, then cooled to room temperature and purified by RP-HPLC (Agilent 1200 Series system equipped with a Synergi 4 μm Polar-RP 80 Å 10 × 150 mm C18 column using a gradient of 90:10 to 50:50 water / acetonitrile + 0.1% TFA over 7 min). The desired fractions were collected and lyophilized to yield a pale yellow solid (2.2 mg, 49%). [ka]

[0206] Synthesis of ESV6-DOTAGA for therapeutic use. (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4-oxobutanoic acid (15 mg, 0.032 mmol, 1.0 equiv.) was dissolved in dry DMSO (400 μL). Dicyclohexylcarbodiimide (9 mg, 0.042 mmol, 1.3 equiv.) and N-hydroxysuccinimide (4.5 mg, 0.039 mmol, 1.3 equiv.) were added, and the reaction was stirred at room temperature overnight, protected from light. 100 μL of a PBS solution containing 2,2',2''-(10-(4-((2-aminoethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (20 mg, 0.039 mmol, 1.2 equiv.) was added, and the reaction was stirred for 2 h. The crude product was purified by reverse-phase HPLC (9.5:0.5 to 2:8 water 0.1% TFA / acetonitrile 0.1% TFA, 20 min) and lyophilized to give a white solid (2.4 mg, 8%). MS (ES+) m / z 960.39 (M+H). + . [ka]

[0207] ESV6-DOTAGA- for therapeutic use 175 Synthesis of Lu. To a solution of ESV6-DOTAGA (0.96 mg, 1 μmol, 1 equiv.) in 300 μL of acetate buffer (aqueous, 1 M, pH 8) was added a freshly prepared solution of LuCl3 hexahydrate (0.78 mg, 2 μmol, 2 equiv.) in 0.05 N HCl (1.5 mL). The resulting mixture was stirred at 95 °C for 10–15 min and then purified by RP-HPLC (90:10 to 0:100 ACN / water + 0.1% TFA for 12 min). The desired fractions were collected and lyophilized to give a white solid (0.8 mg, 71%). MS (ESI+) m / z 1133.3.

[0208] 3. Chemical structure of Tri-ESV6-DOTAGA(6) [ka] - Molecular weight: 2246.23 Da - Exact mass: 2244.8877 Da - Formula: C101H122F6N26O27 - SMILES notation: O=C(NCCNC(CCOCC(COCCC(NCCNC(CCC(NC1=C(N=CC=C2C(NCC(N3CC(F)(F)C[C@H]3C#N)=O)=O)C2=CC=C1)=O)=O)=O)(NC(CCC(N4CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC4)C(O)=O)=O)COCCC(NCCNC(CCC(NC5=CC=CC6=C5N=CC=C6C(NCC(N7CC(F)(F)C[C@H]7C#N)=O)=O)=O)=O)=O)=O)CCC(NC8=CC=CC9=C8N=CC=C9C(NCC(N%10[C@H](C#N)CC(F)(F)C%10)=O)=O)=O

[0209] 4. Chemical structure of Tri-ESV6-DOTA(7)

Chem.

[0210] 5. Chemical structure of Tri-ESV6-PEG12-DOTAGA [ka] - Molecular weight: 4045.38Da - Exact mass: 4042.94 Da - Formula: C182H118F6N26O25 - Smiles notation: O=C(NCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCC(NCCNC(CCOCC(COCCC(NCCNC(CCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCNC (CCC(NC1=CC=CC2=C1N=CC=C2C(NCC(N3[C@H](C#N)CC(F)(F)C3)=O)=O)=O)=O)=O)=O)(NC(CCC(N4CCN(CC(O)=O)CCN(CC(O)=O)CCN (CC(O)=O)CC4)C(O)=O)=O)COCCC(NCCNC(CCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCNC(CCC(NC5=CC=CC6=C5N=CC=C6C(NCC(N7[ C@H](C#N)CC(F)(F)C7)=O)=O)=O)=O)=O)=O)=O)=O)CCC(NC8=CC=CC9=C8N=CC=C9C(NCC(N%10[C@H](C#N)CC(F)(F)C%10)=O)=O)=O

[0211] 6. Synthesis of Tri-ESV6-DOTAGA and Tri-ESV6-DOTA a) Synthesis Route [ka]

[0212] b) Synthesis Experimental Procedure Step 1) (S)-(2-(4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4-oxobutanamido)ethyl)tert-butylcarbamate (2) [ka]

[0213] (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4-oxobutanoic acid (ESV6-COOH, 1, 100 mg, 217 μmol, 1 equiv.), tert-butyl (2-aminoethyl)carbamate (42 mg, 261 μmol, 1.2 equiv.), and HATU (100 mg, 261 μmol, 1.2 equiv.) were suspended in 2.0 mL of DMF. DIPEA (150 μL, 868 μmol, 4 equiv.) was added dropwise, and the reaction was stirred for 15 min. The crude product was purified by CombiFlash Nextgen 300+ (parameters: flow rate 30 mL / min, 24 gr C18 column, water / acetonitrile + 0.1% formic acid 98:2 to 0:100, 30 min) and the collected fractions were lyophilized to give a white solid (104 mg, 176 μmol, 80% yield).

[0214] Process 2) (S)-N 1 -(2-aminoethyl)-N 4 -(4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)succinimide (3) [ka]

[0215] tert-Butyl (S)-(2-(4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4-oxobutanamido)ethyl)carbamate (104 mg, 176 μmol) was dissolved in 2 mL of a 30% v / v solution of TFA in DCM and stirred at room temperature for 1 h. The crude product was purified by CombiFlash Nextgen 300+ (parameters: flow rate 30 mL / min, 24 gr C18 column, water / acetonitrile + 0.1% formic acid 98:2 to 0:100, 30 min), and the collected fractions were lyophilized to give a white solid (66 mg, 133 μmol, 76% yield).

[0216] Step 3) tert-Butyl (1,27-bis((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-14-((3-((2-(4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4-oxobutanamido)ethyl)amino)-3-oxopropoxy)methyl)-1,4,9,19,24,27-hexaoxo-12,16-dioxa-5,8,20,23-tetraazaheptacosan-14-yl)carbamate (4, Tri-ESV6-NHBoc) [ka]

[0217] (S)-N 1 -(2-aminoethyl)-N 4-(4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)succinimide (3, 66 mg, 133 μmol, 5 equiv.), 3,3'-((2-((tert-butoxycarbonyl)amino)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (11 mg, 26 μmol, 1 equiv.), and HATU (34 mg, 91 μmol, 3.5 equiv.) were suspended in 1.0 mL of DMF. DIPEA (40 μL, 260 μmol, 10 equiv.) was added to the mixture, and the reaction was stirred at room temperature for 4 h. The crude product was purified by CombiFlash Nextgen 300+ (parameters: flow rate 15 mL / min, 12 gr C18 column, water / acetonitrile + 0.1% formic acid 98:2 to 0:100, 30 min) and the collected fractions were lyophilized to give a white solid (16 mg, 8.5 μmol, 32% yield).

