Multivalent fibroblast activating protein ligands for targeted delivery applications

Abstract

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

Description

[0001] introduction Technical Field

[0002] The present invention relates to ligands of fibroblast activation protein (FAP) for active delivery of various therapeutic payloads (e.g., cytotoxic drugs, therapeutic radionuclides, proteins, and immunomodulators) at disease sites. In particular, the present invention relates to the development of multivalent FAP ligands for therapeutic applications involving a disease or condition, such as cancer, inflammation, or another disease characterized by overexpression of FAP. Background Art

[0003] Chemotherapy is still widely used to treat cancer patients and other diseases. Traditional anticancer chemotherapeutic agents act on basic mechanisms of cell survival and cannot distinguish between healthy and malignant cells. In addition, these drugs do not effectively accumulate to the disease site when administered systemically. The nonspecific mechanism of action and inefficient localization at the tumor site lead to unsustainable side effects and poor therapeutic efficacy of traditional chemotherapy.

[0004] There is a great need to develop therapeutic drugs that can selectively localize at the site of disease after systemic administration. The strategy for generating such therapeutic drugs is represented by chemical conjugation of therapeutic payloads (such as cytotoxic drugs or therapeutic radionuclides) with ligands specific for disease markers. Disease-specific monoclonal antibodies, peptides, and small ligands have been considered as the ligand of choice in the development of targeted therapeutic drug products. The use of small ligands for therapeutic applications has several advantages over larger molecules such as peptides and antibodies: more rapid and efficient tumor penetration, lower immunogenicity, and lower manufacturing costs.

[0005] Small organic ligands specific for prostate-specific membrane antigen, folate receptor and carbonic anhydrase IX show excellent biodistribution characteristics (profile) in preclinical models and patients of cancer. These ligands have been conjugated with cytotoxic drugs and radionuclides to produce small molecule-drug conjugates and small molecule-radioconjugate products (SMDC and SMRC) for the treatment of cancer. 177-Lu-PSMA-617 represents an example of late SMRC, which is now being studied in a Phase III trial for the treatment of patients with metastatic castrate-resistant prostate cancer (mCRPC) (VISION trial).

[0006] Fibroblast activation protein (FAP) is a membrane-bound gelatinase that promotes tumor growth and progression and is overexpressed in cancer-associated fibroblasts. Due to its low expression in normal organs, FAP represents an ideal target for the development of therapeutic SMDCs and SMRCs.

[0007] WO2019154886 and WO2019154859 describe heterocyclic compounds as fibroblast activation protein-alpha inhibitors for the treatment of different cancer types. WO2019118932 describes substituted N-containing cyclic compounds as fibroblast activation protein alpha inhibitors for the treatment of different pathological conditions. WO2019083990 describes imaging and radiotherapy targeted fibroblast activation protein-alpha (FAP-alpha) compounds as FAP-alpha inhibitors for imaging and radiotherapy of diseases associated with FAP-alpha and treating proliferative diseases, and mentions that the 4-isoquinolinoyl and 8-quinolinoyl derivatives described therein are characterized by very low FAP-affinity.

[0008] WO2013107820 describes substituted pyrrolidine derivatives for treating proliferative disorders (e.g., cancer) and diseases indicated by tissue remodeling or chronic inflammation (e.g., osteoarthritis). WO2005087235 describes pyrrolidine derivatives as dipeptidyl peptidase IV inhibitors for treating type II diabetes. WO2018111989 describes conjugates comprising fibroblast activation protein (FAP) inhibitors, bivalent linkers, and, for example, near-infrared (NIR) dyes, which can be used to remove cancer-associated fibroblasts, image cell populations in vitro, and treat cancer. WO2021 / 160825A1, WO2022 / 171811A1, and WO2021 / 016392A1 describe ligands of FAP for active delivery of various payloads. Zboarski, D., Hoehne, A., Bredenbeck, A. et al., Eur J Nucl Med Mol Imaging 49, 3651-3667 (2022) described FAP-2286 (FAP-binding peptide conjugated to a radionuclide chelator) and reported that radiolabeled FAP-2286 showed high tumor uptake and retention, as well as potent efficacy in FAP-positive tumors.

[0009] However, there remains a need for further improved FAP binding agents for the above-mentioned and other applications.

[0010] The present invention is therefore directed to the problem of providing improved therapeutic binding agents (ligands) of fibroblast activation protein (FAP) suitable for therapeutic applications. The binding agents should be suitable for inhibiting FAP and / or for targeted delivery of therapeutic payloads to sites suffering from or at risk of diseases or conditions characterized by overexpression of FAP (e.g., FAP-positive tumors). Preferably, the binding agents should exhibit improved one or more binding parameters (e.g., FAP inhibitory activity, FAP binding affinity, and / or prolonged binding to FAP-positive cells) while showing a favorable tumor uptake or tumor to organ ratio (e.g., tumor to kidney ratio). Summary of the invention

[0011] The present inventors have discovered a novel trivalent organic ligand ("Tri-ESV6") for fibroblast activation protein (FAP) suitable for therapeutic applications. The compounds according to the invention (also referred to as ligands or binding agents) comprise more than two (e.g. three) small binding moieties A having the following structure:

[0012]

[0013] The compounds according to the present invention can be represented by the following general formula I,

[0014]

[0015] Its individual diastereomer, its hydrate, its solvate, its crystalline form, its individual tautomer or its pharmaceutically acceptable salt, wherein A is a binding moiety; B is a multifunctional moiety comprising a ramification point and covalently links moiety A to C; and C is an atom, molecule or particle and is a therapeutic agent. An exemplary structure is represented by Formula II as defined below.

[0016] Alternatively, moiety C comprises a branch point such that three moieties A are attached to C and B is absent, i.e. moiety C may also serve as a branch point, thus replacing B, preferably when C is a chelator. An exemplary structure is represented by formula III as defined below.

[0017] The present invention also provides a pharmaceutical composition comprising the compound and a pharmaceutically acceptable excipient.

[0018] The present invention also provides the compound or pharmaceutical composition and a method for use thereof in the treatment of human or animal body therapy; and a method for treatment of human or animal body by therapy, which comprises administering a therapeutically effective amount of the compound or pharmaceutical composition to a subject in need thereof.

[0019] The present invention also provides a method for using the compound or pharmaceutical composition for treating a subject suffering from a disease or condition or at risk of a disease or condition; and a method for treating a disease or condition, which comprises administering a therapeutically effective amount of the compound or pharmaceutical composition to a subject suffering from the disease or condition or at risk of the disease or condition.

[0020] The present invention also provides a method for using the compound or pharmaceutical composition for targeted delivery of a therapeutic agent to a subject suffering from a disease or condition or at risk of a disease or condition; and a method for targeted delivery of a therapeutically effective amount of the compound or pharmaceutical composition to a subject suffering from a disease or condition or at risk of a disease or condition.

[0021] Preferably, the above diseases or conditions are characterized by overexpression of FAP and are independently selected from cancer, inflammation, atherosclerosis, fibrosis, tissue remodeling and keloid conditions, preferably, wherein 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, clear cell renal carcinoma, neuroendocrine tumors, carcinogenic osteomalacia, sarcoma, CUP (cancer of unknown primary site), thymic carcinoma, desmoid tumor, glioma, astrocytoma, cervical cancer, skin cancer, kidney cancer and prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Inhibition assay with recombinant hFAP at a concentration of 66 pM. Tri-ESV6-DOTAGA exhibited significantly lower IC compared to ESV6-DOTAGA, Bi-ESV6-DOTAGA, and FAP-2286. 50 .

[0023] Figure 2 : Fluorescence polarization assay with recombinant hFAP and fluorescent ligand at 1 nM concentration. Compared with FAP-2286-PEG2-FITC, both ESV6-PEG2-FITC and Tri-ESV6-PEG2-FITC showed significantly lower K D .

[0024] Figure 3 :use 177 Efflux assay of Lu-labeled ligands on SK-RC-52.hFAP cells. 177 Lu-Tri-E SV6 exhibited a longer retention time in FAP-positive tumor cells, followed by 177 Lu-Bi-ESV6, 177 Lu-ESV6 and 177Lu-FAP2286.

[0025] Figure 4 :use 177 Lu-ESV6, 177 Lu-Bi-ESV6, 177 Lu-Tri-ESV6, 177 Lu-Tetra-ESV6, 177 Lu-Hexa-ESV6, 177 Lu-Octa-ESV6 and 177 Quantitative biodistribution analysis of Lu-FAP-2286 (250 nmol / kg; 50 MBq / kg each) in SK-RC-52.hFAP tumor-bearing mice. Tri-ESV6-DOTAGA showed significantly high tumor uptake even after 48, 72 and 96 hours post-injection. At the same dose, the Tri-ESV6 binder showed the best biodistribution characteristics when considering tumor uptake and uptake in healthy organs over time.

[0026] Figure 5 : In BALB / c nu / nu mice bearing SK-RC-52.hFAP tumors, 177 Lu-ESV6-DOTAGA, 177 Lu-Bi-ESV6-DOTAGA and 177 Therapeutic activity of Lu-Tri-ESV6-DOTAGA or saline as a single agent (single administration on day 8, 250 nmol / kg, 250 MBq / kg). Tumor volume (mm 3 ) to evaluate the efficacy of different treatments. Data points represent mean tumor volume ± SEM (n = 4 per group). ****P value <001; *P <0.1 (two-way ANOVA test followed by Bonferroni post-test). Tri-ESV6-DOTAGA showed significant therapeutic activity.

[0027] Figure 6 : In BALB / c nu / nu mice bearing SK-RC-52.hFAP tumors, 177 Lu-ESV6-DOTAGA, 177 Lu-Bi-ESV6-DOTAGA and 177 Therapeutic activity of Lu-Tri-ESV6-DOTAGA or saline as a single agent (single administration on day 8, 250 nmol / kg, 250 MBq / kg). Tumor volume (mm 3) to evaluate the efficacy of different treatments. The curve represents the tumor growth value of a single mouse. CR = Complete Remission. Tri-ESV6-D OTAGA showed significant therapeutic activity.

[0028] Figure 7 :use 177 Lu-ESV6-DOTAGA, 177 Lu-Bi-ESV6-DOTAGA and 177 Tolerability of the different treatments with Lu-Tri-ESV6-DOTAGA as assessed by evaluating the change in body weight (%) during the experiment. All conjugates were very well tolerated in a comparable manner.

[0029] Figure 8 : Inhibition assay with recombinant hFAP at a concentration of 66 pM. Surprisingly, Tri-ESV6-DOTAGA showed a ∼1000-fold lower IC compared to its derivative with a longer spacer between the ESV6 part and the branch (Tri-ESV6-P EG12-D OTAGA). 50 .

[0030] Fig. 9 : Therapeutic activity in SK-RC-52.hFAP tumor-bearing BALB / c nu / nu mice 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) different doses of 5 MBq / mouse, 15 MBq / mouse, or 30 MBq / mouse as a single agent on day 7 177 Lu-Tri-E SV6, (iii) at a dose of 5 MBq / mouse 177 Lu-Tri-E SV6, followed by three injections of L19-IL2 at a dose of 0.05 mg / mouse on days 8, 10, and 12 177 Lu-Tri-E SV6+L19-IL2 combination or (iv) saline. During and after drug administration, tumor volume (mm 3 ) were used to evaluate the efficacy of different treatments. Data points represent mean tumor volume ± SEM (n = 4 per group).

[0031] 177 Lu-Tri-E SV6 exhibited dose-dependent therapeutic activity.

[0032] Fig.10 : Inhibition assay with recombinant hFAP at a concentration of 66 pM. The compound with the shortest distance between the targeting moiety and the branch point (PEG unit = 0) was surprisingly found to have the lowest IC50.

[0033] Fig.11 : Inhibition assay with recombinant hFAP at a concentration of 66 pM. Until Tetra-ESV6-D OTAGA (tetravalent compound), the inhibitory activity was proportional to the valency. Surprisingly, despite their higher valency, Hexa-ESV6-D OTAGA (hexavalent compound) and Octa-ESV6-D OTAGA (octavalent compound) lost more than 10'000-fold activity compared to the tetravalent derivative. Compounds with alternative linkers (ESV6-L-DOTAGA and Bi-ESV6-D OTAGA) present similar activity to the original derivatives ( Figure 1 ESV6-DOTAGA and Bi-ESV6-DOTAGA in the study had comparable IC50.

[0034] Fig.12 : The preferred immunocytokine for use in the present invention is the L19-IL2 conjugate immunocytokine, wherein in each polypeptide chain, the IL2 polypeptide sequence is fused at its N-terminus to the C-terminus of the VL domain of a single-chain variable fragment (scFv) sequence comprising the VH and VL domains of L19 via a linker, and the scFv unit of one L19-IL2 polypeptide chain forms a homodimer with the scFv unit of another L19-IL2 polypeptide chain ("immunocytokine in scFv2 form").

[0035] Fig.13 : Therapeutic activity in SK-RC-52.hFAP tumor-bearing BALB / c nu / nu mice following intravenous administration of: (i) L19-IL2 as a single agent at a dose of 0.05 mg / mouse on days 8, 10, 12, (ii) L19-IL2 as a single agent at a dose of 5 MBq / mouse on day 7 177 Lu-Tri-E SV6, (iii) 177 Lu-Tri-E SV6+L19-IL2 combination (at a dose of 5 MBq / mouse on day 7) 177 Lu-Tri-E SV6, followed by three administrations of L19-IL2 at a dose of 0.05 mg / mouse on days 8, 10, and 12 (Schedule 1), or three administrations of L19-IL2 at a dose of 0.05 mg / mouse on days 12, 17, and 22 (Schedule 2), or a single administration of L19-IL2 at a dose of 0.05 mg / mouse on day 8 (Schedule 3), or a single administration of L19-IL2 at a dose of 0.05 mg / mouse on day 12 (Schedule 4)) or (iv) saline. Tumor volume (mm) was measured daily during and after drug administration. 3 ) were used to evaluate the efficacy of different treatments. Data points represent mean tumor volume ± SEM (n = 4 per group).177 Lu-Tri-ESV6 exhibited dose-dependent therapeutic activity.

[0036] Fig.14 : In vivo quantitative MMAE released in tumors and healthy organs by ESV6-GlyPro-MMAE (conjugate 58a of EP3891138B1), Bi-ESV6-GlyPro-MMAE (conjugate 11 of WO2022 / 171811) and Tri-ESV6-GlyPro-MMAE (conjugate 9 of the present invention). Each conjugate was injected at a dose of 250nmol / Kg, and MMAE release was recorded at different time points after administration. The values ​​are expressed as a percentage of the dose injected per gram of tissue (%ID / g). Quantification of MMAE released after administration of Tri-ESV6-GlyPro-MMAE reveals a cleaner biodistribution profile, high and prolonged accumulation at the tumor site, and lower release in healthy organs (compared to ESV6-GlyPro-MMAE and Bi-ESV6-GlyPro-MMAE).

[0037] Fig.15 To identify the optimal dose for radiotherapy, 177 Lu-Tri-ESV6-DOTAGA was injected into tumor-bearing mice at eight different doses ranging from 3nmol / Kg to 2250 / Kg. The results showed that at the highest dose (>90nmol / kg), %ID / g in the tumor decreased (A), while the lowest dose (<10nmol / kg) was partially taken up in healthy organs (B). The optimal mouse dose range was between 90 and 250nmol / kg, which is equivalent to 1 to 3mg per human patient.

[0038] Fig.16 : Evaluation of the therapeutic anticancer efficacy of ESV6-GlyPro-MMAE and Tri-ESV6-GlyPro-MMAE in HT-1080.hFAP tumor-bearing mice, tumor volume (A) and survival percentage (B) over time. Tolerability was assessed by evaluation of the change in body weight (%) during the experiment, indicating that all conjugates were well tolerated (C).

[0039] Fig.17 :constitute Fig.16 Datasets underlying the results shown (single graph; one curve per animal): 50 nmol / kg (A) or 125 nmol / kg (B) of ESV6-GlyPro-MMAE; 50 nmol / kg (D) or 125 nmol / kg (E) of Tri-ESV6-GlyPro-MMAE; and saline (C).

[0040] Fig.18 : Comparative assay of hFAP inhibition with trivalent binders 11, 12, 13 and 14 having, among others, different linker groups B and / or different payload groups C.

[0041] Fig.19 :Use binder compound 177 Lu-Tri-ESV6-DOTAGA at different radioisotope molar activities (per nmol ligand) 24 hours after injection 177 MBq of Lu): low (0.2 MBq / nmol) and high (4.8 MBq / nmol, preferred for use in clinical practice) 177 Lu biodistribution results, indicating that altering the molar activity does not significantly interfere with the favorable biodistribution of this binder.

[0042] Fig. 20 : Comparative hFAP inhibition assay with tetravalent binders 10 and 29. DETAILED DESCRIPTION

[0043] The present inventors have identified small molecule binders to fibroblast activation protein (FAP) that are suitable for therapeutic applications.

[0044] As shown by the in vitro data provided herein, therapeutic binding agents according to the present 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 suffering from or at risk of a disease or condition characterized by overexpression of FAP.

[0045] As shown by the in vivo data provided herein, therapeutic binding agents according to the present invention can provide high and prolonged tumor uptake and potent anti-tumor effects. At the same time, significantly high tumor to organ ratios (particularly: tumor to kidney) can be achieved.