[0218] Process 4) N 1 ,N 1 '-(9-amino-9-((3-((2-(4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4-oxobutanamido)ethyl)amino)-3-oxopropoxy)methyl)-4,14-dioxo-7,11-dioxa-3,15-diazaheptadecane-1,17-diyl)bis(N 4 -(4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)succinamide) (5, Tri-ESV6-NH2) [ka]

[0219] (1,27-bis((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-14-((3-((2-(4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino) tert-Butyl (4-oxobutanamido)ethyl)amino)-3-oxopropoxy)methyl)-1,4,9,19,24,27-hexaoxo-12,16-dioxa-5,8,20,23-tetraazaheptacosan-14-yl)carbamate (4, Tri-ESV6-NHBoc, 16 mg, 8.5 μmol) was dissolved in 500 μL of a 30% v / v solution of TFA in DCM and stirred at room temperature for 1 h. The crude product was purified by CombiFlash Nextgen 300+ (parameters: flow rate 15 mL / min, 4 gr C18 column, water / acetonitrile + 0.1% formic acid 98:2 to 0:100, 30 min), and the collected fractions were lyophilized to give a white solid (13 mg, 7.2 μmol, 85% yield).

[0220] Step 5a) 2,2',2''-(10-(19-carboxy-1-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-14,14-bis((3-((2-(4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl) (I)carbamoyl)quinolin-8-yl)amino)-4-oxobutanamido)ethyl)amino)-3-oxopropoxy)methyl)-1,4,9,16-tetraoxo-12-oxa-5,8,15-triazanonadecan-19-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (6,Tri-ESV6-DOTAGA) [ka]

[0221] N 1 ,N 1’-(9-amino-9-((3-((2-(4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4-oxobutanamido)ethyl)amino)-3-oxopropoxy)methyl)-4,14-dioxo-7,11-dioxa-3,15-diazaheptadecane-1,17-diyl)bis(N 4 -(4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)succinamide) (5, Tri-ESV6-NH, 13 mg, 7.2 μmol, 1 equiv.), 2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTA-GA anhydride, 16 mg, 36 μmol, 5 equiv.), and DMAP (5 mg, 36 μmol, 5 equiv.) were suspended in 500 μL of dry DMF. The resulting mixture was stirred at 50 °C for 6 h, then diluted with milliQ water (2 mL) and purified by RP-HPLC (linear gradient from 90:10 to 0:100 ACN / water + 0.1% TFA). The desired fractions were collected and lyophilized to give a white solid (6 mg, 2.7 μmol, 38% yield).

[0222] Step 5b) 2,2',2'-(10-(17-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4,4-bis((3-((2-(4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino) Rubamoyl)quinolin-8-yl)amino)-4-oxobutanamido)ethyl)amino)-3-oxopropoxy)methyl)-2,9,14,17-tetraoxo-6-oxa-3,10,13-triazaheptadecyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (7,Tri-ESV6-DOTA) [ka]

[0223] N 1 ,N 1’ -(9-amino-9-((3-((2-(4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4-oxobutanamido)ethyl)amino)-3-oxopropoxy)methyl)-4,14-dioxo-7,11-dioxa-3,15-diazaheptadecane-1,17-diyl)bis(N 4 -(4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)succinamide) (5, Tri-ESV6-NH, 13 mg, 7.2 μmol, 1 equiv.), 2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTA-GA anhydride, 16 mg, 36 μmol, 5 equiv.), and DMAP (5 mg, 36 μmol, 5 equiv.) were suspended in 500 μL of dry DMF. The resulting mixture was stirred at 50 °C for 6 h, then diluted with milliQ water (2 mL) and purified by RP-HPLC (linear gradient from 90:10 to 0:100 ACN / water + 0.1% TFA). The desired fractions were collected and lyophilized to give a white solid (6 mg, 2.7 μmol, 38% yield).

[0224] 7. Synthesis of Tri-ESV6-PEG12-DOTAGA a) Synthesis Route [ka] [ka]

[0225] b) Synthesis Experimental Procedure Step 1) 2,5-dioxopyrrolidin-1-yl(S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4-oxobutanoate [ka]

[0226] (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4-oxobutanoic acid (ESV6-COOH, 1, 100 mg, 217 μmol, 1 equiv.), 1-hydroxypyrrolidine-2,5-dione (25 mg, 217 μmol, 1 equiv.), and HATU (100 mg, 261 μmol, 1.2 equiv.) were suspended in 1.0 mL of DMF. DIPEA (150 μL, 868 μmol, 4 equiv.) was added dropwise, and the reaction was stirred for 15 min. The crude product was purified by CombiFlash Nextgen 300+ (parameters: flow rate 30 mL / min, 24 gr C18 column, water / acetonitrile + 0.1% formic acid 98:2 to 0:100, 30 min) and the collected fractions were lyophilized to give a white solid (90 mg, 162 μmol, 75% yield).

[0227] Step 2) (S)-44-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-41,44-dioxo-4,7,10,13,16,19,22,25,28,31,34,37-dodecaoxa-40-azatetratetracontanoic acid [ka]

[0228] 2,5-Dioxopyrrolidin-1-yl (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4-oxobutanoate (ESV6-NHS, 90 mg, 162 μmol) and H2N-PEG12-COOH (CAS: 1415408-69-3, 60 mg, 97 μmol) were suspended in 1.0 mL of DMF. DIPEA (200 μL) was added dropwise and the reaction was stirred for 15 min. The crude product was purified by CombiFlash Nextgen 300+ (parameters: flow rate 30 mL / min, 24 gr C18 column, water / acetonitrile + 0.1% formic acid 98:2 to 0:100, 30 min) and the collected fractions were lyophilized to give a yellow oil (100 mg, 94 μmol, 97% yield).

[0229] Step 3) tert-Butyl (S)-(47-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4,44,47-trioxo-7,10,13,16,19,22,25,28,31,34,37,40-dodecaoxa-3,43-diazaheptatetracontyl)carbamate [ka]

[0230] (S)-44-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-41,44-dioxo-4,7,10,13,16,19,22,25,28,31,34,37-dodecaoxa-40-azatetratetracontanoic acid (ESV6-PEG-COOH, 100 mg, 94 μmol), tert-butyl (2-aminoethyl)carbamate (24 mg, 113 μmol, 1.2 equiv.), and HATU (57 mg, 148 μmol, 1.5 equiv.) were suspended in 2.0 mL of DMF. DIPEA (75 μL) was added dropwise, and the reaction was stirred for 15 min. The crude product was purified by CombiFlash Nextgen 300+ (parameters: flow rate 30 mL / min, 24 gr C18 column, water / acetonitrile + 0.1% formic acid 98:2 to 0:100, 30 min) and the collected fractions were lyophilized to give a yellow oil (95 mg, 79 μmol, 84% yield).

[0231] Process 4) (S)-N 1 -(42-amino-39-oxo-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxa-40-azadotetracontyl)-N4-(4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)succinamide [ka]

[0232] tert-Butyl (S)-(47-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4,44,47-trioxo-7,10,13,16,19,22,25,28,31,34,37,40-dodecaoxa-3,43-diazaheptatetracontylcarbamate (95 mg, 79 μmol) was dissolved in 2 mL of a 50% v / v solution of TFA in DCM and stirred at room temperature for 1 h. The crude product was purified using a CombiFlash Nextgen 300+ (parameters: flow rate 30 mL / min, 24 gr The mixture was purified using a C18 column, water / acetonitrile+0.1% formic acid 98:2 to 0:100, 30 minutes, and the collected fractions were lyophilized to give a yellow oil (67 mg, 61 μmol, yield 64%).