[0046] Compared to related prior art FAP-binding agents (such as FAP-2286), the therapeutic binding agents according to the present invention are surprisingly advantageous in terms of one or more of the above and other effects. Figures 1 to 5 As evident from the results in , the 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 ratio (compared to FAP-2286).

[0047] Compared to monovalent FAP-binding agents, such as ESV6-DOTAGA having only one FAP-binding moiety A, the therapeutic binding agents according to the invention are surprisingly advantageous in terms of one or more of the above and other effects. Figure 1 The results are clear, compared to the monovalent binder ESV6-DOTAGA, the trivalent binder (Tri-ESV6-DOTAGA) according to the present invention having three FAP-binding parts A shows a 43-fold improvement in IC50, which is surprising and far exceeds the 3-fold improvement usually expected when assuming additive behavior. Compared to the bivalent binder Bi-ESV6-DOTAGA, the trivalent binder (Tri-ESV6-DOTAGA) according to the present invention also shows a 10-fold improvement in IC50, which is surprising and far exceeds the 1.5-fold improvement usually expected when assuming additive behavior. Therefore, the binders according to the present invention can provide unexpected synergistic improvements in FAP inhibitory activity.

[0048] As from Figure 3 As is evident from the results in Figure 2, trivalent binding agents according to the invention show surprisingly improved efflux duration in FAP-positive tumor cells when compared to bivalent binding agents, which is even further improved when compared to monovalent binding agents. Thus, binding agents according to the invention can therefore provide unexpected improvements in terms of prolonged tumor uptake duration.

[0049] As from Figure 4 As is evident from the results in Tables 1 to 3, 11 to 12, the trivalent binders according to the invention show surprisingly improved in vivo tumor uptake in FAP-positive tumors, while maintaining surprisingly high tumor to organ ratios, in particular tumor to kidney ratios (see also Tables 5 to 7 and 13 to 15), when compared to monovalent, bivalent, hexavalent and octavalent binders (see also Tables 1 to 3 and 11 to 12). This is surprising, as increasing the valency of the binder is generally expected to result in higher organ accumulation and a deterioration of the tumor to organ ratio. For example, at the same dose, the Tri-ESV6 binder exhibits significantly better (cleaner) biodistribution, i.e. a more favorable tumor to organ ratio, compared to the other tested binders (e.g., hexavalent and octavalent).

[0050] Overall, considering tumor uptake over time (i.e., ~49% ID / g, 6 hours after injection) and uptake in healthy organs (e.g., ~0.3% ID / g spleen uptake and ~0.7% ID / g liver uptake, 6 hours after injection), 177Lu-TriESV6-DOTAGA exhibited the best biodistribution profile, highlighting trivalent Tri ESV6-based binders as best-in-class tumor targeting ligands. In addition, trivalent compounds are smaller (lower molecular weight), have higher atom economy, and are easier and cheaper to manufacture than their, for example, tetravalent counterparts.

[0051] Thus, binding agents according to the invention may provide unexpected improvements in tumor uptake while providing a favorable biodistribution and therefore have improved therapeutic potential.

[0052] As from Figure 5 and Figure 6 It is evident from the results that the trivalent binding agents according to the invention show surprisingly improved in vivo therapeutic activity against FAP-positive tumors compared to monovalent and bivalent binding agents, while maintaining tolerability (e.g., Figure 7 Thus, binding agents according to the invention may provide unexpected improvements in therapeutic efficacy and / or targeting specificity.

[0053] As from Figure 8 and Fig.10 It is clear from the results that a short spacer (L)a between the trivalent ESV6 moiety and the branch point confers superior inhibitory activity compared to longer spacers (see also Table 16). This is surprising considering that spacers with sufficient length to reach multiple adjacent FAPs on target cells in multivalent compounds would have been expected to be advantageous.

[0054] 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 are expected to provide a favorable side effect profile (on the one hand due to the lower required dose and on the other hand due to their improved biodistribution, in particular high tumor:kidney ratio and tolerability profile).

[0055] In a preferred embodiment, the compound of the present invention is represented by the following formula II:

[0056]

[0057] Among them, L, B S and B Lis independently at each occurrence a moiety comprising or consisting of a structural unit selected from the group consisting of alkylene, cycloalkylene, arylalkylene, heteroarylalkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, arylalkenylene, heteroarylalkenylene, heteroalkenylene, heterocycloalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, oxoalkylene, dioxyalkylene, aminoalkylene, diaminoalkylene, diacid, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylenealkoxycarbamate, carbamates), disulfides, vinylenes, imines, imidamides, phosphoramides, sugars, phosphates, phosphoramides, carbamates, dipeptides, tripeptides, and tetrapeptides, each of which is substituted or unsubstituted;

[0058] J is a branch point containing moiety comprising or consisting of structural units independently selected from the group consisting of alkylene, cycloalkylene, aralkylene, heteroaralkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, aralkenylene, heteroaralkenylene, heteroalkenylene, heterocycloalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, (oxy)alkylene, dioxoalkylene, aminoalkylene, diaminoalkylene, diacid, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylenealkoxycarbamate, disulfide, 1,2-vinylene, imine, imineamide, phosphoramide, sugar, phosphate, phosphoramide, carbamate, dipeptide, tripeptide, tetrapeptide, each of which is substituted or unsubstituted;

[0059] 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;

[0060] 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;

[0061] Each z is an integer independently selected from 0, 1, 2, 3, 4, and 5; and

[0062] Each a is an integer independently selected from 0, 1, 2, 3, 4 and 5.

[0063] In a further preferred embodiment, moiety C comprises a branch point such that three moieties A are attached to C, and B is absent, i.e. moiety C acts as a branch point, thus replacing 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, wherein all groups and variables are as defined in the claims, and wherein preferably a is 0 at each occurrence:

[0064]

[0065] In this embodiment, when C is a chelating agent with multiple -COOH groups, each A-(L) a The -arm is attached to the -C(O)- group in the chelator structure derived from the corresponding -COOH group. For example, if A-(L) a - is oxygen (ie, the arm has the general formula ARO-), the arm can be attached to the corresponding -COOH group of the chelator, thereby forming an ester (-C(O)ORA); if A-(L) a -, the terminal atom of the arm is an amino nitrogen (i.e., the arm has the general formula AR-NH-), then the arm can be attached to the corresponding -COOH group of the chelator to form an amide (-CONH-RA), where R is (L) in the corresponding arm excluding the terminal atom. a specific structure.

[0066] As used herein, unless otherwise indicated, the terms "branch point," "RP," or Represents a central atom bonded to at least three non-hydrogen atoms, wherein at least two of the non-hydrogen atoms are located on a covalent chain, and the covalent chain represents the shortest path between part A and the central atom. If the path including the minimum number of covalent bonds between two or more parts A does not pass through the same atom but through the same ring system, the geometric center of such a ring system should be considered as a branch point. A molecule comprising three parts A can contain, for example, one or two branch points, and preferably contains only one branch point. If the molecule contains more than one branch point, the number of covalent bonds between each branch point and the nearest branch point is preferably 7 or less, more preferably 5 or less, and most preferably 3 or less.

[0067] As used herein, unless otherwise indicated, the term "distance" or "length" between moieties or groups refers to the longest interatomic (through space) distance in the most extended conformation between two non-hydrogen atoms belonging to the respective group in which the atoms are connected by a path containing the minimum number of covalent bonds. For example, in compound G1 -CH2-CH2-G 2 Middle group G 1 (= 13 CH3) and G 2 The distance between (=OH) will be the distance between atoms in the most extended (stretched) conformation. 13 The longest interatomic (through space) distance between C and atom O.

[0068] Without wishing to be bound by theory, it is believed that multiple FAP-binding moieties A (preferably up to 4, more preferably 3) present in the binding agent molecule according to the present invention participate in binding and rebinding to the very same FAP target molecule and / or establish additional synergistic interactions that a single moiety could not establish. This is believed to be caused by the interaction between the relatively large active pocket of the FAP protein on the one hand and the specific structural features of the binding agent of the present invention on the other hand. Therefore, it is expected that one or more of the advantageous effects described herein are particularly significant when the intramolecular distance (length) between each moiety A and the closest branch point is short, and preferably when the binding agent participates in binding to one FAP target molecule rather than multiple adjacent FAP molecules (e.g., on a target cell). That is, moiety (L) a and J are preferably such that they do not provide the arms of the multivalent compound with a length to reach multiple adjacent FAPs on a target cell.

[0069] This is through Figure 8 The results shown confirm that it shows a long distance (about ) compared to Tri-ESV6-PEG12-DOTAGA, Tri-ESV6-DOTAGA (each part A has a distance of about 1.000 m from the nearest branch point) ) showed significantly stronger FAP inhibition (~1000-fold lower IC 50 In this regard, a particularly suitable distance (d1+d2) between each portion A and the closest branch point is considered to be or less, preferably or less, more preferably or less.

[0070] Without wishing to be bound by theory, it is believed that reducing the distance d1 + d2 helps improve the binding properties.

[0071] Preferably, the distance between each part A and J is or smaller, preferably or less, more preferably or less. This distance represents the length of the spacer connecting the ligand A (which binds to the fibroblast activation protein (FAP) on the target cell) to the multipoint template J (which is connected to multiple arms of the compound) and can also be represented as d1, as illustrated in the schematic representation below. In other words, d1 is the length of the spacer directly bonded to the moiety (L) a The distance between the two atoms at each end.

[0072] Without wishing to be bound by theory, it is believed that reducing the distance d1 helps improve the binding properties.

[0073] Preferably, the distance between each portion L and the closest branch point is or smaller, preferably or less, more preferably or less. This distance represents the length from the branch point to the "first" atom belonging to the moiety L along the chain, which represents the shortest path between the branch point and the corresponding moiety A, and defines a portion of the distance between each moiety A and the closest branch point belonging to the multi-point template J. This distance can also be represented as d2, as shown in the following schematic representation. In other words, d2 is the distance belonging to the radical moiety (L) directly bonded to the moiety J. a (or A if a is 0) is the distance between the atom and the branch point.

[0074] Without wishing to be bound by theory, it is believed that reducing the distance d2 helps improve the binding properties.

[0075]

[0076] Part A

[0077] Without wishing to be bound by any theory, it is believed that some of the surprising technical effects are related to the specific 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 amido group:

[0078]

[0079] It has been previously shown that a higher affinity of a compound for a target protein can lead to longer tumor retention in vivo (Wichert et al., Nature Chemistry 7, 241-249 (2015)). Compared to prior art compounds, the therapeutic compounds of the present invention have increased affinity for FAP, slower off-rates, and prolonged retention at the disease site at therapeutically relevant levels after injection, preferably greater than 1 hour, more preferably greater than 6 hours. Preferably, the highest enrichment is achieved after 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours; and / or the enrichment at the disease site after injection is maintained at therapeutically relevant levels for or 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, more preferably greater than 6 hours, even more preferably greater than 24 hours.

[0080] Part B

[0081] Moiety B is a covalent bond or a moiety comprising a chain of atoms that covalently links moiety A to payload C. Moiety B may be a cleavable or non-cleavable multifunctional moiety that can be used to link one or more payload and / or binding agent moieties to form a targeted conjugate of the invention.

[0082] In particular, moiety B is a multifunctional moiety that links one or more moieties C and / or moiety A. The structure of the compound may comprise 3 moieties A per molecule. The structure of the compound may comprise more than one moiety C per molecule, preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 moieties C. Preferably, the structure of the compound comprises 3 moieties A and 1 moiety C per molecule.

[0083] When a cleavable linker unit is present in part B, the release mechanisms may be identical to those specific for attachment to a cytotoxic payload. In practice, the nature of the binding moiety is independent in this respect. Thus, there is an assumed pH dependency [Leamon, CP et al. (2006) Bioconjugate Chem., 17 , 1226; Casi, G. et al. (2012) J. Am. Chem. Soc., 134 ,5887], reducibility [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]. Under certain circumstances, when functional groups are present on the binding moiety or payload (e.g., thiol, alcohol), linkerless ligation can be established, resulting in the release of the intact payload, which greatly simplifies pharmacokinetic analysis.

[0084] Part B may comprise or consist of the units shown in Table A below, wherein the substituents R and R n R, R1, R2 and R3 are each independently selected from H, OH, SH, NH2, halogen, cyano, carboxyl, 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 Independently selected from H, methyl or ethyl.

[0085] Table A

[0086]

[0087]

[0088] Part B, one or more units B L and / or one or more units B S Disulfide linkages may be suitably included as cleavable bonds, as these linkages are stable to hydrolysis while providing suitable drug release kinetics at the in vivo target, and may provide traceless cleavage of drug moieties including thiol groups.

[0089] Part B, one or more units B L and / or one or more units B SIt can 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 salt, polyethylene glycol, poly(methyl) hydroxyethyl acrylate, polysulfonates, etc. Suitably, the linker may contain a polar or charged peptide portion containing, for example, 2 to 10 amino acid residues. Amino acids may refer to any natural or non-natural amino acids. The peptide linker suitably includes a free thiol group, preferably an N-terminal cysteine, for forming the cleavable disulfide bond with the 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-A sp-Cys.

[0090] In these and other embodiments, moiety B, one or more units B L and / or one or more units B S Specially tailored cleavable or non-cleavable peptide units may be included so that they will be selectively enzymatically cleaved from the drug portion by one or more proteases on the cell surface or extracellular region of the target tissue. The amino acid residue chain length of the peptide unit is appropriately in the range of a single amino acid to about eight amino acid residues. A large number of specific cleavable peptide sequences suitable for the present invention can be designed and optimized for selectivity for enzymatic cleavage by specific tumor-related enzymes (e.g., proteases). Cleavable peptides for use in the present invention include those peptides optimized for proteases MMP-1, 2 or 3 or cathepsin B, C or D. Particularly suitable are peptides that can be cleaved by cathepsin B. Cathepsin B is a ubiquitous cysteine ​​protease. Except under pathological conditions such as metastatic tumors or rheumatoid arthritis, it is an intracellular enzyme. An example of a peptide that can be cleaved by cathepsin B is one that contains the sequence Val-Cit.

[0091] In any of the above embodiments, moiety B, and in particular one or more units B LSuitably, a self-destructive (self-immolative) part is also included, which may or may not be present after the joint. Self-destructive joints are also referred to as electronic grade joints. These joints undergo elimination and cleavage to release active forms, preferably free forms of drugs when the enzymatic cleavage of the peptide is performed. The conjugate is stable outside the cell in the absence of an enzyme that can cleave the joint. However, when exposed to a suitable enzyme, the joint breaks, triggering a spontaneous self-destructive reaction, resulting in the cleavage of the bond that the self-destructive part is covalently connected to the drug, thereby affecting the release of the drug in its non-derivatized or pharmacologically active form. In these embodiments, the self-destructive joint is coupled to the binding part by an enzymatically cleavable peptide sequence, and the enzymatically cleavable peptide sequence provides a substrate for the enzyme to break the amide bond and trigger a self-destructive reaction. Suitably, the drug part is connected to the self-destructive part of the joint via a chemically reactive functional group (e.g., a primary or secondary amine, a hydroxyl, a sulfhydryl or a carboxyl group) suspended on the drug.

[0092] Examples of self-immolative linkers are PABC or PAB (p-aminobenzyloxycarbonyl), attaching the drug moiety to the binding moiety in the 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 carboxyl terminus of the peptide unit and the p-aminobenzyl group of PAB can be a substrate and can be cleaved by certain proteases. The aromatic amine becomes electron-donating and initiates an electron cascade leading to the expulsion of the leaving group, which releases the free drug after the elimination of carbon dioxide (de Groot et al. (2001) Journal of Organic Chemistry 66 (26): 8815-8830). Other self-immolative linkers are described in WO2005 / 082023.

[0093] In other embodiments, the linker comprises a glucuronyl group that can be cleaved by glucuronidases present on the cell surface or extracellular regions of the target tissue. Lysosomal β-glucuronidases have been shown to be released extracellularly at high local concentrations in necrotic regions of human cancers, and this provides a pathway for targeted chemotherapy (Bosslet, K. et al., Cancer Res. 58, 1195-1201 (1998)).

[0094] In any of the above embodiments, part B suitably further comprises a spacer unit. The spacer unit may be unit B S, which may be linked to the binding moiety A, for example via an amide, amine or thioether bond. The spacer unit has a length such that, for example, the cleavable peptide sequence is accessible by a cleavage enzyme (e.g., cathepsin B), and suitably also enables hydrolysis of the amide bond coupling the cleavable peptide to the self-immolative moiety X. The spacer unit may, for example, include divalent groups such as alkylene, arylene, heteroarylene, repeating units of alkoxy (e.g., polyethyleneoxy, PEG, polymethyleneoxy) and repeating units of alkylamino (e.g., polyethyleneamino), or diacids and amides, including succinate, succinamide, diglycolate, malonate and caproamide.

[0095] In any of the embodiments described herein, * represents a point of attachment to moiety A, or a point of attachment where the shortest path to moiety A contains fewer atoms than the shortest path to moiety A, as the case may be; and · represents a point of attachment to therapeutic moiety C, or a point of attachment where the shortest path to therapeutic moiety C contains fewer atoms than the shortest path to therapeutic moiety C, as the case may be. The same applies to the case where a reactive moiety L is present instead of a therapeutic payload moiety C. The following symbols and all have the meaning of a point of attachment of a particular group or atom (e.g., R) to another moiety:

[0096]

[0097] If the structure of interest is a peptide monomer or peptide oligomer, then each * represents an attachment point for which the shortest path to moiety A contains fewer atoms than the shortest path to moiety A; and each · represents an attachment point for which the shortest path to therapeutic moiety C contains fewer atoms than the shortest path to therapeutic moiety C contains *, provided that when n>1 and the corresponding attachment point is indicated in R a , R b and R c If it is present on any one of the attachment points indicated in the corresponding structure, then it can be present independently in one or more of the peptide monomer units, preferably in the one peptide monomer unit farthest from the other attachment point indicated in the corresponding structure.

[0098] In any embodiment described herein, the terms "peptide", "dipeptide", "tripeptide", "tetrapeptide", etc. refer to a peptide monomer or peptide oligomer having a backbone formed by proteinogenic and / or non-proteinogenic amino acids. As used herein, the terms "aminoacyl" or "amino acid" generally refer to any proteinogenic or non-proteinogenic amino acid. Preferably, in any embodiment disclosed herein, the side chain residues of the proteinogenic or non-proteinogenic amino acid are composed of R a , R b and R cAny one of which is selected from the following list:

[0099]

[0100]

[0101] Among them, R 1 , R 2 and R 3 are each independently selected from H, OH, SH, NH2, halogen, cyano, carboxyl, alkyl, cycloalkyl, aryl and heteroaryl, each of which is substituted or unsubstituted;

[0102] Each X is independently selected from NH, NR, S, O and CH2, preferably NH; and

[0103] 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.

[0104] Preferably, in any of the embodiments disclosed herein, the side chain residues of the proteinogenic or non-proteinogenic amino acids are composed of R a , R b and R c Any of the expressions,

[0105] Each of these may be part of a 3-, 4-, 5-, 6- or 7-membered ring. For example, the side chain α, β and / or γ position of the proteinogenic or non-proteinogenic amino acid may be part of a ring structure selected from the group consisting of an azetidine ring, a pyrrolidine ring and a piperidine ring, such as in the following amino acids (proline and hydroxyproline):

[0106]

[0107] Each of them may be independently an unsaturated structure (i.e., wherein the a , R b and R c The geminal H atom is absent), for example:

[0108]

[0109] As used herein, the following notation for peptide sequences refers to the sequence from N to C terminus, and attachment of a group via a horizontal bond (here: portion C) refers to covalent attachment to the peptide backbone via an amide bond to the corresponding terminal amino acid (here: AA3):

[0110]

[0111] As used herein, the following notation for peptide sequences refers to the sequence from N to C terminus, and attachment of a group by a vertical bond (here: C moiety) refers to covalent attachment via the side chain of the corresponding amino acid (here AA3):

[0112]

[0113] Further preferred non-proteinogenic amino acids may be selected from the following list:

[0114]

[0115] Particularly preferred embodiments of moiety B and of the compounds according to the invention are indicated in the following further clauses and in the appended claims.

[0116] Preferably, B comprises the following structure

[0117]

[0118] wherein each x is an integer independently selected from the range of 0 to 100, preferably 0 to 50, more preferably 0 to 30, still more 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;

[0119] Each y is independently selected from an integer ranging from 0 to 30, preferably an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20;

[0120] Each z is independently selected from an integer ranging from 0 to 5, preferably an integer selected from 0, 1, 2, 3 and 4;

[0121] * indicates the point of attachment to part A; and

[0122] • Indicates the point of attachment to treatment portion C.

[0123] L, B S and B Leach of which may independently be a moiety comprising or consisting of a structural unit selected from the group consisting of alkylene, cycloalkylene, aralkylene, heteroaralkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, aralkenylene, heteroaralkenylene, heteroalkenylene, heterocycloalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, oxyalkylene, dioxyalkylene, aminoalkylene, diaminoalkylene, diacid, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylenealkoxycarbamate, disulfide, 1,2-vinylene, imine, imineamide, phosphoramide, sugar, phosphate, phosphoramide, carbamate, dipeptide, tripeptide and tetrapeptide, each of which is substituted or unsubstituted;

[0124] L, B S and B L Each of can be independently selected from:

[0125]

[0126]

[0127]

[0128]

[0129] Where in each of the above structures:

[0130] Each n is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0131] Each m is independently 0, 1, 2, 3 or 4;

[0132] Each R' is independently H or selected from H, SH, NH2, halogen, cyano, carboxyl, C 1-6 -alkyl, O(C 1-6 Alkyl), S(C 1-6 -alkyl), C 2-6 Alkenyl, C 2-6 Alkynyl, 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 halogen,

[0133] Each R c , R d and Re are independently selected from H, optionally substituted C 1-6 Alkyl, (C3-C 10 Carbocyclic)C 1-6 Alkyl, (C6-C 10 Aryl)C 1-6 Alkyl, (C1-C 10 Heterocyclic)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 heteroatoms; preferably side chain residues selected from proteinogenic or non-proteinogenic amino acids;

[0134] Each * indicates that the shortest path to part A contains fewer atoms than the shortest path to part A; and each · indicates that the shortest path to treatment part C contains fewer atoms than the shortest path to treatment part C, provided that when n>1 and the corresponding connection point is marked in R c , R d and R e , then it can be present independently in one or more peptide monomer units, preferably in a peptide monomer unit farthest from the other attachment point indicated in the corresponding structure,

[0135] Each of the above structures optionally includes additional attachment points to part A or C.

[0136] Part J

[0137] Moiety J forms part of the linker to 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, wherein at least two of said non-hydrogen atoms are located on a covalent chain representing the shortest path between moiety A and said central atom. It can therefore be considered as a multi-point template with multiple arms of moiety A being joined together.

[0138] Moiety J and group (L) a Together they are referred to herein as Section K.

[0139] J may be a moiety independently comprising a branch point comprising or consisting of structural units independently selected from the group consisting of alkylene, cycloalkylene, aralkylene, heteroaralkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, aralkenylene, heteroaralkenylene, heteroalkenylene, heterocycloalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, oxyalkylene, dioxyalkylene, aminoalkylene, diaminoalkylene, diacid, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylenealkoxycarbamate, disulfide, 1,2-vinyl, imine, imineamide, phosphoramide, sugar, phosphate, phosphoramide, carbamate, dipeptide, tripeptide, tetrapeptide, each of which is substituted or unsubstituted;

[0140] Section C

[0141] 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.

[0142] The effective load can be a chelator for radiolabeling the therapeutic conjugate with a therapeutic nuclide. Suitably, the radionuclide is not released. Chelators are well known to those skilled in the art, and for example, include chelators such as sulfur colloids, 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 one of the preferred chelator structures described in the following further clauses or in the appended claims.

[0143] The therapeutic payload may be a radioactive group comprising or consisting of a therapeutic radioisotope, including isotopes such as 223 Ra, 89 Sr. 90 Y. 121 Sn, 177 Lu, 131 I. 211 At 225 Ac, 188 Re, 149 Tb, 161 Tb and 227Th, preferably 90 Y. 225 Ac or 177 Lu, more preferably 177 Lu; it may not be used for diagnostic applications.

[0144] The therapeutic payload may be a chelate of a therapeutic radioisotope (preferably an isotope listed above) and a chelator (preferably a chelator listed above or any of the preferred chelator structures described further below); or a group selected from the structures listed further below.

[0145] The effective load can be a cytotoxic agent and / or a cytostatic agent. Such an agent can inhibit or prevent the function of a cell and / or cause the destruction of a cell. Examples of cytotoxic agents include radioactive therapeutic isotopes, chemotherapeutic agents and toxins, such as small molecule toxins or enzyme active toxins of bacterial, fungal, plant or animal origin, including synthetic analogs and derivatives thereof. The cytotoxic agent can be selected from the group consisting of: auristatin, DNA minor groove binder, DNA minor groove alkylating agent, enediyne, lexitropsin, duocarmycin, taxane, puromycin, dolastatin, maytansinoid and vinca alkaloid, or a combination of two or more thereof. Preferred cytotoxic effective load parts and / or cytostatic effective load parts are listed in further clause 8 (e) below.

[0146] In one embodiment, the payload is a therapeutic chemotherapeutic agent selected from the group consisting of: topoisomerase inhibitors, alkylating agents (e.g., nitrogen mustards; ethyleneimine; alkyl sulfonates; triazenes; piperazines; and nitrosoureas), antimetabolites (e.g., mercaptopurine, thioguanine, 5-fluorouracil), antibiotics (e.g., anthracyclines, actinomycin, bleomycin, adriamycin, mithramycin, actinomycin), mitotic disruptors (e.g., plant alkaloids - such as vincristine and / or microtubule antagonists - such as paclitaxel), DNA methylating agents, DNA intercalators. In some embodiments, the active agent (e.g., carboplatin and / or cisplatin, daunomycin and / or doxorubicin and / or bleomycin and / or thalidomide), a DNA synthesis inhibitor, a DNA-RNA transcription regulator, an enzyme inhibitor, a gene regulator, a hormone response regulator, a hypoxia selective cytotoxin (e.g., tirapazamine), an epidermal growth factor inhibitor, an anti-vascular agent (e.g., xanthone 5,6-dimethylxanthone-4-acetic acid), a radiation-activated prodrug (e.g., nitroarylmethyl quaternary ammonium (NMQ) salt) or a bioreductive drug, or a combination of two or more thereof. In some embodiments, the payload (i.e., portion C) is not derived from an anthracycline antibiotic, preferably not derived from PNU 159682.

[0147] The therapeutic chemotherapeutic agent may be selected from the group consisting of: Erlotinib Bortezomib Fulvestrant Sutent (SU11248), letrozole Imatinib mesylate PTK787 / ZK222584, Oxaliplatin 5-FU (5-fluorouracil), folinic acid, sirolimus Lapatinib (GSK572016), lonafarnib (SCH66336), sorafenib (BAY43-9006), and gefitinib AG1478, AG1571 (SU5271; Sugen), or a combination of two or more thereof.

[0148] The therapeutic chemotherapeutic agent may be an alkylating agent - such as thiotepa, and / or cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan and / or piposulfan; aziridines, such as benzodopa, carboquone, meturedopa and / or uredopa; ethyleneimine and / or methylamelamine, such as hexamethylmelamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphamide and / or trimethylomelamine; acetogenins, such as acetogenins, ... ) - such as bullatacin and / or bullatacinone; camptothecin; bryostatin; callystatin; cryptophycins; dolastatin; duocarmycin; eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard mustard - for example, chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and / or uracil mustard mustard; nitrosoureas - such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and / or ranimnumine; dynemicin; bisphosphonates - such as clodronate; esperamicin; neocarzinostatin chromophore;aclainomycin, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Doxorubicin - such as morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and / or deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin - such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomycin, streptozocin, tuberculin, ubenimex, zinostatin, zorubicin, bicin; antimetabolites - such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs - such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs - such as fludarabine (zorubicin), 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs - such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens - such as calusterone, dromostanolone propionate, propionate, epitiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamide; demecolcine; diaziquone;elformithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidinine; macrocyclic depsipeptides, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; riaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes - such as verracurin A, roridin A A) and / or anguidine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside; cyclophosphamide; thiotepa; taxoids - e.g. Paclitaxel, albumin-bound paclitaxel (abraxane), and / or Docetaxel; Chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs - such as cisplatin and carboplatin; vinblastine; platinum; etoposide; ifosfamide; mitoxantrone; vincristine; Vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, derivatives, or any combination of two or more of the foregoing.

[0149] The therapeutic payload can be a microtubule disrupting agent, including but not limited to: taxanes - such as paclitaxel and docetaxel, vinca alkaloids, discodermolide, epothilone A and B, desooxycerthilone, cryptophycin, cruacin A, combretastatin A-4-phosphate, BMS247550, BMS184476, BMS188791; LEP, RPR109881A, EPO906, TXD258, ZD6126, vinflunine, LU103793, dolastatin 10, E7010, T138067 and T900607, colchicine, phenstatin, chalcone, indanocine, T138067, oncocidin, vincristine, vinblastine, vinorelbine, vinflunine, halichondrin B, isohomohalichondrin B, ER-86526, pironetin, spongistatin 1, spiket P, cryptophycin 1, LU103793 (cematodin or cemadotin), rhizoxin, sarcodictyin, eleutherobin, lauliamide, VP-16 and D-24851 and pharmaceutically acceptable salts, acids, derivatives, or any combination of two or more of the foregoing.

[0150] The therapeutic payload can be a DNA intercalator, including but not limited to: acridine, actinomycin, anthracycline, benzothiopyranoindazole, pixanone, crisnatol, brostallicin, CI-958, doxorubicin (adriamycin), dactinomycin D, daunorubicin (daunomycin), bleomycin, idarubicin, mitoxantrone, cyclophosphamide, melphalan, mitomycin C, bizelesin, etoposide, mitoxantrone, SN-38, carboplatin, cisplatin, dactinomycin D, amsacrine, DACA, pyrazoloacridine, irinotecan and topotecan and pharmaceutically acceptable salts, acids, derivatives thereof, or a combination of any two or more of the foregoing.

[0151] The therapeutic payload can be an anti-hormonal agent, which is used to regulate or inhibit the effects of hormones on tumors - such as antiestrogens and selective estrogen receptor modulators, including but not limited to tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and / or fareston toremifene and pharmaceutically acceptable salts, acids, derivatives, or any combination of two or more of the above. The therapeutic payload can be an aromatase inhibitor that inhibits the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, formstanie, fadrozole, Vorozole, Letrozole and and / or anastrozole and a pharmaceutically acceptable salt, acid, derivative, or a combination of two or more of any of the foregoing.

[0152] The therapeutic payload can be an anti-androgen, such as flutamide, nilutamide, bicalutamide, leuprolide, goserelin and / or troxacitabine and pharmaceutically acceptable salts, acids, derivatives, or a combination of two or more of any of the foregoing.

[0153] The therapeutic payload may be a protein or an antibody.Preferably, the payload is a cytokine (eg, an interleukin such as IL2, IL10, IL12, IL15; a member of the TNF superfamily; or an interferon such as interferon gamma).

[0154] Any therapeutic payload can be used in unmodified or modified form. Combinations of therapeutic payloads, some of which are unmodified and some of which are modified, can 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.

[0155] In a preferred embodiment, the payload moiety C is a topoisomerase inhibitor; preferably camptothecin (CPT) or a derivative thereof; more preferably derived (e.g., by replacement of a hydrogen atom) from topotecan, irinotecan, silatecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan, rubitecan, deruxtecan, DXd; even more preferably exatecan; even more preferably

[0156] wherein each n is 0, 1, 2, 3, 4, 5 or 6; most preferably

[0157] In a preferred embodiment, the therapeutic moiety C is an auristatin (ie, has a structure derived from a member of the auristatin compound family) or an auristatin derivative. More preferably, the therapeutic moiety C has a structure according to the formula:

[0158]

[0159] in:

[0160] R 1d Independently H or C1-C6 alkyl; preferably H or CH3;

[0161] R 2dIndependently C1-C6 alkyl; preferably CH3 or iPr;

[0162] R 3d Independently H or C1-C6 alkyl; preferably H or CH3;

[0163] 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;

[0164] R 5d are independently H, OH, C1-C6 alkyl; preferably H or OH; and

[0165] R 6d Independently for C3-C 10 Aryl or C3-C 10 Heteroaryl; preferably optionally substituted phenyl or pyridinyl.

[0166] More preferably, the therapeutic moiety C is derived from MMAE or MMAF.

[0167] In a preferred embodiment, the therapeutic moiety C has a structure according to the following formula:

[0168]

[0169] in:

[0170] n is 0, 1, 2, 3, 4 or 5; preferably 1;

[0171] R 1e is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH;

[0172] R 2e are independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH;

[0173] Each R 3e is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH;

[0174] R 4e is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH; and

[0175] X is O, NH or S; preferably O.

[0176] In a preferred embodiment, the therapeutic moiety C has a structure according to the following formula:

[0177]

[0178] in:

[0179] n is 0, 1, 2, 3, 4 or 5; preferably 1

[0180] R 1f is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH;

[0181] R 2f is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH;

[0182] R 3f is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH; and

[0183] X is O, NH or S; preferably O.

[0184] Particularly preferred embodiments of the therapeutic moiety C and of the compounds according to the invention are indicated in the following further clauses and in the appended claims.