[0233] Step 5) tert-Butyl(1,107-bis((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-54-(53-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-5,10,50,53-tetraoxo-2,13,16,19,22,25,28,31,34,37,40,43,46-tridecaoxa-6,9, 49-triazatripentacontyl-1,4,44,49,59,64,104,107-octaoxo-8,11,14,17,20,23,26,29,32,35,38,41,52,56,67,70,73,76,79,82,85,88,91,94,97,100-hexacosaoxa-5,45,48,60,63,103-hexaazaheptahectan-54-yl)carbamate [ka]

[0234] (S)-N1 -(42-amino-39-oxo-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxa-40-azadotetracontyl)-N 4 -(4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)succinamide (ESV6-PEG-EtNH, 60 mg, 54 μmol, 4.5 equiv.), 3,3′-((2-((tert-butoxycarbonyl)amino)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (5.3 mg, 12 μmol, 1 equiv.), and HATU (21 mg, 54 μmol, 4.5 equiv.) were suspended in 0.5 mL of DMF. DIPEA (40 μL, 260 μmol, 10 equiv.) was added to the mixture, and the reaction was stirred at room temperature for 4 h. The crude product was purified by CombiFlash Nextgen 300+ (parameters: flow rate 15 mL / min, 12 gr C18 column, water / acetonitrile + 0.1% formic acid 98:2 to 0:100, 30 min) and the collected fractions were lyophilized to give a yellow oil (35 mg, 9.4 μmol, 78% yield).

[0235] Process 6) N 1 ,N 1’ -(49-amino-49-(53-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-5,10,50,53-tetraoxo-2,13,16,19,22,25,28,31,34,37,40,43,46-tridecaoxa-6,9,49- Triazatripentacontyl)-39,44,54,59-tetraoxo-3,6,9,12,15,18,21,24,27,30,33,36,47,51,62,65,68,71,74,77,80,83,86,89,92,95-hexacosaoxa-40,43,55,58-tetraazaheptanonacontane-1,97-diyl)bis(N 4 -(4-((2-(( S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)succinamide) [ka]

[0236] tert-Butyl(1,107-bis((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-54-(53-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-5,10,50,53-tetraoxo-2,13,16,19,22,25,28,31,34,37,40,43,46-tridecaoxa-6,9,49-triazatripe (Antacontyl)-1,4,44,49,59,64,104,107-octaoxo-8,11,14,17,20,23,26,29,32,35,38,41,52,56,67,70,73,76,79,82,85,88,91,94,97,100-hexacosaoxa-5,45,48,60,63,103-hexaazaheptahectan-54-yl)carbamate (Tri-ESV6-PEG-NHBoc, 30 mg, 8.1 μmol) was dissolved in 500 μL of a 50% v / v solution of TFA in DCM and stirred at room temperature for 1 h. The crude product was purified by CombiFlash Nextgen 300+ (parameters: flow rate 15 mL / min, 4 gr C18 column, water / acetonitrile + 0.1% formic acid 98:2 to 0:100, 30 min) and the collected fractions were lyophilized to give a white solid (15 mg, 4.2 μmol, 52% yield).

[0237] Step 7) 2,2',2''-(10-(1-carboxy-26-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-6,6-bis(20-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinoline-8 -yl)amino)-5,10,17,20-tetraoxo-2,13-dioxa-6,9,16-triazaicosyl)-4,11,16,23,26-pentaoxo-8,19-dioxa-5,12,15,22-tetraazahexacosyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (Tri-ESV6-PEG12-DOTAGA) [ka]

[0238] N 1 ,N 1’ -(49-amino-49-(53-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-5,10,50,53-tetraoxo-2,13,16,19,22,25,28,31,34,37,40,43,46-tridecaoxa-6,9,49- Triazatripentacontyl)-39,44,54,59-tetraoxo-3,6,9,12,15,18,21,24,27,30,33,36,47,51,62,65,68,71,74,77,80,83,86,89,92,95-hexacosaoxa-40,43,55,58-tetraazaheptanenonacontane-1,97-diyl)bis(N 4-(4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)succinamide) (Tri-ESV6-PEG-NH, 10 mg, 2.8 μmol, 1 equiv.), 2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTA-GA anhydride, 6.5 mg, 14 μmol, 5 equiv.), and DMAP (2 mg, 14 μmol, 5 equiv.) were suspended in dry DMF (500 μL). The resulting mixture was stirred at 50 °C for 6 h, then diluted with milliQ water (2 mL) and purified by RP-HPLC (linear gradient from 90:10 to 0:100 ACN / water + 0.1% TFA). The desired fractions were collected and lyophilized to give a white solid (7 mg, 1.6 μmol, 56% yield).

[0239] 8. Chemical Structure and Synthesis of Multivalent ESV6-DOTAGA Compounds a) Chemical structure of Tetra-ESV6-DOTAGA (10) [ka] - Molecular weight: 3062.0652Da - Exact mass: 3060.2291Da - Formula: C138H169F8N35O37 - Smiles notation: O=C(NCCNC(CCOCC(NC(CCOCC(NC(CCC(N1CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC1)C(O)=O)=O)COCCC(NC(COCCC(NCCNC(CCC(NC2=C(N=CC=C3C(NCC(N4CC(F)(F)C[C@H]4C#N)=O)=O)C3=CC=C2)=O)=O)=O)COCCC(NCCNC(CCC(NC5=CC=CC6=C5N=CC=C6C(NCC(N7[C@H](C#N)CC(F)(F)C7)=O)=O)=O)=O)=O)=O)=O)COCCC(NCCNC(CCC(NC8=C(N=CC=C9C(NCC(N%10CC(F)(F)C[C@H]%10C#N)=O)=O)C9=CC=C8)=O)=O)=O)=O)CCC(NC%11=CC=CC%12=C%11N=CC=C%12C(NCC(N%13[C@H](C#N)CC(F)(F)C%13)=O)=O)=O

[0240] b) Chemical structure of Hexa-ESV6-DOTAGA [Chemical formula] - Molecular weight: 4233.2028 Da - Exact mass: 4230.6586 Da - Formula: C192H227F12N49O49 - SMILES notation: O=C(NCCNC(CCOCC(NC(CCOCC(NC(CCC(N1CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC1)C(O)=O)=O)COCCC(NC(COCCC(NCCNC(CCC(NC2=C(N=CC=C3C(NCC(N4CC(F)(F)C[C@H]4C#N)=O)=O)C3=CC=C2)=O)=O)=O)(COCCC(NCCNC(CCC(NC5=C(N=CC=C6C(NCC(N7CC(F)(F)C[C@H]7C#N)=O)=O)C6=CC=C5)=O)=O)=O)COCCC(NCCNC(CCC(NC8=CC=CC9=C8N=CC=C9C(NCC(N%10[C@H](C#N)CC(F)(F)C%10)=O)=O)=O)=O)=O)=O)=O)(COCCC(NCCNC(CCC(NC%11=C(N=CC=C%12C(NCC(N%13CC(F)(F)C[C@H]%13C#N)=O)=O)C%12=CC=C%11)=O)=O)=O)COCCC(NCCNC(CCC(NC%14=C(N=CC=C%15C(NCC(N%16CC(F)(F)C[C@H]%16C#N)=O)=O)C%15=CC=C%14)=O)=O)=O)=O)CCC(NC%17=CC=CC%18=C%17N=CC=C%18C(NCC(N%19[C@H](C#N)CC(F)(F)C%19)=O)=O)=O

[0241] c) Chemical structure of Octa-ESV6-DOTAGA

Chem.

[0242] d) Synthetic Routes for the Synthesis of Tetra-ESV6-DOTAGA and Octa-ESV6-DOTAGA

Chem.