[0185] As described above, a preferred embodiment when C is a chelator is represented by Formula III. An exemplary, particularly preferred structure of this type is represented by Formula IIIa, wherein all variables and groups are as defined in the claims, and which may exist in the form of a complex with any therapeutic radioisotope disclosed herein (preferably 90-yttrium, 225-actinium or 177-lutetium, more preferably 177-lutetium):

[0186]

[0187] Other aspects

[0188] Fragment ((B S ) x (B L ) y ) z Can be represented by one of the following structures:

[0189] Single bond, (BS)x,

[0190]

[0191]

[0192] wherein each of AA3, AA4, AA5, AA6, AA7 and AA8 represents a proteinogenic or non-proteinogenic amino acid, or is absent;

[0193] Among them, preferably:

[0194] Each proteinogenic or non-proteinogenic amino acid is preferably independently represented by one of the following structures:

[0195]

[0196] and / or AA4 is an amino acid having a charged side chain, and AA7 is an amino acid having an aliphatic side chain;

[0197] Among them, more preferably:

[0198] AA3 is selected from Asp, Glu and Lys, or absent; preferably Asp;

[0199] AA4 is selected from Arg, HomoArg, Lys, Asp and Glu, or absent; preferably Lys or Arg;

[0200] AA5 is selected from Asp, Glu and Lys; preferably Asp;

[0201] AA6 is selected from Cys, Lys, Gly and Val; preferably Cys or Lys;

[0202] AA7 is selected from the group consisting of Gly, Ala, Val, Arg, Ile, Pro; and

[0203] AA8 is selected from Pro and citrulline (Cit);

[0204] Even more preferably according to one of the sequences shown in the following table:

[0205]

[0206] or

[0207] Fragment ((B S ) x (B L ) y ) z Can be represented by one of the following structures:

[0208] single bond,

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217] In some embodiments, the compound can be represented by one of the following structures:

[0218]

[0219]

[0220] Preferred compounds according to the present invention can be represented by:

[0221]

[0222]

[0223] Among them B S , B L , x, y and n and the remaining groups are as defined elsewhere herein,

[0224] wherein each of AA3, AA4, AA5, AA6, AA7 and AA8 represents a proteinogenic or non-proteinogenic amino acid, or is absent;

[0225] Preferably, each (B S ) x and (B L ) y are independently represented by: bond, –NHC(O)(CH2) n C(O)–, –NH(CH2) n C(O)–,–NHC(O)(CH2 CH2O) 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(CH2CH2O) 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)—, and wherein each n and m is independently an integer, preferably selected from 0, 1, 2, 3, 4, 5 and 6;

[0226] More preferably

[0227]

[0228] Most preferably

[0229]

[0230] Preferred compounds according to the present invention can also be represented by:

[0231] More preferably

[0232] Most preferably:

[0233]

[0234] Among them B S , BL , x, y and n and the remaining groups are as defined elsewhere herein,

[0235] Preferably, each (B S ) x and (B L ) y are independently represented by: 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(CH2CH2O) 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)—, and wherein each n and m is independently an integer, preferably selected from 0, 1, 2, 3, 4, 5 and 6.

[0236] Moiety C is a therapeutic agent, which can 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 and a chelator; (d) a cytotoxic therapeutic agent and / or a cytostatic therapeutic agent; (f) an immunomodulator; or (g) a protein, preferably:

[0237] (a) a chelator group suitable for radiolabeling with a therapeutic nuclide selected from the group consisting of sulfur colloid, 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),

[0238]

[0239]

[0240] Having a structure according to the formula:

[0241]

[0242] in:

[0243] n is 0, 1, 2, 3, 4 or 5; preferably 1;

[0244] R 1e is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH;

[0245] R 2e is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH;

[0246] Each R 3e is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH;

[0247] R 4e is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH; and

[0248] X is O, NH or S; preferably O; or

[0249] Having a structure according to the formula:

[0250]

[0251] in:

[0252] n is 0, 1, 2, 3, 4 or 5; preferably 1

[0253] R 1f is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH;

[0254] R 2f is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH;

[0255] R 3f is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH; and

[0256] X is O, NH or S; preferably O;

[0257] (b) the therapeutic radioactive group comprises a radioisotope selected from the group consisting of 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, preferred 90 Y. 225 Ac or 177 Lu, more preferably 177 Lu; it may not be used for diagnostic applications;

[0258] (c) the chelate of the therapeutic radioisotope is a chelate of the isotope listed in (b) above and / or a chelate with the chelator listed in (a) above; or the moiety C is a group selected from any one of the following structures:

[0259]

[0260] Wherein M is a therapeutic radioisotope, preferably selected from the list in (b) above; more preferably:

[0261]

[0262] (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, anti-vascular agents and combinations of two or more thereof, preferably selected from the following structures:

[0263]

[0264]

[0265]

[0266]

[0267] Where each n is 0, 1, 2, 3, 4, 5 or 6;

[0268]

[0269]

[0270]

[0271] Moiety C is an auristatin, preferably having a structure according to the following formula:

[0272]

[0273] in:

[0274] R 1d Independently H or C1-C6 alkyl; preferably H or CH3;

[0275] R 2d Independently C1-C6 alkyl; preferably CH3 or iPr;

[0276] R 3d Independently H or C1-C6 alkyl; preferably H or CH3;

[0277] 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 10Heteroaryl; preferably H, CH3, COOH, COOCH3 or thiazolyl;

[0278] R 5d are independently H, OH, C1-C6 alkyl; preferably H or OH; and

[0279] R 6d Independently for C3-C 10 Aryl or C3-C 10 heteroaryl; preferably optionally substituted phenyl or pyridyl,

[0280] Wherein preferably, part C is derived from MMAE or MMAF;

[0281] (e) immunomodulator therapeutic agents are selected from molecules known to modulate the immune system, such as ligands of CD3, CD25, TLR, STING, 4-1BBL, 4-1BB, PD-1, mTor, PDL-1, NKG-2D IMiD, where the ligands can be agonists and / or antagonists; or

[0282] (f) The protein is a therapeutic protein selected from cytokines such as IL2, IL10, IL12, IL15, TNF, interferon gamma, or a therapeutic antibody.

[0283] treat

[0284] The compounds described herein can be used to treat disease. Treatment can be therapeutic treatment, the purpose of which is to prevent, reduce or stop undesirable physiological changes or conditions. Treatment can prolong survival compared to the expected survival without treatment. The disease treated by the compound can be any disease that may benefit from treatment. This includes chronic and acute conditions or diseases, including those pathological conditions that predispose to the condition.

[0285] The terms "cancer" and "cancerous" are used in their broadest sense to mean the physiological condition in mammals that is typically characterized by unregulated cell growth. A tumor includes one or more cancer cells. When treating cancer, the observed therapeutic effect can be a reduction in the number of cancer cells; a reduction in tumor size; an inhibition or delay in the infiltration of cancer cells into peripheral organs; an inhibition of tumor growth; and / or amelioration of one or more symptoms associated with cancer.

[0286] In animal models, efficacy can be assessed by physical measurements of tumors during treatment and / or by determining partial and complete remission of the cancer. For cancer treatment, efficacy can be measured, for example, by assessing time to disease progression (TTP) and / or determining response rate (RR).

[0287] Particularly preferred embodiments relating to the method of treatment of the invention are set out in the following further clauses and in the appended claims.

[0288] Also disclosed herein are methods for treating a human or animal body implemented on the human or animal body, such as by therapy, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or pharmaceutical composition as described herein. More specifically, disclosed herein are methods for treating (e.g., by therapy) a subject suffering from a disease or condition or at risk of a disease or condition; methods for targeted delivery of a therapeutic agent to a subject suffering from a disease or condition or at risk of a disease or condition. In the above method, the disease or condition can be independently selected from cancer, inflammation, atherosclerosis, fibrosis, tissue remodeling and keloid conditions, 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, clear cell renal carcinoma, neuroendocrine tumors, carcinogenic osteomalacia, sarcoma, CUP (cancer of unknown primary site), thymic carcinoma, desmoid tumor, glioma, astrocytoma, cervical cancer, skin cancer, kidney cancer and prostate cancer. When used in the methods disclosed herein, the compounds have prolonged retention at the disease site at therapeutically relevant levels, e.g., 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 more than 1 hour, more preferably more than 6 hours, and even more preferably more than 24 hours.

[0289] The disclosure of all compounds, compositions or combinations disclosed herein for use in any method of treating the human or animal body is also intended to extend to corresponding methods for treating a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the compound, composition or combination; and to the corresponding use of the compound, composition or combination in the preparation of a medicament for treating the corresponding disease or condition.

[0290] dose

[0291] The inventors have surprisingly discovered that compounds, when administered at specific doses, can provide optimal tumor uptake and / or tumor to organ ratios, thereby exhibiting minimal capture in other organs (ie, a particularly clean biodistribution profile).

[0292] Compounds, pharmaceutical compositions or combinations described herein can be administered to subjects 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, most preferably 90-125 nmol / kg (expressed as mouse doses) or corresponding human equivalent doses of compounds of Formula I, II, III, IV, V, VI or VII. Compounds, pharmaceutical compositions or combinations described herein can be administered to human subjects 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, most preferably 7-10 nmol / kg.

[0293] like Fig.15 As shown, in mice, it was found that particularly clean biodistribution characteristics and optimal tumor uptake can be achieved at doses preferably in the range of 10 to 500 nmol / kg, particularly at doses of 90 nmol / kg or more. Above 250 nmol / kg, a saturation effect was observed. Therefore, a range of 90 nmol / kg or more, preferably 90 to 250 nmol / kg (expressed as mouse doses) is particularly preferred. A range of 7 nmol / kg or more, preferably 7-20 nmol / kg (expressed as human doses) is particularly preferred.

[0294] Mouse doses can be converted to human equivalent doses (HEDs), for example, considering the recommendations reported by the FDA in USFDA. Guidance for Industry: Estimating the Maximum Safe Starting Dose in Adult Healthy Volunteer. Rockville, MD: US Food and Drug Administration; 2005, the mouse dose (nmol / kg) must be divided by a factor of 12.3 to obtain the corresponding HED (nmol / kg). For conversion to human equivalent doses expressed in nmol or mg, a human reference weight of 60, 70 or 80 kg, preferably 70 kg, can be used. This conversion takes into account the surface area differences between the two different species (Mus musculus and Homo sapiens saiens).

[0295] 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 a 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.

[0296] In view of the above, a human dose of 1 mg or more, preferably 1 mg to 3 mg per administration is particularly advantageous.

[0297] Pharmaceutical composition

[0298] The compounds described herein can be in the form of pharmaceutical compositions, which can be used for human or animal therapeutic purposes in human and / or veterinary medicine, and will generally include any one or more of a pharmaceutically acceptable diluent, carrier or excipient. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field and are described in, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro, ed. 1985). The selection of pharmaceutical carriers, excipients or diluents can be selected according to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may include any suitable one or more binding agents, one or more lubricants, one or more suspending agents, one or more coating agents, one or more solubilizing agents as or in addition to a carrier, excipient or diluent.

[0299] Preservatives, stabilizers, dyes and even flavoring agents may be provided in the therapeutic pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

[0300] Depending on the different delivery systems, there may be different composition / formulation requirements. For example, a therapeutic pharmaceutical composition may be formulated for administration using a micropump or by a mucosal route, such as as a nasal spray or aerosol for inhalation or an ingestible solution, or for parenteral administration, wherein the composition is formulated in an injectable form for delivery by, for example, intravenous, intramuscular or subcutaneous routes. Alternatively, the therapeutic formulation may be designed to be administered by a number of routes.

[0301] If a pharmaceutical agent is to be administered transmucosally across the gastrointestinal mucosa, it should be able to remain stable during transit through the gastrointestinal tract; for example, it should resist proteolytic degradation, be stable at acidic pH, and resist the detergent effects of bile.

[0302] Where appropriate, the therapeutic pharmaceutical composition may be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by the use of a skin patch, orally in the form of a tablet containing an excipient (e.g., starch or lactose), or in the form of a capsule or ovule alone or mixed with an excipient, or in the form of an elixir, solution or suspension containing a flavoring or coloring agent, or the pharmaceutical composition may be injected parenterally (e.g., intravenously, intramuscularly or subcutaneously). For parenteral administration, the therapeutic composition may be best used in the form of a sterile aqueous solution which may contain other substances, such as sufficient salts or monosaccharides to render the solution isotonic with the blood. For buccal or sublingual administration, the therapeutic composition may be administered in the form of tablets or lozenges which can be formulated in conventional manner.

[0303] 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, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, gluconates, glucuronates, sugarates, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and pamoates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts.

[0304] Routes for administration (delivery) may include, but are not limited to, one or more of the following: oral (e.g., as a tablet, capsule, or as an ingestible solution), topical, mucosal (e.g., as a nasal spray or aerosol for inhalation), nasal, parenteral (e.g., by injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ocular (including intravitreal or intracameral), transdermal, rectal, buccal, vaginal, epidural, sublingual.

[0305] Typically, the physician will determine the actual therapeutic dose most suitable for an individual subject. The specific dosage level and dosage frequency for any particular patient may vary and will depend on a variety of factors, including the activity of the specific compound employed, the metabolic stability and length of action of the compound, age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, severity of the particular condition, and the individual undergoing treatment.

[0306] Therapeutic preparations can be packaged in unit dose or multidose containers, such as sealed ampoules and vials, and can be stored in freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier, such as water, for administration. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the aforementioned types. Exemplary unit dose formulations contain a daily dose or a unit daily subdose of the active ingredient or an appropriate portion thereof.

[0307] Drug combinations

[0308] As used herein, the term "pharmaceutical combination" refers to, for example, a combination in which the individual components contained can be administered simultaneously or sequentially in the same dosage form or as separate dosage forms; preferably, the subject is exposed to a therapeutically or prophylactically effective amount of each of the individual components of the combination at a certain time point. For example, a pharmaceutical combination can contain two or more individual components in the same formulation, or contain two or more individual components as separate formulations that are intended (e.g., as part of a therapeutic dosage regimen / plan, or by instructions for use) or arranged (e.g., as a kit of parts) for concomitant or sequential administration.

[0309] The compounds described herein can 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 a scFv polypeptide sequence capable of binding to the epitope represented by SEQ ID NO: 40 (and further preferably an L19-IL2 immunocytokine, which further comprises linked VL and VH domains derived from antibody L19, optionally capable of homodimerization in the form of scFv2), as described below.

[0310] Combination with immune cytokines

[0311] Provided is a pharmaceutical combination comprising a compound or pharmaceutical composition as disclosed herein and an immunocytokine.

[0312] As used herein, the term "immunocytokine" refers to a conjugated protein or fusion protein comprising a cytokine and an antibody, an antibody fragment or an antibody derivative. A fusion protein is a polypeptide of a translation product produced by fusing two or more genes or nucleic acid coding sequences to an open reading frame (ORF). The fusion expression product of two genes or ORFs can be conjugated by a linker. As used herein, the terms conjugated protein or fusion protein are generally used interchangeably. The fusion protein may also include a signal peptide sequence, which is generally located upstream (5') of the specific binding member and subunit.

[0313] Immune cytokines including IL2

[0314] Preferably, the immunocytokine comprises a sequence having IL2 activity, i.e. an IL2 polypeptide, i.e. a cytokine IL2 or a functional fragment thereof. Preferably, each polypeptide chain of the immunocytokine comprises only one (i.e., a single) IL2 polypeptide. In this article, the terms "IL2" and "IL2 polypeptide" are used interchangeably.

[0315] IL2 can be derived from any animal, such as humans, rodents (e.g., rats, mice), horses, cattle, pigs, sheep, dogs, etc. Human IL2 is preferably used in a conjugate for human administration. The amino acid sequence of human IL2 is listed in SEQ ID NO: 21. The immunocytokine conjugate preferably comprises a single IL2 polypeptide. The IL2 polypeptide in the immunocytokine of the present invention maintains the biological activity of IL2, such as promoting the proliferation and / or differentiation of activated T and B lymphocytes and natural killer (NK) cells and / or inducing cytotoxic T cell (CTL) activity and / or inducing NK / lymphokine activated killer (LAK) cell anti-tumor cytotoxicity.

[0316] Antibodies, fragments or derivatives thereof contained in immunocytokines

[0317] In addition to cytokines (e.g., IL2), immunocytokines include antibodies or antibody fragments or antibody derivatives. Preferably, the immunocytokines herein include antibody derivatives. Antibody derivatives may, for example, include single-chain variable fragments (scFv), diabodies or single-chain diabodies (scDb) or Fab or Fab2 or nanobodies or "SIP" (WO2003 / 076469) or "Crab"

[0318] (Neri et al., (1995) J Mol Biol, 246, 367-73). The immunocytokine may comprise an IgG antibody or an IgG derivative.

[0319] Preferably, the antibody derivative comprises an scFv. As is known in the art, an scFv comprises a VH domain and a VL domain, wherein these domains are connected by a linker that allows the VH and VL domains to associate to form an antigen binding site. The scFv can be stabilized by the incorporation of a disulphide bridge connecting the VH and VL domains

[48] .

[0320] Single-chain Fv (scFv) antibody polypeptide sequences are particularly preferred for incorporation into immunocytokines (e.g., together with another polypeptide sequence having IL2 activity) due to their small size in scFv form, which provides physiological and therapeutic advantages for the use of immunocytokine conjugates in vivo. In addition, scFv lacks the Fc region, potentially reducing anti-idiotypic responses, and also minimizes undesirable properties associated with complement activation and interaction with Fc receptors (which may hinder tumor targeting and cause nonspecific cell activation).

[0321] The linker connecting the VH and VL domains in the scFv chain can be a peptide linker sequence that is not long enough to allow the VH and VL domains to be paired in the same scFv polypeptide chain. Therefore, the homodimer of scFv is alternatively formed, in which the VH of one scFv chain is paired with the VL of another scFv chain (and vice versa). This general form can be referred to as the "scFv2" form, or alternatively, in some cases, is referred to as a "double antibody". Examples of suitable short linker sequences are GSSGG (SEQ ID NO: 26) and GGSGG (SEQ ID NO: 27). Preferably, the 12-residue linker of the linker such as SEQ ID NO: 22.