[0243] e) Synthetic Route for the Synthesis of Hexa-ESV6-DOTAGA [ka]

[0244] 9. In vitro tests and assays a) Inhibition assay The enzymatic activity of hFAP toward the Z-Gly-Pro-AMC substrate was measured at room temperature using a microtiter plate reader, monitoring fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. The reaction mixture contained substrate (20 μM), protein (constant 66 pM), assay buffer (50 mM Tris, 100 mM NaCl, and 1 mM EDTA, pH 7.4), and a 1:2 serial dilution of inhibitor from 167 nM to 80 fM in a total volume of 20 μL. Experiments were performed in triplicate, and mean fluorescence values ​​were fitted using Prism 7. This value is defined as the concentration of inhibitor required to reduce enzyme activity by 50% after the addition of substrate (Figures 1 and 8).

[0245] Following the same procedure, further comparative inhibition assays using trimeric binders, particularly those with different lengths of linker group B and / or different payload groups C, were performed using compounds Tri-ESV6-ValCit-MMAE (14), Tri-ESV6-Linker-DOTAGA (13), and diastereoisomers of Tri-ESV6-DOTAGA (11, 12) [Figure 18]. Comparative hFAP inhibition assays were also performed using tetravalent binders 10 and 29 [Figure 20].

[0246] b) Fluorescence polarization assay Fluorescence polarization experiments were performed in 384-well plates (non-binding, PS, F-bottom, black, high-volume, final volume 30 μL). A stock solution of hFAP was serially diluted (1:2) in buffer (50 mM Tris, 100 mM NaCl, and 1 mM EDTA, pH = 7.4), while the final concentration of binder was kept constant at 1 nM. Fluorescence anisotropy was measured using a Tecan microtiter plate reader. Experiments were performed in triplicate, and average anisotropy values ​​were fitted using Prism 7 ((Y = m1 + m2 × 0.5 × ((X + k + m3) - sqrt((X + k + m3)^2 - 4 × X × k)), where k is the concentration of fluorescent binder). The data are shown in Figure 2.

[0247] c) Cell excretion SK-RC-52.hFAP cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic. Cells were seeded into 24-well plates (200,000 cells / well, 0.5 mL / well) and incubated overnight at 37°C and 5% CO2. The medium was then removed, and the cells were washed with PBS (2 x 0.5 mL). 177 Lu-ESV6, 177 Lu-Bi-ESV6, 177 Lu-Tri-ESV6 or 177 The cells were incubated with a solution of Lu-FAP-2286 (10 KBq, 1 pmol, 0.5 mL). After 1 h, the medium was removed, the cells were washed with PBS (2 × 0.5 mL), and then incubated with fresh RPMI medium without supplements. The fraction of the culture medium was measured at different time points using a gamma counter (Packard Cobra). The percentage of bound compound was calculated as a percentage of the total activity, as shown in Figure 3. 177 Corrections were made for the decay of Lu.

[0248] d) LogD 7.4 Measurement of 177 Lu-ESV6, 177 Lu-Bi-ESV6, 177 Lu-Tri-ESV6 and 177The lipophilicity of Lu-FAP-2286 was measured as follows: A 100 μL aliquot of the radioligand (approximately 1 MBq) in PBS buffer was added to 500 μL of PBS buffer (pH 7.4) and 600 μL of 1-octanol. The two-phase mixture was vigorously shaken on a vortex mixer for 10 minutes and then centrifuged at 700 rpm for 5 minutes to facilitate separation. 100 μL aliquots of both phases were measured on a Packard Cobra Gamma Counter, and the partition coefficient was calculated by dividing cpm (octanol) by cpm (PBS), and the LogD 7.4 is shown as. LogD 7.4 ( 177 Lu-ESV6):-4.13 LogD 7.4 ( 177 Lu-Bi-ESV6):-3.75 LogD 7.4 ( 177 Lu-Tri-ESV6):-3.10 LogD 7.4 ( 177 Lu-FAP-2286):-3.05

[0249] 10. Animal Experiments All animal experiments were performed in accordance with Swiss animal protection laws and regulations under license number ZH006 / 2021 granted by Veterinaeramt des Kantons Zuerich.

[0250] SK-RC-52.hFAP cells were grown to 80% confluence in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic and then detached with 0.05% trypsin-EDTA (ethylenediaminetetraacetic acid). The tumor cells were resuspended in Hank's balanced salt solution. An aliquot of 5 million cells (100 μL of suspension) was injected subcutaneously into the right flank of female athymic Balb / c AnNRj-Foxn1 mice (6–8 weeks old).

[0251] a) 177 Radiolabeling with Lu Radiolabeling of ESV6-DOTAGA, Bi-ESV6-DOTAGA, Tri-ESV6-DOTAGA, Tetra-ESV6-DOTAGA, Hexa-ESV6-DOTAGA, Octa-ESV6-DOTAGA and FAP-2286 with lutetium-177 (a therapeutic radionuclide) was performed at different specific activities for three different studies (biodistribution, therapeutic and dose optimization).

[0252] Prior to biodistribution studies, the precursor (100 nmol) was dissolved in 100 μL of milliQ water and diluted with 200 μL of sodium acetate (1 M in water, pH = 4.5). 177 Lu solution was added and the mixture was heated at 90°C for 10 min and then diluted with 1600 μL of PBS to a final volume of 2 mL (20 doses of 100 μL each).

[0253] Prior to the therapeutic test, the precursor (25 nmol) was dissolved in 25 μL of milliQ water, followed by the addition of sodium acetate buffer (75 μL, 1 M in water) and 25, 75, or 150 MBq of 177Lu solution. The mixture was heated at 90 °C for 1 min and subsequently diluted with 400 μL of PBS to a final volume of 500 μL (five doses of 100 μL each). Quality control of the radiosynthesis was performed using radio-HPLC.

[0254] Prior to dose optimization studies for radiotherapy applications, the precursors (0, 24–180 nmol) were dissolved in milliQ water and diluted with sodium acetate (1 M in water, pH = 4.5). 177 Lu solution (4 MBq) was added and the mixture was heated at 90°C for 10 min and then diluted with PBS to a final volume of 400 μL (four doses of 100 μL each).

[0255] Prior to biodistribution studies at different specific activities (FIG. 19), the precursor (20 nmol) was dissolved in milliQ water and diluted with sodium acetate (1 M in water, pH=4.5). 177Lu solution (4 or 96 MBq) was added, and the mixture was heated at 90°C for 10 min and then diluted with PBS to a final volume of 400 μL (four doses of 100 μL each, corresponding to 0.2 MBq / nmol and 4.8 MBq / nmol, respectively).

[0256] b) Quantitative biodistribution analysis in tumor-bearing mice ESV6-DOTAGA, Bi-ESV6-DOTAGA, Tri-ESV6-DOTAGA, Tetra-ESV6-DOTAGA, Hexa-ESV6-DOTAGA, Octa-ESV6-DOTAGA and FAP-2286 were prepared as described above. 177 The tumors were radiolabeled with Lu. 3 The mice were randomized and allowed to grow to an average volume of 1000 mg / kg. 177 Lu-ESV6, 177 Lu-Bi-ESV6, 177 Lu-Tri-ESV6, Tetra-ESV6-DOTAGA, Hexa-ESV6-DOTAGA, Octa-ESV6-DOTAGA or 177 A radiolabeled formulation of Lu-FAP-2286 (250 nmol / kg; 50 MBq / kg) was intravenously injected. Mice were euthanized by CO2 asphyxiation at different time points after intravenous injection. Tumors, organs, and blood were harvested, weighed, and radioactivity was measured using a Packard Cobra Gamma Counter. Values ​​are expressed as %ID / g ± SD [Figure 4]. The %ID / g in the tumor was corrected to account for the SK-RC-52.hFAP tumor growth rate.