[0322] In the 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 connected into a single chain via a peptide linker ("scDb linker") as follows:

[0323] (VH-VL)-Linker-(VH-VL), wherein "(VH-VL)" represents a scFv unit consisting of a set of VH and VL domains connected by a short linker, as described above.

[0324] The scDb linker sequence is long and / or flexible enough to allow the VH domain of one scFv unit (i.e., the first VH and VL-containing polypeptide) to pair with the VL domain of another complementary scFv unit (i.e., the second VH and VL-containing polypeptide) (or vice versa) within a single polypeptide chain. Typically, long and / or flexible linkers that allow two complementary VH and VL-containing polypeptides to dimerize within a single polypeptide chain in this manner are 10 to 20 amino acids in length.

[0325] Antigen Binding Specificity of Immune Cytokines

[0326] The antibody, antibody fragment or antibody derivative suitably specifically binds to extracellular matrix (ECM) components associated with tumor growth and / or angiogenesis. The antibody, its fragment or derivative (e.g., scFv, diabody or single-chain antibody) comprises an antigen binding site with a complementary determining region (CDR), or a VH and / or VL domain of an antibody that can specifically bind to a target antigen. In particular, it can include one or more CDRs or VH and / or VL domains of an antibody that can specifically bind to an ECM antigen.

[0327] The antigen binding sites of an antibody, antibody fragment or antibody derivative (e.g., scFv, diabody or scFv2 or single-chain diabody) may be the same or different, but are preferably the same (e.g., the L19 antigen binding site of antibody L19, see below). Each of the antigen binding sites can 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, which still bind to the same antigen or epitope. Alternatively, an 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 can bind to two different antigens mentioned herein, such as two different antigens of the extracellular matrix, or two different domains of a specific antigen (e.g., fibronectin or tenascin-C).

[0328] The antigen may be an antigen preferentially expressed by tumor or tumor neovascular cells or an antigen associated with ECM. As described above, such antigens include fibronectin and tenascin C.

[0329] As used herein, the term "specific binding" means that one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s). The term also applies, for example, to an antigen binding site that is specific for a particular epitope present on many different antigens, in which case an antibody, antibody fragment or antibody derivative carrying the antigen binding site will be able to bind to a variety of antigens carrying the epitope.

[0330] 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 alternative splicing isoforms A-FN and B-FN, comprising domains ED-A or ED-B, respectively, which are known markers of angiogenesis. Preferably, the antibody, fragment or derivative thereof binds to fibronectin isoform B-FN, for example, most preferably it binds 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 from the UniProt database entry P02751 Residues 1266-1356 of (human fibronectin) are provided herein as SEQ ID NO:39.

[0331] 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 (eg, scFv polypeptide sequence) binds to the epitope represented by SEQ ID NO:40.

[0332] The fibronectin isoform B-FN is one of the best known markers of angiogenesis (WO1997 / 045544). An additional domain "ED-B" of 91 amino acids is present in the B-FN isoform and is identical in mice, rats, rabbits, dogs and humans. B-FN accumulates around new vascular structures in invasive tumors and other tissues undergoing angiogenesis (such as the endometrium in the proliferative stage and some ocular structures under pathological conditions), but cannot be detected in normal adult tissues.

[0333] 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, wherein preferably the scFv sequence binds to the epitope of SEQ ID NO:39, for example, the epitope represented by SEQ ID NO:40.

[0334] Also preferably, the antibody, antibody fragment or antibody derivative (eg, scFv sequence) that specifically binds to the alternatively spliced ​​ED-B domain of the fibronectin isoform B-FN comprises an antigen binding site derived from the antibody L19. Antibody L19 specifically binds to the alternatively spliced ​​ED-B domain of the fibronectin isoform B-FN, wherein 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. Therefore, an example of a most preferred antibody derivative is L19 scFv, which has been previously described (WO1999 / 058570; WO2006 / 119897 or WO2003 / 076469); see also SEQ ID NO: 15 in WO2020 / 070150 for immunocytokines and Figure 1 B).

[0335] 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 NOs: 15 and 20 in WO2020 / 070150). Figure 1 B, and SEQ ID NO: 12 herein).

[0336] Antigen binding sites and sequences

[0337] The antigen binding site may, for example, comprise one, two, three, four, five or six CDRs of antibody L19. The amino acid sequences of the CDRs of L19 are: 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).

[0338] SEQ ID NOs: 13-15 are the amino acid sequences of the VH CDR regions (1-3, respectively) of the human monoclonal antibody L19. SEQ ID NOs: 16-18 are the amino acid sequences of the VL CDR regions (1-3, respectively) of the human monoclonal antibody L19.

[0339] The antigen binding sites 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: 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).

[0340] The amino acid sequences of the VH and VL domains of antibody L19 correspond to SEQ ID NOs: 19 and 20, respectively.

[0341] Preferably, the antibody derivative comprises a VH domain having an amino acid sequence comprising VH CDR1, VH CDR2 and / or VH CDR3 of L19 and a VL domain having an amino acid sequence comprising VL CDR1, VLCDR2 and / or VL CDR3 of L19.

[0342] The antibody derivatives as described above may comprise a VH domain having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (preferably 80%) identical to the amino acid sequence of the L19 VH domain as set forth in SEQ ID NO: 22, and / or may comprise a VL domain having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (preferably 80%) identical to the amino acid sequence of the L19 VL domain as set forth in SEQ ID NO: 23.

[0343] Preferably, the antibody derivative as described above is a L19 scFv (i.e., a 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 a L19 VH domain (SEQ ID NO: 22) and / or a L19 VL domain (SEQ ID NO: 23), e.g., both L19 VH and L19 VL.

[0344] In the scFv unit, the VH and VL domains are connected by a joint. The joint can be a peptide joint sequence that is not long enough to allow the VH and VL domains to pair. Therefore, the homodimer of scFv can be formed alternatively, in which the VH of one scFv chain is paired with the VL of another scFv chain (and vice versa). This general form can be referred to as the "scFv2" form, alternatively, in some cases, it is referred to as a "double antibody". Examples of suitable short joint sequences are GSSGG (SEQ ID NO: 26) and GGSGG (SEQ ID NO: 27). Preferably, the joint is a 12-residue joint SEQ ID NO: 22.

[0345] The antibody derivative can be an L19 diabody having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (preferably 80%) sequence identity with the amino acid sequence listed in SEQ ID NO:28, or an L19 scDb having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (preferably 80%) sequence identity with the amino acid sequence listed in SEQ ID NO:29.

[0346] In a preferred embodiment, the antibody derivative is a scFv (L19) having an amino acid sequence that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (preferably 80%) sequence identity with the amino acid sequence set forth in SEQ ID NO:38.

[0347] Linking of segments within immune cytokines

[0348] Immunocytokines comprise cytokines (e.g., IL2 polypeptides) and are conjugated to antibodies, antibody fragments, or antibody derivatives. The conjugation can be achieved by any suitable covalent bond or linker moiety (e.g., disulfide or 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

[0349] GSLDGAGGSAGADGG (SEQ ID NO: 25). One or more different linkers may be used. Preferably, the linker may be the 17-residue linker of SEQ ID NO: 23.

[0350] Thus, the antibody, antibody fragment or antibody derivative and IL2 can be produced and / or secreted as a single chain polypeptide.

[0351] IL2 is preferably linked to the C-terminus of the antibody or antibody fragment or antibody derivative. Preferably, the link may be through a peptide linker sequence, as disclosed herein. When IL2 is conjugated to the C-terminus, the N-terminus of the antibody or antibody fragment or antibody derivative is preferably free. "Free" herein means that the N-terminus is not connected or otherwise conjugated to another moiety, such as IL2.

[0352] Preferred L19-IL2 immunoconjugates .

[0353] Preferably, the immunocytokine comprises an L19-derived scFv unit linked to an IL2 polypeptide in a conjugated polypeptide chain that forms a homodimer (according to the scFv2 or diabody format) due to the complementary pairing between the VH and VL domains of the scFv units in the two polypeptide chains. 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 forms a homodimer via the scFv unit in this manner. More preferably, the immunocytokine comprises the sequence of SEQ ID NO: 12 (i.e., the amino acid sequence of the L19-IL2 polypeptide in scFv format).

[0354] The production and purification of L19-IL2 constructs can be carried out as described in WO01 / 062298. A schematic diagram of the L19-IL2 conjugate in scFv form is shown in Fig.12 Shown in.

[0355] Other definitions involving immune cytokines

[0356] Sequence identity is usually 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, which maximizes the number of matches and minimizes the number of gaps. Typically, default parameters are used, with a gap creation penalty of 12 and a gap extension penalty of 0.

[0357] =4. The use of GAP may be preferred, but other algorithms may also be used, such as BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses 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) mentioned above, usually using default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 253389-3402) may be used.

[0358] Variants of these VH and VL domains and CDRs may also be employed in antibody molecules used in conjugates as described herein. Suitable variants may be obtained by methods of sequence alteration or mutation and screening.

[0359] Specific variants used as described herein may include one or more amino acid sequence changes (additions, deletions, substitutions and / or insertions of amino acid residues), which may be less than about 20 changes, less than about 15 changes, less than about 10 changes, or less than about 5 changes, 4, 3, 2 or 1 change.

[0360] Precursor compounds

[0361] In one aspect of the present invention, disclosed herein are compounds, individual diastereomers thereof, hydrates thereof, solvates thereof, crystalline forms thereof, individual tautomers thereof, or salts thereof, wherein the compound (precursor compound) comprises three moieties A and a reactive moiety G capable of reacting with a conjugation partner and forming a covalent bond. After conjugation (i.e., reaction and formation of a covalent bond), the original precursor compound is bound to the original conjugation partner, which in turn is bound to the therapeutic payload moiety C. The conjugation partner may be an atom, a molecule, a particle, a therapeutic agent. Preferably, the conjugate is a therapeutic agent and may correspond to the payload moiety already described in detail above with respect to the therapeutic conjugate according to the present invention.

[0362] Each moiety A preferably has the structure A as defined above 1 or A 2 .

[0363] Preferably, the precursor compound is represented by the formula:

[0364]

[0365] wherein B is a covalent bond or a multifunctional moiety that covalently attaches moiety A to G.

[0366] Preferably, G is capable of forming an amide, ester, carbamate, hydrazone, thiazolidine, methylene alkoxy carbamate, disulfide, alkylene, cycloalkylene, aralkylene, heteroaralkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, aralkenylene, heteroaralkenylene, heteroalkenylene, heterocycloalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, aminoalkylene, diacid, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylene alkoxy carbamate, disulfide, 1,2-vinylene, imine, imineamide, phosphoramide, sugar, phosphate, phosphoramide, carbamate, dipeptide, tripeptide or tetrapeptide linking group after reaction; and / or

[0367] Part B preferably has a structure as described in detail above in relation to the conjugates according to the invention.

[0368] The moiety G is preferably capable of forming an amide, ester, carbamate, hydrazone, thiazolidine, methylene alkoxy carbamate, disulfide, alkylene, cycloalkylene, aralkylene, heteroaralkylene, heteroalkylene, cycloheteroalkylene, alkenylene, cycloalkenylene, aralkenylene, heteroaralkylene, heteroalkenylene, heterocycloalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, aminoalkylene, diacid, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylene alkoxy carbamate, disulfide, 1,2-vinylene, imine, imineamide, phosphoramide, sugar, phosphate, phosphoramide, carbamate, dipeptide, tripeptide or tetrapeptide linking group after reaction. As will be appreciated by the skilled person, there are numerous possibilities how to provide a reactive group capable of reacting with a conjugation partner to form a linking group according to the preceding list, and they are all encompassed by the present disclosure.

[0369] Part B can be a cleavable or non-cleavable multifunctional part that can be used to connect one or more reactive parts and / or binding agent parts to form a conjugate precursor of the present invention. In some embodiments, the structure of the compound independently contains more than one A part per molecule, preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 parts A; and / or more than one part G, preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 parts G. Preferably, the structure of the compound contains 3 parts A and 1 part G per molecule; or 3 parts A and 2 parts G.

[0370] The moiety G is preferably selected from H, NH2, OH, N3, COOH, SH, Hal,

[0371]

[0372] 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 independently selected from carboxyl, alkyl, cycloalkyl, aryl and heteroaryl (wherein each of the foregoing is substituted or unsubstituted), halogen and cyano.

[0373] Other compounds

[0374] Also disclosed herein is a novel tetravalent organic ligand of fibroblast activation protein (FAP) suitable for therapeutic applications ("Tetra-ESV6") comprising four small binding moieties A as defined above.

[0375] The compounds according to the present invention can be represented by the following general formula IV,

[0376]

[0377] Its individual diastereomers, its hydrates, its solvates, its crystalline forms, its individual tautomers, or its pharmaceutically acceptable salts, wherein A is a binding moiety; B is a multifunctional moiety that contains a branch point and covalently links moiety A to C; and C is an atom, molecule or particle and is a therapeutic agent.

[0378] Alternatively, moiety C comprises a branch point such that four moieties A are attached to C, and B is absent, i.e. moiety C may also serve as a branch point, thus replacing B, preferably when C is a chelator. An exemplary structure is represented by general formula V:

[0379]

[0380] All exemplary structures, aspects, embodiments and definitions disclosed for the trivalent binding agents of Formulas I and III disclosed in this specification apply to Formulas IV and V with appropriate modifications.

[0381] For example, a tetravalent binding agent may be represented by Formula VI or VII, wherein all groups and variables are the same as those defined for Formula II or III:

[0382]

[0383] Exemplary structures of portion J' include:

[0384]

[0385] wherein all groups and variables are otherwise the same as defined herein.

[0386] The specific structure of part J' includes:

[0387]

[0388] wherein each i and j is an integer independently selected from 0, 1, 2, 3 and 4; preferably wherein each i is 1 or 2, and each j is 1, 2 or 3.

[0389] Preferred structures of J' include:

[0390]

[0391] Particularly preferred structures of fragment ((L)a)4J' include:

[0392]

[0393] Particularly preferred tetravalent compounds are shown in Table 17.

[0394] Methods of preparing therapeutic conjugates

[0395] In one aspect of the invention, disclosed herein is a method for preparing a therapeutic conjugate, the method comprising the step of conjugating a precursor compound as described above with a conjugation partner. Preferably, the precursor compound is conjugated to the conjugation partner by reacting with the conjugation partner to form a covalent bond. Preferably, the conjugate so obtained is a therapeutic conjugated compound as described elsewhere in this specification.

[0396] The conjugation partner may be an atom, a molecule or a particle which is a therapeutic agent and may correspond to the therapeutic payload moiety which has been described in detail above with respect to the conjugate according to the invention.

[0397] Preferably, the method also includes formulating the conjugate as a therapeutic pharmaceutical composition. The pharmaceutical composition can be used for human or animal therapy in human and animal medicine, and will generally include any one or more of a pharmaceutically acceptable diluent, carrier or excipient. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field and are described in, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edited 1985). The selection of carriers, excipients or diluents can be selected according to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may include any suitable one or more binding agents, one or more lubricants, one or more suspending agents, one or more coating agents, one or more solubilizing agents as or in addition to carriers, excipients or diluents. All formulation details and aspects disclosed above in the section "pharmaceutical composition" are also fully applicable to this.

[0398] General techniques

[0399] Unless otherwise indicated, the practice of the present invention employs conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology and pharmacology known to those skilled in the art. These techniques are fully explained 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: ALaboratory Manual, 2nd edition, Vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel, FM et al., eds. (1999) Short Protocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream et al., eds. (1998) 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.

[0400] Chemical synthesis

[0401] Compounds as described herein can be prepared by chemical synthesis techniques. It is obvious to those skilled in the art that during the synthesis of a compound, sensitive functional groups may need to be protected and deprotected. This can be achieved by conventional techniques, such as in TW Greene and P GM Wuts in "Protective Groups in Organic Synthesis", John Wiley and Sons Inc. (1991) and PJ Kocienski in "Protecting Groups", GeorgThieme Verlag (1994). It is possible that during some reactions, under certain conditions, epimerization may occur in any stereocenter present, such as if a base is used in the reaction of a substrate with an optical center comprising a base-sensitive group. As is well known in the art, potential problems should be avoided by selecting a reaction sequence, conditions, reagents, protection / deprotection schemes, etc.

[0402] definition

[0403] Antibodies. The term "antibody" is used in its broadest sense and encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), recombinant spliced ​​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 are capable of recognizing and binding to specific antigens. Target antigens typically have many binding sites, also known as epitopes, which are recognized by CDRs on a variety of antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, an antigen may have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules containing antigen binding sites that immunospecifically bind to a target target or a portion thereof. Antibodies can be of any type - such as IgG, IgE, IgM, IgD, and IgA - any class - such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 - or its subclass. The antibodies may be or may be derived from murine, human, rabbit or other species.

[0404] Antibody fragment. The term "antibody fragment" refers to a portion of a full-length antibody, typically its antigen-binding or variable region. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-domain antibodies, including dAbs, camelid V HHAntibodies and IgNAR antibodies of cartilaginous fish. Antibodies and their fragments can be replaced by binding molecules based on alternative non-immunoglobulin scaffolds, peptide aptamers, nucleic acid aptamers, structured polypeptides containing polypeptide loops located on non-peptide backbones, natural receptors or their domains.

[0405] Derivatives. Derivatives include chemical modifications of compounds. Examples of such modifications include replacement of hydrogen by halogen groups, alkyl groups, acyl groups, 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 interactions.

[0406] Analog: This term includes any enantiomers, racemates and stereoisomers, and all pharmaceutically acceptable salts and hydrates of these compounds.

[0407] Unless otherwise indicated, the following definitions apply to chemical terms relating to the compounds of the present invention and compositions containing such compounds.

[0408] Alkyl refers to a branched or unbranched saturated hydrocarbon group. Suitably, the alkyl group contains 1 to 100, preferably 3 to 30 carbon atoms, more preferably 5 to 25 carbon atoms. Preferably, the alkyl group refers to methyl, ethyl, propyl, butyl, pentyl or hexyl.

[0409] Alkenyl refers to a branched or unbranched hydrocarbon group containing one or more carbon-carbon double bonds. Suitably, the alkenyl group contains from 2 to 30 carbon atoms, preferably from 5 to about 25 carbon atoms.

[0410] Alkynyl refers to a branched or unbranched hydrocarbon 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.

[0411] Halogen refers to fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.

[0412] Cycloalkyl refers to an alicyclic moiety, suitably having 3, 4, 5, 6, 7 or 8 carbon atoms. The group may be a bridged or polycyclic ring system. More commonly cycloalkyl is monocyclic. The term includes reference to groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo[2.2.2]octyl, etc.

[0413] Aryl refers to an aromatic carbocyclic ring system suitably containing 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring carbon atoms. Aryl can 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, anthracenyl, and the like.

[0414] The prefix (hetero) herein indicates that one or more carbon atoms of the group may be substituted by nitrogen, oxygen, phosphorus, silicon or sulfur. Heteroalkyl includes, for example, alkoxy and alkylthio. The heterocycloalkyl or heteroaryl 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. In particular, 3-10 rings or ring systems, and more particularly 5 or 6 rings, may be saturated or unsaturated. For example, selected from oxirane, azirinyl, 1,2-oxathiolanyl, imidazolyl, thienyl, furanyl, tetrahydrofuranyl, pyranyl, thiopyranyl, thianthrenyl, isobenzofuranyl, benzofuranyl, chromenyl, 2H-pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyrimidinyl, piperidinyl, piperazinyl, pyridazinyl, morpholinyl, thiomorpholinyl, especially thiomorpholinyl, indolizinyl, 1,3-dioxo-1,3-dihydro-isoindolyl, 3H-indolyl, indolyl, benzimidazolyl, coumarin (c umaryl), indazolyl, triazolyl, tetrazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolyl, benzofuranyl, dibenzofuranyl, benzothiophenyl, dibenzothiophenyl, phthalazinyl, naphthyridinyl, quinoxalinyl, 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.

[0415] "Substituted" means that one or more, particularly up to 5, more particularly 1, 2 or 3 hydrogen atoms in the part are replaced by the corresponding number of substituents independently of each other. The term "optionally substituted" used herein includes substituted or unsubstituted. Of course, it should be understood that the substituents are only at their chemically possible positions, and those skilled in the art can determine whether a specific substitution is possible (experimentally or theoretically) without undue effort. For example, if combined with a carbon atom with an unsaturated (e.g., olefinic) bond, an amino or hydroxyl group with free hydrogen may be unstable. Preferably, the term "substituted" means that one or more hydrogen atoms in the part, particularly up to 5, more particularly 1, 2 or 3 are replaced by the corresponding number of substituents selected from OH, SH, NH2, halogen, cyano, carboxyl, alkyl, cycloalkyl, aryl and heteroaryl independently of each other. In addition, the substituents described herein themselves can be replaced by any substituent, subject to the above-mentioned restrictions on appropriate substitution recognized by the technician. Preferably, any of the above-mentioned substituents can be further replaced by any of the above-mentioned substituents, and each of the above-mentioned substituents can be further replaced by any of the above-mentioned substituents.

[0416] Substituents may suitably include halogen atoms and halomethyl groups, such as CF3 and CCl3; oxygen-containing groups, such as oxo, hydroxyl, carboxyl, carboxylalkyl, alkoxy, alkanoyl, alkanoyloxy, aryloxy, aroyl and aroyloxy; nitrogen-containing groups, such as amino, alkylamino, dialkylamino, cyano, azide and nitro; sulfur-containing groups, such as mercaptan, alkylthiol, sulfonyl and sulfoxide; heterocyclic groups, which themselves may be substituted; alkyl, which itself may be substituted; and aryl, which itself may be substituted, such as phenyl and substituted phenyl. Alkyl includes substituted and unsubstituted benzyl.

[0417] Where two or more moieties are described as each independently selected from a list of atoms or groups, this means that the moieties may be the same or different. Thus, the identity of each moiety is independent of the identity of one or more other moieties.

[0418] Example

[0419] 1. General Comments and Procedures

[0420] Unless otherwise stated, yields refer to chromatographically purified compounds.

[0421] Mass spectrometry (LC-ESI-MS) spectra were recorded on an Agilent 6100 Series Single Quadrupole MS system combined with an Agilent 1200 Series LC system using an InfinityLab Poroshell 120E C-C18 column, 4.6 mm × 56 mm, at 2 mL min-1 or using an InfinityLab Poroshell 120E C-C18 column, 2.7 μm, 4.6×50 mm, flow rate 0.8 mL / min, from 10% ACN to 100% ACN in 0.1% HCOOH aqueous solution in 3 or 10 min.

[0422] 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-Class system with a PDAUV detector, using an ACQUITY UPLC BEH C18 column, 1.7 μm, 2.1 mm × 50 mm, at 0.6 mL min -1 The reaction was performed with a linear gradient of solvents A and B (A = Millipore water containing 0.1% FA, B = MeCN containing 0.1% FA) at a flow rate of 2.5 Å.

[0423] Preparative reversed-phase high-pressure liquid chromatography (RP-HPLC) was performed on an Agilent 1200 series system using a Phenomenex 5μm N X-C18 semi-preparative column, 150mm×10mm, at 5mL min -1 The HPLC was performed with a linear gradient of solvents A and B (A = Millipore water containing 0.1% trifluoroacetic acid [TFA], B = MeCN containing 0.1% trifluoroacetic acid [TFA]) at a flow rate of 1.5 %; or on an Agilent 1200 series RP-HPLC with a PDA UV detector using a Synergi 10 μm, MAX-R A 10 x 250 mm C18 column was run at a flow rate of 5 mL / min with a linear gradient of solvents A and B (A = Millipore water with 0.1% TFA, B = ACN with 0.1% TFA).

[0424] 2. Synthesis of precursor and reference compounds

[0425]

[0426] Synthesis of ESV6-Succinic-COOH(P4) for therapeutic use

[0427] 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 minutes, 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 hour, washed with water, dried over Na2SO4, filtered and concentrated to give a brown crude product as a viscous oil. The residue was purified by flash chromatography (DCM / MeOH from 91:1 to 90:10) to give the pure product (24.8 mg, 68.9 μmol, 69% yield) as a brown oil. MS (ES+) m / z 360 (M+H) + .

[0428] 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 solution (0 °C) of P3 (26.8 mg, 70.0 μmol, 1.0 equiv) in DCM, followed by 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) + .

[0429]

[0430] Alternative Synthesis of ESV6-Succinic-COOH(P4) for Therapeutic Use

[0431] Step 1: 8-(4-(tert-Butoxy)-4-oxobutanamido)quinoline-4-carboxylic acid

[0432] At 0 ℃, SOCl2 (24 μ L, 0.33mmol, 1.2 equivalents) is slowly added to anhydrous THF (1.5mL) solution of 4-(tert-butoxy)-4-oxobutanoic acid (57mg, 0.33mmol, 1.2 equivalents). Then DMF (500 μ L) solution of 8-aminoquinoline-4-formic acid (50mg, 0.25mmol, 1 equivalent) is added, and the mixture is stirred at 0 ℃ for 30 minutes. The crude product is concentrated under reduced pressure and purified by reversed-phase MPLC (98:2 to 0:100AcCN / water+0.1% HCOOH, 45 minutes). Collect fractions and freeze-dry to obtain a white solid (50mg, 0.15mmol, 57%).

[0433] Step 2: (S)-tert-butyl 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 eq), (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride (33 mg, 0.15 mmol, 1 eq) and HATU (55 g, 0.15 mmol, 1 eq) were suspended in 2.0 mL of DMF. DIPEA (100 μL, 0.6 mmol, 4 eq) was added dropwise and the reaction was stirred for 15 minutes. The crude was diluted with DCM, washed with water, dried over anhydrous Na2SO4, filtered and the solvent evaporated under vacuum. 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%).

[0434] 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 eq) was dissolved in DCM (600 μL) and TFA (285 μL, 3.85 mmol, 50 eq) 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%).

[0435]

[0436] Synthesis of Bi-ESV6-COOH (P16) for therapeutic use. 2-Chlorotrityl resin (300 mg) was added to a solid phase synthesis syringe and then swollen in dried DCM for 15 minutes. Fmoc-l-Lys(Fmoc)-OH (89 mg, 0.15 mmol, 1 eq) and 4-methylmorpholine (45 μL, 0.40 mmol, 2.7 eq) were added to the resin in sequence and the mixture was reacted for 3 hours. Next, a capping step with methanol / 4-methylmorpholine / DCM (1:2:7 ratio, 5 mL, 30 min) was performed, followed by washing with DMF, and Fmoc was removed by a 20% piperidine solution in DMF (10 mL). The resin was then treated with a solution of ESV6-Succinic-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 hour. After multiple washes with DMF, the resin was cleaved with a 30% TFA solution in DCM (10 mL) for 1 hour. The cleaved solution was recovered, concentrated under vacuum and purified by RP chromatography (gradient: water / acetonitrile + 0.1% FA 98:2 to 0:100, 45 minutes). The fractions were collected and lyophilized to give Bi-ESV6-COOH (P16) (30 mg, 0.029 mmol, 19% yield) as a white solid. MS (ES+) m / z 1029.3 (M+H) +

[0437]

[0438] Synthesis of Bi-ESV6-DOTAGA for therapeutic use. 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 to a DMF solution (500 μL) of Bi-ESV6-COOH (P16, 12 mg, 0.012 mmol). After 30 minutes, an aqueous solution (500 μL) of DOTA-GA-NH2 (12 mg, 0.023 mmol, 2.0 equiv.) was added dropwise. The reaction mixture was stirred at room temperature for another 30 minutes and then analyzed by RP-HPLC (Agilent 1200 series system equipped with Synergi 4 μm 10×150 mm C18 column, using a gradient of 90:10 to 0:100 water / acetonitrile + 0.1% TFA, 12 minutes). The 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) + .

[0439]

[0440] Bi-ESV6-DOTAGA- for therapeutic use 175 Synthesis of Lu. Bi-ESV6-DOTAGA (4.0 mg, 2.6 μmol, 1 equivalent) was dissolved in acetate buffer, pH = 8 (300 μL). Subsequently, a solution of LuCl3 hexahydrate (2.0 mg, 5.2 μmol, 2 equivalents) dissolved in 0.05N HCl (1.50 mL) was added. The reactants were stirred at 90 ° C for 20 minutes and then cooled to room temperature. Purified by RP-HPLC (Agilent 1200 series system equipped with Synergi 4μm Polar-RP10×150mm C18 column, using a gradient of 90:10-50:50 water / acetonitrile + 0.1% TFA, 7 minutes). The desired fractions were collected and lyophilized to give a light yellow solid. (2.2 mg, 49%).

[0441]

[0442] 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 anhydrous 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 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 hours. The crude product was purified by reverse phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA 9.5:0.5 to 2:8, 20 minutes) and lyophilized to give a white solid (2.4 mg, 8%). MS (ES+) m / z 960.39 (M+H) +

[0443]

[0444] ESV6-DOTAGA- for therapeutic use 175 Synthesis of Lu. To a solution of ESV6-DOTAGA (0.96 mg, 1 μmol, 1 equivalent) in 300 μL acetate buffer (aqueous solution, 1 M, pH 8), a 0.05N HCl (1.5 mL) solution of freshly prepared LuCl3 hexahydrate (0.78 mg, 2 μmol, 2 equivalents) was added. The resulting mixture was stirred at 95 ° C for 10-15 minutes and then purified via RP-HPLC (90: 10 to 0: 100 ACN / water + 0.1% TFA, 12 minutes). The desired fractions were collected and lyophilized to give a white solid. (0.8 mg, 71%). MS (ESI+) m / z 11333.

[0445] 3. Chemical structure of Tri-ESV6-DOTAGA (6)

[0446]

[0447] -Molecular weight: 2246.23Da

[0448] - Exact mass: 2244.8877Da

[0449] Chemical formula: C101H122F6N26O27

[0450] -SMILES:

[0451] 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

[0452] 4. Chemical structure of Tri-ESV6-DOTA(7)

[0453]

[0454] Molecular weight: 2174.17Da

[0455] - Exact mass: 2177.87Da

[0456] Chemical formula: C98H118F6N26O25

[0457] -SMILES:

[0458] 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(CN4CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC4)=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

[0459] 5. Chemical structure of Tri-ESV6-PEG12-DOTAGA

[0460]

[0461] -Molecular weight: 4045.38Da

[0462] - Exact mass: 4042.94Da

[0463] -Chemical formula: C182H118F6N26O25

[0464] -SMILES:

[0465] O=C(NCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCC(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

[0466] 6. Synthesis of Tri-ESV6-DOTAGA and Tri-ESV6-DOTA

[0467] a) Synthesis route

[0468]

[0469] b) Experimental procedures for synthesis

[0470] Step 1) 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 (2)

[0471]

[0472] (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 eq), tert-butyl (2-aminoethyl)carbamate (42 mg, 261 μmol, 1.2 eq) and HATU (100 mg, 261 μmol, 1.2 eq) were suspended in 2.0 mL DMF. DIPEA (150 μL, 868 μmol, 4 eq) was added dropwise and the reaction was stirred for 15 minutes. 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 obtain a white solid (104 mg, 176 μmol, 80% yield).

[0473] Step 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)

[0474]

[0475] (S)-tert-butyl (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 30% v / v TFA in DCM and stirred at room temperature for 1 hour. The crude product was purified by CombiFlash Nextgen300+ (parameters: flow rate 30 mL / min, 24 gr C18 column, water / acetonitrile+0.1% formic acid 98:2 to 0:100, 30 minutes), and the collected fractions were lyophilized to obtain a white solid (66 mg, 133 μmol, 76% yield).

[0476] 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)

[0477]

[0478] (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 eq), 3,3'-((2-((tert-butoxycarbonyl)amino)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropanoic acid (11 mg, 26 μmol, 1 eq) and HATU (34 mg, 91 μmol, 3.5 eq) were suspended in 1.0 mL of DMF. DIPEA (40 μL, 260 μmol, 10 eq) was added to the mixture and the reaction was stirred at room temperature for 4 hours. The crude product was purified by CombiFlash Nextgen300+ (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 obtain a white solid (16 mg, 8.5 μmol, 32% yield).

[0479] Step 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-oxobutyramido)ethyl)amino)-3-oxopropoxy)methyl)-4,14-dioxo-7,11-dioxa-3,15-diazaheptadecan-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)

[0480]

[0481] 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, 16 mg, 8.5 μmol) was dissolved in 500 μL of 30% v / v TFA in DCM solution and stirred at room temperature for 1 hour. The crude product was purified via CombiFlash Nextgen300+ (parameters: flow rate 15 mL / min, 4 gr C18 column, water / acetonitrile+0.1% formic acid 98:2 to 0:100, 30 minutes), and the collected fractions were lyophilized to obtain a white solid (13 mg, 7.2 μmol, 85% yield).

[0482] 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)carbamoyl)quinolin-8-yl)amino)-4-oxobutyramido)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-D OTAGA)

[0483]

[0484] 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-oxobutyramido)ethyl)amino)-3-oxopropoxy)methyl)-4,14-dioxo-7,11-dioxa-3,15-diazaheptadecan-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, 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 m g, 36 μmol, 5 equivalents) and DMAP (5 mg, 36 μmol, 5 equivalents) were suspended in anhydrous DMF (500 μL). The resulting mixture was stirred at 50 ° C for 6 hours, then diluted with milliQ water (2 mL) and purified via RP-HPLC (linear gradient of ACN / water + 0.1% TFA from 90:10 to 0:100). The desired fractions were collected and lyophilized to give a white solid. (6 mg, 2.7 μmol, yield 38%).

[0485] 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)-4-oxobutanamido)ethyl)amino)-3-oxopropoxy)methyl)-2,9,14,17-tetraoxo-6-oxa-3,10,13-triazaheptadecanyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (7,Tri-ESV6-DOTA)

[0486]

[0487] 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-oxobutyramido)ethyl)amino)-3-oxopropoxy)methyl)-4,14-dioxo-7,11-dioxa-3,15-diazaheptadecan-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, 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 m g, 36 μmol, 5 equivalents) and DMAP (5 mg, 36 μmol, 5 equivalents) were suspended in anhydrous DMF (500 μL). The resulting mixture was stirred at 50 ° C for 6 hours, then diluted with milliQ water (2 mL) and purified via RP-HPLC (linear gradient of ACN / water + 0.1% TFA from 90:10 to 0:100). The desired fractions were collected and lyophilized to give a white solid. (6 mg, 2.7 μmol, yield 38%).