[0257] At different specific activities: low activity (0.2MBq / nmol) and high activity (4.8MBq / nmol) 177 Lu Tri-ESV6-DOTAGA 177 The results of the Lu biodistribution study are shown in FIG.

[0258] Values ​​(mean ± standard deviation): At different time points after intravenous administration (250 nmol / kg, 50 MBq / kg) to SK-RC-52.hFAP tumor-bearing mice 177 Lu-ESV6-DOTAGA,177 Lu-Bi-ESV6-DOTAGA, 177 Lu-Tri-ESV6-DOTAGA, 177 The quantitative in vivo biodistribution of Lu-FAP-2286, Tetra-ESV6-DOTAGA, Hexa-ESV6-DOTAGA, and Octa-ESV6-DOTAGA is shown in Tables 1 to 4 and 10 to 12. Data are presented as %ID / g ± standard deviation.

[0259] Tumor-to-organ ratio: At different time points after intravenous administration (250 nmol / kg, 50 MBq / kg) to SK-RC-52.hFAP tumor-bearing mice 177 Lu-ESV6-DOTAGA, 177 Lu-Bi-ESV6-DOTAGA, 177 Lu-Tri-ESV6-DOTAGA, 177 The tumor-to-organ ratios for Lu-FAP-2286, Tetra-ESV6-DOTAGA, Hexa-ESV6-DOTAGA, and Octa-ESV6-DOTAGA are shown in Tables 5-8 and 13-15. Avg. = mean; Std.d = standard deviation. Particularly advantageous results (e.g., kidney / tumor ratios) for Tri-ESV6-DOTAGA are shown in Table 7.

[0260] c) Evaluation of therapeutic efficacy in tumor-bearing mice 177 Lu-ESV6, 177 Lu-Bi-ESV6 and 177 The anticancer effect of Lu-Tri-ESV6 was evaluated in athymic Balb / c AnNRj-Foxn1 mice bearing SK-RC-52.hFAP tumors in the right flank. 177 Lu-ESV6, 177 Lu-Bi-ESV6 or 177 Lu-Tri-ESV6 was administered intravenously at a dose of 250 nmol / kg and 250 MBq / kg (a single dose administered 9 days after tumor implantation). The treatment experiment was conducted in mice with established tumors with an average volume of 100–150 mm. 3The study began when the tumor volume reached 100 mm. Animal weights and tumor volumes were measured daily and recorded. Tumor size was measured using electronic calipers, and tumor volume was calculated using the formula (longer side, mm) x (shorter side, mm) x (shorter side, mm) x 0.5. Animals were euthanized when they reached one or more of the termination criteria specified in the experimental permit. Data analysis was performed using Prism 7 software (GraphPad Software). The results are shown in Figures 5, 6, and 7.

[0261] d) Dose Escalation for Radiotherapy Applications Female athymic Balb / c AnNRj-Foxn1 mice bearing subcutaneous SK-RC-52.hFAP tumors were administered eight different doses ranging from 3 nmol / kg to 2250 nmol / kg. 177 Lu-Tri-ESV6-DOTAGA was injected intravenously. 24 hours later, mice were sacrificed, and tumors and healthy organs were harvested and measured using a gamma counter. Results, reported as %ID / g, are shown in Figure 15A. Results, reported as tumor-to-organ ratios, are shown in Figure 15B. Because the best tumor-to-organ ratios were obtained at high doses in the range of 90-250 nmol / kg, we predict that the ideal dose in human patients for radiotherapy applications would be between 1-3 milligrams per patient.

[0262] e) Therapeutic efficacy evaluation in tumor-bearing mice with dose escalation and in combination with L19-IL2 Anticancer effect of L19-IL2 (3 × 0.05 mg / mouse) as a single agent, 177 Anticancer effects of Lu-Tri-ESV6 at different doses (5MBq / mouse, 15MBq / mouse, 30MBq / mouse) 177 The combination of Lu-Tri-ESV6+L19-IL2 (5MBq / mouse) 177 The anti-cancer effects of Lu-Tri-ESV6 (followed by three injections of L19-IL2 at 0.05 mg / mouse) or saline were evaluated in athymic Balb / c AnNRj-Foxn1 mice bearing SK-RC-52.hFAP tumors on the right flank. Treatment experiments were performed on mice with established tumors with a mean volume of 100–150 mm. 3The study began when the tumor volume reached 100 mm. Animal weights and tumor volumes were measured daily and recorded. Tumor size was measured using electronic calipers, and tumor volume was calculated using the formula: (long side, mm) x (short side, mm) x (short side, mm) x 0.5. Animals were euthanized when they reached one or more of the termination criteria specified in the experimental permit. Data analysis was performed using Prism 7 software (GraphPad Software). The results are shown in Figure 9. 177 All mice receiving the Lu-Tri-ESV6+L19-IL2 combination were cured after treatment.

[0263] The L19-IL2 immunocytokine conjugate used in this example comprises a human IL2 polypeptide having the sequence of SEQ ID NO:21 fused at its N-terminus to the C-terminus of the VL domain of a single-chain variable fragment (scFv) molecule comprising the VH (SEQ ID NO:19) and VL (SEQ ID NO:20) domains of the antibody L19, which specifically binds to the extra domain B (ED-B) of fibronectin, via a 17-amino acid linker (SEQ ID NO:23). The bound epitope is the sequence of SEQ ID NO:40 (see Figure 4C in Fattorusso et al., Structure 7:381-390). In the immunocytokine used in this example, pairing of the VH domain of one L19-IL2 molecule with the VL domain of another L19-IL2 molecule allows for the formation of a homodimer via the scFv portion of the immunocytokine, essentially in the form of an "scFv2" or "diabody," as shown in Figure 12.

[0264] The amino acid sequence of the L19-IL2 immunocytokine conjugate is set forth in SEQ ID NO:12. EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGDGSGSGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSG SGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKEFSSSSGSSSSGSSSGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0265] These results demonstrate a surprising synergistic increase in therapeutic activity compared to the sum of the effects achieved by the individual components of the combination (SMDC and L19-IL2). Without wishing to be bound by any theory, it is believed that the mechanism of action of this combination involves the activation of NK cells within the tumor, which enhances the antitumor effect.

[0266] f) Therapeutic efficacy evaluation in tumor-bearing mice (optimization of the schedule for combination with L19-IL2) Anticancer efficacy was evaluated in athymic Balb / cAnNRj-Foxn1 mice bearing SK-RC-52.hFAP tumors on the right flank. Treatment experiments were performed in mice with established tumors with a mean volume of 100–150 mm. 3The treatment started when the tumor volume reached 1000 mg / mouse. Animal weights and tumor volumes were measured daily and recorded. Tumor size was measured with an electronic caliper, and tumor volume was calculated using the formula (longer side, mm) x (shorter side, mm) x (shorter side, mm) x 0.5. Animals were euthanized when they reached one or more of the termination criteria specified in the experimental permit. Data analysis was performed using Prism 7 software (GraphPad Software). Different groups were treated with (i) L19-IL2 as a single agent at a dose of 0.05 mg / mouse on days 8, 10, and 12; (ii) L19-IL2 as a single agent at a dose of 5 MBq / mouse on day 7; 177 treated with Lu-Tri-ESV6, (iii) 177 Treatment with the combination of Lu-Tri-ESV6+L19-IL2 (at a dose of 5MBq / mouse on day 7) 177 After Lu-Tri-ESV6 administration, mice were treated with either three doses of L19-IL2 at a dose of 0.05 mg / mouse on days 8, 10, and 12 (Schedule 1), three doses of L19-IL2 at a dose of 0.05 mg / mouse on days 12, 17, and 22 (Schedule 2), one dose of L19-IL2 at a dose of 0.05 mg / mouse on day 8 (Schedule 3), or one dose of L19-IL2 at a dose of 0.05 mg / mouse on day 12 (Schedule 4), or (iv) saline. The results are shown in Figure 13.

[0267] g) Quantitative in vivo MMAE release from different therapeutic conjugates Monomeric, dimeric, or trimeric ESV6 binding moieties were conjugated to the same linker-payload structure based on a glycine-proline linker and an MMAE cytotoxic moiety. Three conjugates: (i) ESV6-GlyPro-MMAE, corresponding to conjugate 58a in EP 3891138, (ii) Bi-ESV6-GlyPro-MMAE, corresponding to conjugate 11 in WO 2022 / 171811, and (iii) Tri-ESV6-GlyPro-MMAE, corresponding to conjugate 9 of the present invention, were compared in a tumor-bearing mouse model to examine the efficacy of delivery of the MMAE cytotoxic moiety to the tumor.

[0268] HT-1080.hFAP tumors were implanted into the right flank of athymic Balb / c AnNRj-Foxn1 mice and grew to approximately 200 mm 3Mice were injected with the three conjugates at a dose of 250 nmol / kg and sacrificed at different time points after administration. Fresh blood was collected into lithium heparin tubes (BD Microcontainer LH Tubes), vortexed, and centrifuged (15,000 g, 15 min). Plasma was frozen and stored at -80°C. Healthy organs and tumors were then excised, frozen on dry ice, and stored at -80°C. Frozen plasma (50 μL) and mouse tissue (approximately 50 mg) were thawed and 500 μL of PBS was added. The samples were kept on ice, and 50 μL of internal standard solution (d8-MMAE, 50 nM) was added. The samples were then homogenized using a tissue lyser at 4°C, 30 Hz, for 2 min, for four cycles. After homogenization, the samples were centrifuged (21,000 g, 10 min). Afterwards, 100 μL of the supernatant was collected and added to 900 μL of acetonitrile (ACN) to precipitate the protein. After centrifugation (21,000 g, 10 min), 800 μL of the supernatant was collected and dried at room temperature using a vacuum centrifuge. The pellet was then resuspended in 20 μL of an aqueous solution containing 3% acetonitrile and 0.1% HCOOH, and 5 μL was injected into the UHPLC-MS system. Chromatographic separation was performed on a Hypersil Gold C18 column (100 mm × 2.1 mm, 1.9 μm particle size, 175 Å pore size). The column temperature was set at 50 °C, the flow rate was 700 μL / min, and the gradient program was 95% A (water + 0.1% HCOOH), 5% B (ACN + 0.1% HCOOH) to 35% A in 2.5 min, 35% A to 5% A in 0.4 min, and a 1.3 min hold at 5% A before readjusting to 95% A. The LC system was coupled to a Q-Exactive mass spectrometer via an Ion Max HESI Source. Ionization was performed with a spray voltage of 3.5 kV; sheath gas 40 units; auxiliary gas 10 units; capillary temperature 380 °C; auxiliary gas temperature 450 °C; and S-lens RF level 60. The mass spectrometer was operated in targeted single ion monitoring mode (t-SIM) tracking the molecular ion 718.5113 m / z.The detector was operated in positive ionization mode with the following parameters: resolution 70,000 (FWHM at 200 m / z); AGC target 5 × 104; maximum injection time 200 ms; isolation window 14 m / z; isolation offset 5 m / z. The peak areas of the analyte and internal standard were integrated, and the corresponding ratio was calculated. This ratio was then converted to pmol per gram of wet tissue using a single-concentration external calibration point and corrected for the total weight of the analyzed sample. Finally, the percentage of administered dose per gram (%ID / g) was calculated by normalizing the values ​​based on the total dose administered to the mice. Data analysis was performed using Skyline v22.2.0.351. The results are shown in Figure 14.

[0269] h) Therapeutic efficacy evaluation of ESV6-GlyPro-MMAE and Tri-ESV6-GlyPro-MMAE in HT-1080 hFAP tumor-bearing mice (schedule optimization) The anti-cancer effects of (i) ESV6-GlyPro-MMAE (50 or 125 nmol / kg), corresponding to conjugate 58a of EP 3891138, and (ii) Tri-ESV6-GlyPro-MMAE (50 or 125 nmol / kg) according to the present invention were evaluated in athymic Balb / c AnNRj-Foxn1 mice bearing HT-1080.hFAP tumors on the right flank. Treatment experiments were performed on mice with established tumors with a mean volume of 80-100 mm. 3 The study began when the tumor volume reached 100 mm. Animal weights and tumor volumes were measured daily and recorded. Tumor size was measured using electronic calipers, and tumor volume was calculated using the formula: (long side, mm) x (short side, mm) x (short side, mm) x 0.5. Animals were euthanized when they reached one or more of the termination criteria specified in the experimental permit. Data analysis was performed using Prism 7 software (GraphPad Software). The results are shown in Figures 16 and 17.

[0270] 11. Chemical Structure and Synthesis of Tri-ESV6-DOTAGA Compound with PEG Spacer a) Chemical structure of Tri-ESV6-PEG2-DOTAGA [ka] - Molecular weight: 2723.7874Da - Exact mass: 2722.1563 Da - Formula: C122H161F6N29O36 - SMILES notation: O=C(NC(COCCC(NCCNC(CCOCCOCCNC(CCC(NC1=C(N=CC=C2C(NCC(N3CC(F)(F)C[C@H]3C#N)=O)=O)C2=CC=C1)=O)=O)=O)=O)(COCCC(NCCNC(CCOCCOCCNC(CCC(NC4=C(N=CC=C5C(NCC(N6CC(F)(F)C[C@H]6C#N)=O)=O)C5=CC=C4)=O)=O)=O)=O)COCCC(NCCNC(CCOCCOCCNC(CCC(NC7=C(N=CC=C8C(NCC(N9CC(F)(F)C[C@H]9C#N)=O)=O)C8=CC=C7)=O)=O)=O)=O)CCC(N%10CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC%10)C(O)=O

[0271] b) Chemical structure of Tri-ESV6-PEG4-DOTAGA [Chemical formula] - Molecular weight: 2988.1054 Da - Exact mass: 2986.3136 Da - Formula: C134H185F6N29O42 - SMILES notation: O=C(NC(COCCC(NCCNC(CCOCCOCCOCCOCCNC(CCC(NC1=C(N=CC=C2C(NCC(N3CC(F)(F)C[C@H]3C#N)=O)=O)C2=CC=C1)=O)=O)=O)=O)(COCCC(NCCNC(CCOCCOCCOCCOCCNC(CCC(NC4=C(N=CC=C5C(NCC(N6CC(F)(F)C[C@H]6C#N)=O)=O)C5=CC=C4)=O)=O)=O)=O)COCCC(NCCNC(CCOCCOCCOCCOCCNC(CCC(NC7=C(N=CC=C8C(NCC(N9CC(F)(F)C[C@H]9C#N)=O)=O)C8=CC=C7)=O)=O)=O)=O)CCC(N%10CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC%10)C(O)=O

[0272] c) Chemical structure of Tri-ESV6-PEG6-DOTAGA

Chem.