[0488] 7. Synthesis of Tri-ESV6-P EG12-DOTAGA

[0489] a) Synthesis route

[0490]

[0491] b) Synthesis Experimental Procedure

[0492] Step 1) (S)-2,5-dioxopyrrolidin-1-yl 4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4-oxobutanoate

[0493]

[0494] (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 eq), 1-hydroxypyrrolidine-2,5-dione (25 mg, 217 μmol, 1 eq) and HATU (100 mg, 261 μmol, 1.2 eq) were suspended in 1.0 mL DMF. DIPEA (150 μL, 868 μmol, 4 eq) was added dropwise and the reaction was stirred for 15 minutes. The crude product was purified by CombiFlash Nextgen300+ (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 obtain a white solid (90 mg, 162 μmol, 75% yield).

[0495] Step 2) (S)-4-((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-dodeca-40-azatetradecanoic acid

[0496]

[0497] (S)-2,5-dioxopyrrolidin-1-yl 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 DMF. DIPEA (200 μL) was added dropwise and the reaction was stirred for 15 minutes. The crude product was purified by CombiFlash Nextgen300+ (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 obtain a yellow oil (100 mg, 94 μmol, 79% yield).

[0498] Step 3) Tert-butyl (S)-(4,7-((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-dodecano-3,43-diazatetratetradecanoyl)carbamate

[0499]

[0500] (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-dodeca-40-azatetradecanoic acid (ESV6-PEG12-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 DMF. DIPEA (75 μL) was added dropwise and the reaction was stirred for 15 minutes. The crude product was purified via CombiFlash Nextgen300+ (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 obtain a yellow oil (95 mg, 79 μmol, 84% yield).

[0501] Step 4) (S)-N 1 -(42-amino-39-oxo-3,6,9,12,15,18,21,24,27,30,33,36-dodeca-40-azatetradecanoyl)-N4-(4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)succinamide

[0502]

[0503] (S)-tert-Butyl(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-dodecano-3,43-diazatetratetradecanoyl)carbamate (95 mg, 79 μmol) was dissolved in 2 mL of 50% v / v TFA in DCM and stirred at room temperature for 1 hour. The crude product was purified by CombiFlash Nextgen300+ (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 obtain a yellow oil (67 mg, 61 μmol, 64% yield).

[0504] Step 5) (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 tert-butyl ester of 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-hexadecanoic acid-5,45,48,60,63,103-hexaazahexadecanoic acid-54-yl)

[0505]

[0506] (S)-N 1 -(42-amino-39-oxo-3,6,9,12,15,18,21,24,27,30,33,36-dodecano-40-azatetradecanoyl)-N 4-(4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)succinamide (ESV6-PEG12-EtNH2, 60 mg, 54 μmol, 4.5 equiv), 3,3'-((2-((tert-butoxycarbonyl)amino)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropanoic acid (5.3 mg, 12 μmol, 1 equiv) and HATU (21 mg, 54 μmol, 4.5 equiv) were suspended in 0.5 mL DMF. DIPEA (40 μL, 260 μmol, 10 equiv) was added to the mixture and the reaction was stirred at room temperature for 4 hours. The crude product was purified via CombiFlash Nextgen300+ (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 obtain a yellow oil (35 mg, 9.4 μmol, 78% yield).

[0507] Step 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-trideca-6,9,49- triazatricosyl)-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-hexacosoxa-40,43,55,58-tetraazatricosyl)bis(N-(triazatricosyl)-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 4 -(4-((2-(( S )-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)succinamide)

[0508]

[0509] (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-trideca-6,9,49-triazapentazone Tert-butyl)-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-cosixaoxa-5,45,48,60,63,103-hexaazahexadecane-54-yl)carbamate (Tri-ESV6-PEG12-NHBoc, 30 mg, 8.1 μmol) was dissolved in 50% v / v TFA in DCM and stirred at room temperature for 1 hour. The crude product was purified via CombiFlash Nextgen300+ (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 obtain a white solid (15 mg, 4.2 μmol, 52% yield).

[0510] 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)quinolin-8-yl) (amino)-5,10,17,20-tetraoxo-2,13-dioxa-6,9,16-triazaeicosyl)-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)

[0511]

[0512] 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-trideca-6,9,49- triazapentacontriacontane-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-hexacosine-40,43,55,58-tetraazapentacontriacontane-1,97-diyl)bis(N-(triazapentacontriacontane)) 4 -(4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)succinamide)(Tri-ESV6-PEG12-NH2, 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. 5mg, 14μmol, 5 equivalents) and DMAP (2mg, 14μmol, 5 equivalents) were suspended in anhydrous DMF (500μL). The resulting mixture was stirred at 50°C for 6 hours, then diluted with milliQ water (2mL) and purified via RP-HPLC (linear gradient of ACN / water+0.1% TFA from 90:10 to 0:100). The desired fractions were collected and lyophilized to give a white solid. (7mg, 1.6μmol, 56% yield).

[0513] 8. Chemical structures and synthesis of multivalent ESV6-DOTAGA compounds

[0514] a) Chemical structure of Tetra-ESV6-DOTAGA (10)

[0515]

[0516] -Molecular weight: 3062.0652Da

[0517] - Exact mass: 3060.2291Da

[0518] -Chemical formula: C138H169F8N35O37

[0519] -SMILES:

[0520] OC(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(N6CC)C3C(NCC(N4CC(F)(F)C[C@H]4C#N)0O)O)C 3CCCC2)O)0O)O)COCCC(NCCNC(CCC(NC5)CCCC6C5NCCCC6 C(NCC(N7[C@H](C#N)CC(F)(F)C7)O)0)O)O)O)O)O)O)CO CCC(NCCNC(CCC(NC8)C(N0CC)C9C(NCC(N10CC(F)(F)C[C@H]1 0C#N)(O)(O)C9(CCC8)(O)(O)(O)(O)CCC(NC11)CCCCC12C N11N6CCC12C(NCC(N13[C@H](C#N)CC(F)(F)C13)0O)0O)0O

[0521] b) Chemical structure of Hexa-ESV6-DOTAGA

[0522]

[0523] -City:4233.2028Da

[0524] -Database:4230.6586Da

[0525] -Specifier: C192H227F12N49O4

[0526] -SMILES:

[0527] OC(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(N6CC)C3C(NCC(N4CC(F)(F)C[C@H]4C#N)O )(O)C3(CCC2)(O)(O)(O)(COCCC(NCCNC(CCC(NC5)C(N)CC)C6C(NCC(N7CC(F)(F)C[C@H]7 C#N)O)O)C6CCCC5)O)0O)O)COCCC(NCCNC(CCC(NC8)CCCCC9C8NCCCC9C(NCC(N10[C @H](C#N)CC(F)(F)C(10)(O)(O)(O)(O)(O)(O)(COCCC(NCCNC(CCC(NC11)C(N6CC)C1 2C(NCC(N13CC(F)(F)C[C@H]13C#N)0O)0)C120CCCC11)0)0O)0)COCCC(NCCNC(CCC( NC14C(N0CC)C15C(NCC(N16CC(F)(F)C[C@H]16C#N)(O)(O)C15(CC(C14)(O)O)O) NO)CCC(NCC170CCCCC18C17N6CCC18C(NCC(N19[C@H](C#N)CC(F)(F)C19)SO)O)0O

[0528] c) Chemical structure of Octa-ESV6-DOTAGA

[0529]

[0530] -India:5864.8685Da

[0531] -Picture Number:5861.3414Da

[0532] - Part Number: C266H321F16N67O69

[0533] -SMILES:

[0534] O=C(NCCNC(CCOCC(NC(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(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)COCCC(NC(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)COCCC(NCCNC(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)=O)=O)=O)=O)=O)COCCC(NC(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)COCCC(NCCNC(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)=O)=O)=O)=O)COCCC(NCCNC(CCC(NC%20=C(N=CC=C%21C(NCC(N%22CC(F)(F)C[C@H]%22C#N)=O)=O)C%21=CC=C%20)=O)=O)=O)=O)CCC(NC%23=CC=CC%24=C%23N=CC=C%24C(NCC(N%25[C@H](C#N)CC(F)(F)C%25)=O)=O)=O

[0535] d) Synthetic routes for the synthesis of 4-ESV6-DOTAGA and Octa-ESV6-DOTAGA

[0536]

[0537]

[0538] 9. In vitro testing and assays

[0539] a) Inhibition assay

[0540] The enzymatic activity of hFAP on the Z-Gly-Pro-AMC substrate was measured on a microtiter plate reader at room temperature, 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 inhibitors, which were serially diluted from 167 nM to 80 fM in a 1:2 ratio in a total volume of 20 μL. The experiment was performed in triplicate and the average fluorescence value was fitted using Prism 7. This value is defined as the inhibitor concentration required to reduce enzyme activity by 50% after the addition of substrate [ Figure 1 and Figure 8 ].

[0541] According to the same procedure, the compounds Tri-ESV6-ValCit-MMAE (14), Tri-ESV6-linker-DOTAGA (13) and diastereomers of Tri-ESV6-DOTAGA (11, 12) [ Fig.18 ] Other comparative inhibition assays with trimeric binders were performed, particularly with different lengths of linker groups B and / or different payload groups C. Comparative hFAP inhibition assays with tetravalent binders 10 and 29 were also performed [ Fig. 20 ].

[0542] b) Fluorescence polarization measurement

[0543] Fluorescence polarization experiments were performed in 384-well plates (non-binding, ps, f-bottom, black, high capacity, 30 μL final volume). The stock solution of hFAP was serially diluted (1:2) from buffer (50 mM Tris, 100 mM NaCl and 1 mM EDTA, pH = 7.4), while the final concentration of the binder was kept constant at 1 nM. Fluorescence anisotropy was measured on a Tecan microtiter plate reader. The experiment was performed in triplicate and the average anisotropy value was 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 the fluorescent binder). Data are available at Figure 2 Report in.

[0544] c) Cell effluent

[0545] SK-RC-5hFAP cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic. Cells were seeded in 24-well plates (200'000 cells / well, 0.5 mL / well) and incubated overnight at 37°C, 5% CO2. The medium was then removed, the cells were washed with PBS (2×0.5 mL), and the cells were incubated with 177 Lu-ESV6, 177 Lu-Bi-ESV6, 177 Lu-Tri-ESV6 or 177 Lu-FAP-2286 (10 KBq, 1 pmol, 0.5 mL) solution was incubated. After 1 hour, the medium was removed, the cells were washed with PBS (2×0.5 mL), and incubated with fresh RPMI medium without supplements. A portion of the medium was measured at different time points using a gamma counter (Packard Cobra). The percentage of compound binding was calculated as a fraction of the total activity and was calculated according to 177 Lu attenuation is corrected, such as Figure 3 shown.

[0546] d)LogD 7.4 Determination of

[0547] 177 Lu-ESV6, 177 Lu-Bi-ESV6, 177 Lu-Tri-ESV6 and 177 The lipophilicity of Lu-FAP-2286 was determined as follows. A 100 μL aliquot of the radioligand (~1 MBq) in PBS buffer was added to PBS buffer (500 μL, pH 7.4) and 1-octanol (600 μL). The two-layer mixture was shaken vigorously on a vortex mixer for 10 minutes and then centrifuged at 700 rpm for 5 minutes to facilitate separation. A 100 μL aliquot of the two layers was measured in a Packard Cobra gamma counter, and the partition coefficient was determined by dividing cpm (octanol) by cpm (PBS) and expressed as Log D 7.4 .

[0548] LogD 7.4 ( 177 Lu-ESV6): -4.13

[0549] LogD 7.4 ( 177 Lu-Bi-ESV6): -3.75

[0550] LogD 7.4 ( 177Lu-Tri-ESV6): -3.10

[0551] LogD 7.4 ( 177 Lu-FAP-2286): -3.05

[0552] 10. Animal research

[0553] All animal experiments were performed under license number ZH006 / 2021 issued by the Veterinary Office of the Canton of Zurich (Veterinarmt des Kanton Zürich) and in compliance with Swiss animal welfare laws and regulations.

[0554] SK-RC-52.hFAP was grown to 80% confluence in RPMI-1640 containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic and detached by 0.05% trypsin-EDTA (ethylenediaminetetraacetic acid). Tumor cells were resuspended in Hanks balanced salt solution medium. Aliquots of 5 million cells (100 μL suspension) were injected subcutaneously in the right flank of female athymic Balb / cAnNRj-Foxn1 mice (6-8 weeks of age).

[0555] a) Use 177 Lu radiolabeling

[0556] ESV6-DOTAGA, Bi-ESV6-DOTAGA, Tri-ESV6-DOTAGA, Tetra-ESV6-DOTAGA, Hexa-ESV6-DOTAGA, Octa-ESV6-DOTAGA and FAP-2286 were radiolabeled with Lutetium-177 (a therapeutic radionuclide) with different specific activities for three different studies (biodistribution, therapeutic and dose optimization).

[0557] 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 aqueous solution, pH = 4.5). 20 MBq of 177 Lu solution, and the mixture was heated at 90°C for 10 min, then diluted with 1600 μL of PBS to achieve a final volume of 2 mL (20 doses, 100 μL each).

[0558] Prior to treatment studies, the precursor (25 nmol) was dissolved in 25 μL of milliQ water, followed by the addition of sodium acetate buffer (75 μL, 1 M aqueous solution) and 25, 75, or 150 MBq of 177Lu solution. The mixture was heated at 90°C for 1 min and then diluted with 400 μL of PBS to provide a final volume of 500 μL (5 doses of 100 μL each). Quality control of the radiosynthesis was performed using radio-HPLC.

[0559] Prior to dose optimization studies for radiotherapy applications, the precursors (0,24 to 180 nmol) were dissolved in milliQ water and diluted with sodium acetate (1 M aqueous solution, pH = 4.5). 177 Lu solution (4 MBq), and the mixture was heated at 90°C for 10 min and then diluted with PBS to obtain a final volume of 400 μL (4 doses, 100 μL each).

[0560] Prior to the biodistribution studies at different specific activities ( Fig.19 ), the precursor (20 nmol) was dissolved in milliQ water and diluted with sodium acetate (1 M aqueous solution, pH = 4.5). 177 Lu solution (4 or 96 MBq), and the mixture was heated at 90°C for 10 min and then diluted with PBS to obtain a final volume of 400 μL (4 doses of 100 μL each, corresponding to 0.2 MBq / nmol and 4.8 MBq / nmol, respectively).

[0561] b) Quantitative biodistribution analysis in tumor-bearing mice

[0562] ESV6-DOTAGA, Bi-ESV6-DOTAGA, Tri-ESV6-DOTAGA, Tetra-ESV6-DOTAGA, Hexa-ESV6-D OTAGA, Octa-ESV6-DOTAGA and FAP-2286 were used as described above. 177 Lu radiolabeled. Allow tumors to grow to 300 mm 3 The mice were randomly assigned and injected i.v. 177 Lu-ESV6, 177 Lu-Bi-ESV6, 177 Lu-Tri-ESV6, Tetra-ESV6-DOTAGA, Hexa-ESV6-DOTAGA, Octa-ESV6-DOTAGA or 177 Radiolabeled preparation of Lu-FAP-2286 (250 nmol / kg; 50 MBq / kg). Mice were euthanized at different time points after intravenous CO2 asphyxiation. Tumors, organs, and blood were collected, weighed, and radioactivity was measured using a Packard Cobra gamma counter. Values ​​are expressed as percent ID / g ± SD [ Figure 4]. The %ID / g in tumors was corrected for SK-RC-52.hFAP tumor growth rate.

[0563] Fig.19 The 177 Lu Tri-ESV6-DOTAGA was carried out at different specific activities: low (0.2MBq / nmol) and high (4.8MBq / nmol). 177 Results of Lu biodistribution studies.

[0564] Values ​​(mean ± standard deviation):

[0565] Tables 1 to 4 and 10 to 12 show the results of intravenous administration (250 nmol / kg, 50 MBq / kg) in mice bearing SK-RC-52.hFAP tumors. 177 Lu-ESV6-DOTAGA, 177 Lu-Bi-ESV6-DOTAGA, 177 Lu-Tri-ESV6-DOTAGA, 177 Quantitative in vivo biodistribution of Lu-FAP-2286, Tetra-ESV6-DOTAGA, Hexa-ESV6-DOTAGA, and Octa-ESV6-DOTAGA at different time points. Data are reported as %ID / g ± standard deviation.

[0566] Tumor to Organ Ratio:

[0567] Tables 5 to 8 and 13 to 15 show the results of intravenous administration (250 nmol / kg, 50 MBq / kg) in mice bearing SK-RC-52.hFAP tumors. 177 Lu-ESV6-DOTAGA, 177 Lu-Bi-ESV6-DOTAGA, 177 Lu-Tri-ESV6-DOTAGA, 177 Tumor to organ ratios at different time points for Lu-FAP-2286, Tetra-ESV6-DOTAGA, Hexa-ESV6-DOTAGA, and Octa-ESV6-DOTAGA. Avg. = average; Std.d = standard deviation. Particularly favorable results for Tri-ESV6-DOTAGA (e.g., kidney / tumor ratio) are reported in Table 7.