[0273] d) Chemical structure of Tri-ESV6-PEG8-DOTAGA

Chem.

[0274] e) Synthetic routes for the synthesis of TriOncoFAP-PEG2-DOTAGA, TriOncoFAP-PEG4-DOTAGA, TriOncoFAP-PEG6-DOTAGA and TriOncoFAP-PEG8-DOTAGA [ka]

[0275] The enzymatic activity of hFAP toward the Z-Gly-Pro-AMC substrate was measured at room temperature using a microtiter plate reader, monitoring fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. The reaction mixture contained substrate (20 μM), protein (constant 66 pM), assay buffer (50 mM Tris, 100 mM NaCl, and 1 mM EDTA, pH 7.4), and a 1:2 serial dilution of inhibitor from 167 nM to 80 fM in a total volume of 20 μL. Experiments were performed in triplicate, and mean fluorescence values ​​were fitted using Prism 7. This value is defined as the inhibitor concentration required to reduce enzyme activity by 50% after the addition of substrate. The results are shown in Figure 10. Surprisingly, the compound with the shortest distance between the targeting moiety and the branch point (0 PEG units) was found to have the lowest IC50 (Table 16).

[0276] 11. Chemical Structures and Synthesis of Compounds with Alternative Linkers a) Chemical structure of ESV6-L-DOTAGA

Chem.

[0277] b) Chemical structure of Bi-ESV6-L-DOTAGA

Chem.

[0278] c) Synthetic Routes for the Synthesis of ESV6-L-DOTAGA and Bi-ESV6-L-DOTAGA

Chem.

[0279] e) Inhibition assay using Tri-ESV6-DOTAGA, Tetra-ESV6-DOTAGA, Hexa-ESV6-DOTAGA, Octa-ESV6-DOTAGA, ESV6-L-DOTAGA, and BI-ESV6-L-DOTAGA The enzymatic activity of hFAP toward the Z-Gly-Pro-AMC substrate was measured at room temperature using a microtiter plate reader, monitoring fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. The reaction mixture contained substrate (20 μM), protein (66 pM, constant), assay buffer (50 mM Tris, 100 mM NaCl, and 1 mM EDTA, pH 7.4), and a 1:2 serial dilution of inhibitor from 167 nM to 80 fM in a total volume of 20 μL. Experiments were performed in triplicate, and mean fluorescence values ​​were fitted using Prism 7. This value is defined as the concentration of inhibitor required to reduce enzyme activity by 50% after substrate addition. The results are shown in Figure 11. Inhibitory activity is directly proportional to the valency up to Tetra-ESV6-DOTAGA (a tetravalent compound). Surprisingly, despite their superior valency, Hexa-ESV6-DOTAGA (hexavalent compound) and Octa-ESV6-DOTAGA (octavalent compound) lose over 10,000-fold less activity than the tetravalent compound. Compounds with alternative linkers (ESV6-L-DOTAGA and Bi-ESV6-DOTAGA) exhibit IC50 values ​​comparable to those of the parent derivatives (ESV6-DOTAGA and Bi-ESV6-DOTAGA in Figure 1).

[0280] 13. Additional Compounds a) Chemical structure of an alternative version of Tri-ESV6-DOTAGA(8). [ka] - Molecular weight: 2071.0484Da - Exact mass: 2069.8032 Da - Formula: C94H109F6N25O23 - Smiles notation: O=C(CCC(NC1=C(N=CC=C2C(NCC(N3CC(F)(F)C[C@H]3C#N)=O)=O)C2=CC=C1)=O)NCCNC(CCC(N4CCN(C(C(O)=O)CCC(NCCNC(CCC(NC5=C(N=CC=C6C(NCC(N7CC(F)(F)C[C@H]7C#N)=O)=O)C6=CC=C5)=O)=O)=O)CCN(CC(O)=O)CCN(C(CCC(NCCNC(CCC(NC8=C(N=CC=C9C(NCC(N%10CC(F)(F)C[C@H]%10C#N)=O)=O)C9=CC=C8)=O)=O)=O)C(O)=O)CC4)C(O)=O)=O

[0281] b) Synthetic pathway for the synthesis of an alternative version of TriOncoFAP-DOTAGA (8)

Chem.

[0282] c) Chemical structure of Tri-ESV6-GlyPro-MMAE (9)

Chem.

[0283] Chemical structure of (S,S)-Tri-ESV6-DOTAGA(11) [Chemical] - Molecular weight: 2246.2324 Da - Exact mass: 2244.8877 Da - Formula: C101H122F6N26O27 - SMILES notation: O=C(NCCNC(CCOCC(COCCC(NCCNC(CCC(NC1=C(N=CC=C2C(NCC(N3CC(F)(F)C[C@H]3C#N)=O)=O)C2=CC=C1)=O)=O)=O)(NC(CC[C@H](N4CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC4)C(O)=O)=O)COCCC(NCCNC(CCC(NC5=CC=CC6=C5N=CC=C6C(NCC(N7CC(F)(F)C[C@H]7C#N)=O)=O)=O)=O)=O)=O)CCC(NC8=CC=CC9=C8N=CC=C9C(NCC(N%10[C@H](C#N)CC(F)(F)C%10)=O)=O)=O

[0284] Chemical structure of (S,R)-Tri-ESV6-DOTAGA(12)

Chem.

[0285] In all experiments described in this specification, unless otherwise indicated, Tri-ESV6-DOTAGA was used as its (S,R)-diastereomer.

[0286] Chemical structure of Tri-ESV6-linker-DOTAGA(13) [Chemical formula] - Molecular weight: 2581.6294 Da - Exact mass: 2580.0821 Da - Formula: C116H151F6N27O34 - SMILES notation: O=C(NCCNC(CCOCC(COCCC(NCCNC(CCC(NC1=C(N=CC=C2C(NCC(N3CC(F)(F)C[C@H]3C#N)=O)=O)C2=CC=C1)=O)=O)=O)(NC(CCOCCOCCOCCOCCOCCOCCNC(CCC(N4CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC4)C(O)=O)=O)=O)COCCC(NCCNC(CCC(NC5=CC=CC6=C5N=CC=C6C(NCC(N7CC(F)(F)C[C@H]7C#N)=O)=O)=O)=O)=O)=O)CCC(NC8=CC=CC9=C8N=CC=C9C(NCC(N%10[C@H](C#N)CC(F)(F)C%10)=O)=O)=O

[0287] Chemical structure of Tri-ESV6-ValCit-MMAE (14) [Chemical formula] - Molecular weight: 3665.9774 Da - Exact mass: 3663.6397 Da - Formula: C171H228F6N38O44S - SMILES notation: O=C1CC(SC[C@@H](C(O)=O)NC([C@H](CC(O)=O)NC([C@H](CCCCN)NC([C@H](CC(O)=O)NC(CCC(NC(COCCC(NCCNC(CCC(NC2=CC=CC3=C2N=CC=C3C(NCC(N4CC(F)(F)C[C@H]4C#N)=O)=O)=O)=O)=O)(COCCC(NCCNC(CCC(NC5=C(N=CC=C6C(NCC(N7CC(F)(F)C[C@H]7C#N)=O)=O)C6=CC=C5)=O)=O)=O)COCCC(NCCNC(CCC(NC8=CC=CC9=C8N=CC=C9C(NCC(N%10[C@H](C#N)CC(F)(F)C%10)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)C(N1CCCCCC(N[C@@H](C(C)C)C(N[C@H](C(NC%11=CC=C(COC(N(C)[C@@H](C(C)C)C(N[C@@H](C(C)C)C(N([C@@H]([C@H](C)CC)[C@H](OC)CC(N%12[C@H]([C@H](OC)[C@@H](C)C(N[C@H](C)[C@@H](O)C%13=CC=CC=C%13)=O)CCC%12)=O)C)=O)=O)=O)C=C%11)=O)CCCNC(N)=O)=O)=O)=O

[0288] 14. Preferred conjugates For ease of reference, the numbers and structures of some compounds of the present invention are summarized in Tables 9 and 17 below. [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] Table 9 Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 Table 17 Table 18 Table 19 Table 20 Table 21 Table 22 Table 23 Table 24 Table 25 Table 26 Table 27 Table 28

Claims

1. A compound, an individual diastereoisomer thereof, a hydrate thereof, a solvate thereof, a crystal thereof, an individual tautomer thereof, or a pharmaceutically acceptable salt thereof, wherein the compound has the following formula: 【Chemistry 1】 【Chemistry 2】 (where each BS or BL is independently a bond, -NHC(O)(CH ２ ) ｎ C(O)-, -NH(CH ２ ) ｎ C(O)-, -NHC(O)(CH ２ CH ２ O) ｍ (CH ２ ) ｎ -, -C(O)(CH ２ CH ２ O) ｍ (CH ２ ) ｎ -, -C(O)(CH ２ CH ２ O) ｍ (CH ２ ) ｎ NH-, -(CH ２ CH ２ O) ｍ (CH ２ ) ｎ -, -(CH ２ CH ２ O) ｍ (CH ２ ) ｎ NH-, -(CH ２ CH ２ O) ｍ (CH ２ ) ｎ NHC(O)-, -(CH ２ ) ｎ O(CH(CH ２ CH ２ O) ｍ (CH ２ ) ｎ -, -(CH ２ ) ｎ O(CH ２ CH ２ O) ｍ (CH ２ ) ｎ NH-, -(CH ２ CH ２ O) ｍ (CH ２ ) ｎ NHC(O)-, -C(O)(CH ２ ) ｎ O(CH ２ CH ２ O) ｍ (CH ２ ) ｎ -, -C(O)(CH ２ ) ｎ O (CH ２ CH ２ O) ｍ (CH ２ ) ｎ NH-, -(CH ２ ) ｎ O (CH ２ CH ２ O) ｍ (CH ２ ) ｎ NH-, -C(O)(CH ２ ) ｎ C(O)-, -C(O)(CH ２ ) ｎ -, -C(O)(CH ２ ) ｎ NH- or -(CH ２ ) ｎ C(O)-; each n and m is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; each x is an integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; each y is an integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; each z is independently an integer selected from 0, 1, 2, 3, 4, and 5; each m ３ is independently 0, 1, 2, 3, or 4; each l ３ is independently 0 or 1; and Part C is (a) a compound of the formula: 【Transformation 3】 (In the formula, n is 0, 1, 2, 3, 4 or 5; R １ｅ is independently H, COOH, aryl-COOH, or heteroaryl-COOH; R ２ｅ is independently H, COOH, aryl-COOH, or heteroaryl-COOH; Each R ３ｅ is independently H, COOH, aryl-COOH, or heteroaryl-COOH; R ４ｅ is independently H, COOH, aryl-COOH, or heteroaryl-COOH; R １ｆ is independently H, COOH, aryl-COOH, or heteroaryl-COOH; R ２ｆ is independently H, COOH, aryl-COOH, or heteroaryl-COOH; R ３ｆ is independently H, COOH, aryl-COOH, or heteroaryl-COOH; and X is O, NH, or S); (b) diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane-N,N',N'',N''''-tetraacetic acid (TETA), iminodiacetic acid, bis(carboxymethylimidazole)glycine, 6-hydrazinopyridine-3-carboxylic acid (HYNIC), 【Chemistry 4】 【Transformation 5】 a chelator group suitable for radiolabeling with a therapeutic nuclide selected from: (c) ２２３ Ra, ８９ Sr, ９０ Y. １２１ Sn, １７７ Lu, １３１ I, ２１１ At, ２２５ Ac, １８８ Re, １４９ Tb, １６１ Tb and ２２７ a therapeutic radioactive group comprising a therapeutic radioisotope, which may not be used for diagnostic purposes; and (d) chelates of the isotopes listed in (c) above and / or chelates with the chelating agents listed in (a) or (b) above. a compound, an individual diastereoisomer thereof, a hydrate thereof, a solvate thereof, a crystal thereof, an individual tautomer thereof, or a pharmaceutically acceptable salt thereof, wherein

2. Moiety C has the following structure: 【Transformation 6】 wherein M is a therapeutic radioisotope.

3. Part C is 【Transformation 7】 2. The compound of claim 1, an individual diastereoisomer thereof, a hydrate thereof, a solvate thereof, a crystal thereof, an individual tautomer thereof, or a pharmaceutically acceptable salt thereof, selected from:

4. below: 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 a compound represented by a structure selected from the following: an individual diastereoisomer thereof, a hydrate thereof, a solvate thereof, a crystal thereof, an individual tautomer thereof, or a pharmaceutically acceptable salt thereof.

5. The compound of claim 1, wherein the compound has the following structure: 【Chemistry 14】 2. The compound of claim 1, an individual diastereoisomer thereof, a hydrate thereof, a solvate thereof, a crystal thereof, an individual tautomer thereof, or a pharmaceutically acceptable salt thereof, wherein

6. A compound according to claim 1 or 4, an individual diastereoisomer thereof, a hydrate thereof, a solvate thereof, a crystal thereof, an individual tautomer thereof, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable excipient.

7. A pharmaceutical composition as described in claim 6, for use in administration in combination with administration of an immunocytokine to a subject for the treatment of a human or animal.

8. 8. The pharmaceutical composition of claim 7, wherein the immunocytokine specifically binds to the alternatively spliced ​​ED-B domain of fibronectin isoform B-FN.

9. 9. The pharmaceutical composition of claim 8, wherein the immunocytokine specifically binds to the epitope sequence represented by SEQ ID NO:

40.

10. The pharmaceutical composition of claim 7 , wherein the immunocytokine comprises a sequence having IL2 activity.

11. The pharmaceutical composition of claim 7, wherein the immunocytokine comprises one or more CDRs of any one of SEQ ID NOs: 13-18.

12. The pharmaceutical composition of claim 11, wherein the immunocytokine comprises all of the CDRs of SEQ ID NOs: 13-18.

13. The pharmaceutical composition of claim 11 , wherein the immunocytokine comprises the VH sequence of SEQ ID NO: 19 and / or the VL sequence of SEQ ID NO:

20.

14. The pharmaceutical composition of claim 7 , wherein the immunocytokine comprises an scFv sequence; a human monoclonal scFv sequence; or the sequence of SEQ ID NO:

12.

15. (i) A compound according to claim 1 or 4, an individual diastereoisomer thereof, a hydrate thereof, a solvate thereof, a crystal thereof, an individual tautomer thereof, or a pharmaceutically acceptable salt thereof; or a pharmaceutical composition comprising a compound according to claim 1 or 4, an individual diastereoisomer thereof, a hydrate thereof, a solvate thereof, a crystal thereof, an individual tautomer thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient; and (ii) A kit comprising an immunocytokine.

Images