[0568] c) Evaluation of therapeutic efficacy in tumor-bearing mice

[0569] 177 Lu-ESV6, 177 Lu-Bi-ESV6 and 177The anticancer efficacy of Lu-Tri-ESV6 was evaluated in athymic Balb / c AnNRj-Foxn1 mice bearing SK-RC-52.hFAP tumors in the right flank. Lu-Tri-ESV6 was administered intravenously at a dose of 250 nmol / kg, 250 MBq / kg 177 Lu-ESV6, 177 Lu-Bi-ESV6 or 177 Lu-Tri-ESV6 (single administration on day 9 after tumor implantation). When the average volume of established tumors reached 100-150 mm 3 The treatment experiment started at 14:00 p.m. The body weight and tumor volume of the animals were measured and recorded daily. Tumor size was measured by electronic calipers, and tumor volume was calculated by the formula (long side, mm) × (short side, mm) × (short side, mm) × 0.5. When one or more termination criteria indicated by the experimental license were reached, the animals were euthanized. Prism 7 software (GraphPad Software) was used for data analysis. The results are shown in Figure 5 , Figure 6 and Figure 7 Shown in.

[0570] d) Dose escalation in radiotherapy

[0571] Female athymic Balb / c AnNRj-Foxn1 mice bearing subcutaneous SK-RC-52.hFAP tumors were injected intravenously with eight different doses (ranging from 3 nmol / kg to 2250 nmol / kg) of 177 Lu-Tri-E SV6-DOTAGA. After 24 hours, the mice were sacrificed and the tumors and healthy organs were collected and measured using a gamma counter. The results reported as %ID / g are shown in Fig.15 Results reported as tumor-to-organ ratios are shown in A. Fig.15 B. Since the best tumor-to-organ ratios were obtained at high doses ranging from 90 to 250 nmol / kg, we anticipate that the ideal dose for human patients for radiotherapy applications would be 1 to 3 mg / patient. e) Evaluation of therapeutic efficacy in tumor-bearing mice in dose escalation and in combination with L19-IL2

[0572] L19-IL2 as a single agent (3x0.05 mg / mouse), different doses of 177 Lu-Tri-ESV6 (5MBq / mouse, 15MBq / mouse, 30MBq / mouse), 177Lu-Tri-ESV6+L19-IL2 combination (5MBq / mouse 177The anticancer efficacy of Lu-Tri-ESV6 followed by three injections of 0.05 mg / mouse L19-IL2) or saline was evaluated in athymic Balb / cAnNRj-Foxn1 mice bearing SK-RC-52.hFAP tumors in the right flank. When the mean volume of established tumors reached 100-150 mm 3 The treatment experiment started at 14:00 p.m. The body weight and tumor volume of the animals were measured and recorded daily. Tumor size was measured by electronic calipers, and tumor volume was calculated by the formula (long side, mm) × (short side, mm) × (short side, mm) × 0.5. Animals were euthanized when one or more termination criteria indicated by the experimental license were reached. Prism 7 software (GraphPad Software) was used for data analysis. The results are shown in Fig. 9 In. Application 177 All mice in the Lu-Tri-ESV6+L19-IL2 combination were cured after treatment.

[0573] The L19-IL2 immunocytokine conjugate used in this example comprises a human IL2 polypeptide having a sequence of SEQ ID NO: 21 fused at its N-terminus via a 17 amino acid linker (SEQ ID NO: 23) 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 antibody L19, which specifically binds to the extra domain B (ED-B) of fibronectin, as described in WO2001 / 062298. The bound epitope is the sequence of SEQ ID NO: 40 (see Fattorusso et al., Structure 7: 381-390 in Figure 4 C). In the immunocytokines used in this example, the pairing of the VH domain of one L19-IL2 molecule with the VL domain of another L19-IL2 molecule allows the formation of homodimers via the scFv portion of the immunocytokine, essentially in the form of "scFv2" or "diabody", e.g. Fig.12 shown.

[0574] The amino acid sequence of the L19-IL2 immunocytokine conjugate is listed in SEQ ID NO 12:

[0575] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGDGSSGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSG SGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKEFSSSSSGSSSSGSSSSGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT.

[0576] The results show 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 the combination involves the activation of NK cells in the tumor, which enhances the anti-tumor efficacy.

[0577] f) Evaluation of therapeutic efficacy in tumor-bearing mice (planned optimization of combination with L19-IL2)

[0578] Anticancer efficacy was evaluated in athymic Balb / c AnNRj-Foxn1 mice bearing SK-RC-52.hFAP tumors in the right flank. When the mean volume of established tumors reached 100-150 mm 3 The treatment experiment was started at 14:00 pm. The body weight and tumor volume of the animals were measured and recorded daily. Tumor size was measured by electronic calipers, and tumor volume was calculated by the formula (long side, mm) × (short side, mm) × (short side, mm) × 0.5. Animals were euthanized when one or more termination criteria indicated by the experimental license were reached. Prism7 software (GraphPad Software) was used for data analysis. 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) 177 Lu-Tri-ESV6, (iii) 177 Lu-Tri-ESV6+L19-IL2 combination (at a dose of 5 MBq / mouse on day 7) 177Lu-Tri-ESV6, followed by three administrations of L19-IL2-Plan 1- at a dose of 0.05 mg / mouse on days 8, 10, and 12, or three administrations of L19-IL2-Plan 2- at a dose of 0.05 mg / mouse on days 12, 17, and 22, or a single administration of L19-IL2-Plan 3- at a dose of 0.05 mg / mouse on day 8, or a single administration of L19-IL2-Plan 4- at a dose of 0.05 mg / mouse on day 12, or (iv) saline or (iv) saline. The results are shown in Fig.13 middle.

[0579] g) Quantitative MMAE release from different therapeutic conjugates in vivo

[0580] The ESV6 binding moiety in monomeric, dimer or trimer form is conjugated to the same linker-payload structure based on a glycine-proline linker and an MMAE cytotoxic moiety. Three conjugates were compared in a tumor mouse model: (i) ESV6-GlyPro-MMAE-corresponding to conjugate 58a of EP3891138B1, (ii) Bi-ESV6-GlyPro-MMAE-corresponding to conjugate 11 of WO2022 / 171811A1, and (iii) Tri-ESV6-GlyPro-MMAE-corresponding to conjugate 9 of the present invention to study the efficacy of the MAE cytotoxic moiety released into the tumor.

[0581] HT-1080.hFAP tumors were implanted into the right flank of athymic Balb / c AnNRj-Foxn1 mice and allowed to grow to approximately 200 mm 3The average volume of . Mice were injected with 250nmol / kg doses of three conjugates and sacrificed at different time points after administration. Fresh blood was collected in heparin lithium tubes (BD microcontainer LH tubes), vortexed, and centrifuged (15,000g, 15 minutes). Plasma was frozen and stored at -80°C. Healthy organs and tumors were subsequently removed, frozen with dry ice, and stored at -80°C. After thawing of frozen plasma (50μL) and mouse tissue (~50mg), 500μL of PBS was added. The samples were stored on ice and 50μL of internal standard solution (d8-MMAE, 50nM) was added. The samples were then homogenized at 4°C for 2 minutes at 30Hz with a tissue grinder (tissue lyser) for 4 cycles. After homogenization, the samples were centrifuged (21'000g, 10 minutes). Subsequently, 100μL of supernatant was collected and added to 900μL of acetonitrile (ACN) to induce protein precipitation. After centrifugation (21'000g, 10 minutes), 800 μL of supernatant was collected and dried in a vacuum centrifuge at room temperature. The precipitate 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 x 2.1 mm, 1.9 μm particle size, pore size), the column temperature was set at 50°C and the flow rate was 700 μL / min, the gradient program was 95% A (water + 0.1% HCOOH), 5% B (ACN + 0.1% HCOOH) to 35% A in 2.5 minutes, 35% A to 5% A in 0.4 minutes, 5% A was maintained for 1.3 minutes, and then re-adjusted at 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; Aux gas 10 units; capillary temperature of 380°C; Aux gas temperature 450°C; S-lens RF level 60. The mass spectrometer was operated in targeted single ion monitoring mode (t-SIM) after 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 of 5×104; maximum injection time of 200 ms; separation window 14 m / z; separation offset 5 m / z. The peak areas of the analyte and the internal standard were integrated and the corresponding ratios were calculated. The ratios were then converted to pmol / g wet tissue using a single concentration external calibration point and corrected by the total weight of the sample analyzed. The percentage of injected dose / gram (%ID / g) was finally calculated by normalizing the values ​​based on the total dose injected into the mice. Data analysis was performed using Skyline v22.2.0.351. The results are shown in Fig.14 middle.

[0582] h) ESV6-GlyPro-MMAE and Tri-ESV6-GlyPro-MMAE in mice bearing HT-1080.hFAP tumors Treatment efficacy evaluation in (plan optimization)

[0583] The anticancer efficacy of (i) ESV6-GlyPro-MMAE (50 or 125 nmol / kg) - conjugate 58a corresponding to EP3891138B1 and (ii) Tri-ESV6-GlyPro-MMAE (50 or 125 nmol / kg) according to the present invention was evaluated in athymic Balb / c AnNRj-Foxn1 mice bearing HT-1080.hFAP in the right flank. When the average volume of established tumors reached 80-100 mm 3 The treatment experiment started at 14:00 p.m. The body weight and tumor volume of the animals were measured and recorded daily. Tumor size was measured by electronic calipers, and tumor volume was calculated by the formula (long side, mm) × (short side, mm) × (short side, mm) × 0.5. Animals were euthanized when one or more termination criteria indicated by the experimental license were reached. Prism 7 software (GraphPad Software) was used for data analysis. The results are shown in Fig.16 and Fig.17 Shown in.

[0584] 11. Chemical Structure and Synthesis of Tri-ESV6-DOTAGA Compounds with PEG Spacer

[0585] a) Chemical structure of Tri-ESV6-PEG2-DOTAGA

[0586]

[0587] -Molecular weight: 2723.7874Da

[0588] - Exact mass: 2722.1563Da

[0589] -Chemical formula: C122H161F6N29O36

[0590] -SMILES:

[0591] O(C(NC(COCCC(NCCNC(CCOCCOCCNC(CCC(NC1)C(N)CCC2C(NCC(N3CC(F)(F)C[C@H]3C#N) / O)+O)C2(CCC1)O)0 O)O)O)(COCCC(NCCNC(CCOCCOCCNC(CCC(NC4)C(N6CCC5C(NCC(N6CC(F)(F)C[C@H]6C#N)O)O)C5CCCC4)O )O)(O)O)COCCC(NCCNC(CCOCCOCCNC(CCC(NC7)(N)CCC8C(NCC(N9CC(F)(F)C[C@H]9C#N)O)O)C8CCCC7)D O)(O)(O)(O)CCC(N10CCN(CC(O)(O)CCN(CC(O)(O)CCN(CC(O)(O)CC10)C(O)Ob)Tri-ESV6-PEG4-DOTAGA is a strong solvent

[0592]

[0593] -Spring:2988.1054Da

[0594] -Picture Number:2986.3136Da

[0595] -Synthesis: C134H185F6N29O4

[0596] -SMILES:

[0597] OC(NC(COCCC(NCCNC(CCOCCOCCOCCOCCNC(CCC(NC1)C(N6CCC2C(NCC(N3CC(F)(F)C[C@H]3C#N)O)O)C2CCC NC1)0O)0O)0O)O)(COCCC(NCCNC(CCOCCOCCOCCOCCNC(CCC(NC4)C(N0CC)C5C(NCC(N6CC(F)(F)C[C@H]6C#N)6 O)O)C5(CCC4)O)0)O)0)O)COCCC(NCCNC(CCOCCOCCOCCOCCNC(CCC(NC7)(N)CCC8C(NCC(N9CC(F)(F)C[C @H]9C#N)0O)0)C80CCC7)0O)0)0)0)CCC(N10CCN(CC(O)O)CCN(CC(O)O)CCN(CC(O)O)CC10)C(O)O

[0598] c) Chemical structure of Tri-ESV6-PEG6-DOTAGA

[0599]

[0600] -India: 3252.4234Da

[0601] -Specification number:3250.4709Da

[0602] -Synthesis: C146H209F6N29O4

[0603] -SMILES:

[0604] OC(NC(COCCC(NCCNC(CCOCCOCCOCCCOCCCOCCCNC(CCC(NC1)C(N6CCC2C(NCC(N3CC(F)(F)C[C@H]3C#N)O)O)C2C CC(C1)(O)(O)(O)(O)(COCCC(NCCNC(CCOCCOCCOCCOCCOCCOCCNC(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(CCOCCOCCOCCCOCCCOCCCNC(CCC(NC7)C(NCCC)C8C(NCC(N9CC(F)( F)C[C@H]9C#N)(O)(O)C8(CCC7)(O)(O)(O)O)CCC(N510CCN(CC(O)O)CCN(CC(O)O)CCN(CC(O)O)CCC10)C(O)O

[0605] d) Chemical structure of Tri-ESV6-PEG8-DOTAGA

[0606]

[0607] -Spring: 3516.7414Da

[0608] -Database:3514.6282Da

[0609] -Synthesis: C158H233F6N29O54

[0610] -SMILES:

[0611] O=C(NC(COCCC(NCCNC(CCOCCOCCOCCOCCOCCOCCOCCOCCNC(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(CCOCCOCCOCCOCCOCCOCCOCCOCCNC(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(CCOCCOCCOCCOCCOCCOCCOCCOCCNC(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

[0612] e) used to synthesize TriOncoFAP-PEG2-DOTAGA, TriOncoFAP-PEG4-DOTAGA, TriOncoFAP- Synthetic routes of PEG6-DOTAGA and TriOncoFAP-PEG8-DOTAGA

[0613]

[0614] The enzymatic activity of hFAP on the Z-Gly-Pro-AMC substrate was measured on a microtiter plate reader at room temperature, 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 inhibitor, which was serially diluted from 167 nM to 80 fM in a 1:2 ratio in a total volume of 20 μL. The experiment was performed in triplicate and the average fluorescence value was fitted using Prism 7. This value is defined as the inhibitor concentration required to reduce the enzyme activity by 50% after the addition of the substrate. The results are shown in Fig.10 It is shown in Table 16. Surprisingly, it was found that the compound with the shortest distance between the targeting moiety and the branch point (PEG unit = 0) had the lowest IC50 [Table 16].

[0615] 11. Chemical structures and syntheses of compounds with alternative linkers

[0616] a) Chemical structure of ESV6-L-DOTAGA

[0617]

[0618] -Molecular weight: 1075.0948Da

[0619] - Exact mass: 1074.4582Da

[0620] -Chemical formula: C47H64F2N12O15

[0621] -SMILES:

[0622] O=C(NCCNC(CCOCCNC(CCC(N1CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC1)C(O)=O)=O)=O)CCC(NC2=CC=CC3=C2N=CC=C3C(NCC(N4[C@H](C#N)CC(F)(F)C4)=O)=O)=O

[0623] b) Chemical structure of Bi-ESV6-L-DOTAGA

[0624]

[0625] -Molecular weight: 1660.6636Da

[0626] - Exact mass: 1659.6730Da

[0627] -Chemical formula: C74H93F4N19O21

[0628] -SMILES:

[0629] O=C(NCCNC(CCOCC(NC(CCC(N1CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC1)C(O)=O)=O)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)=O)CCC(NC5=CC=CC6=C5N=CC=C6C(NCC(N7[C@H](C#N)CC(F)(F)C7)=O)=O)=O

[0630] c) Synthetic route for synthesizing ESV6-L-DOTAGA and Bi-ESV6-L-DOTAGA

[0631]

[0632] 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 on the Z-Gly-Pro-AMC substrate was measured on a microtiter plate reader at room temperature, 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 inhibitor, which was serially diluted from 167 nM to 80 fM in a ratio of 1:2 in a total volume of 20 μL. The experiment was performed in triplicate and the average fluorescence value was fitted using Prism 7. This value is defined as the inhibitor concentration required to reduce the enzyme activity by 50% after the addition of the substrate. The results are shown in Fig.11 It is shown in Figure 2. Until Tetra-ESV6-DOTAGA (quadrivalent compound), the inhibitory activity was proportional to the valence. Surprisingly, despite their superior valence, Hexa-ESV6-DOTAGA (hexavalent compound) and Octa-ESV6-DOTAGA (octavalent compound) lost more than 10'000-fold activity compared to the tetravalent compound. Compounds with alternative linkers (ESV6-L-DOTAGA and Bi-ESV6-DOTAGA) presented similar results to the original derivatives ( Figure 1 ESV6-DOTAGA and Bi-ESV6-DOTAGA in the study had comparable IC50.

[0633] 13. Additional compounds

[0634] a) Chemical structures of alternative forms of Tri-ESV6-DOTAGA (8)

[0635]

[0636] -Molecular weight: 2071.0484Da

[0637] - Exact mass: 2069.8032Da

[0638] Chemical formula: C94H109F6N25O23

[0639] -SMILES:

[0640] 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

[0641] b) Alternative Synthetic Route for Synthesis of TriOncoFAP-DOTAGA (8)

[0642]

[0643] c) Chemical structure of Tri-ESV6-GlyPro-MMAE (9)

[0644]

[0645] -Molecular weight: 3563.8404Da

[0646] - Exact mass: 3561.5603Da

[0647] -Chemical formula: C167H218F6N36O43S

[0648] -SMILES:

[0649] 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(NCC(N%11[C@H](C(NC%12=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%13[C@H]([C@H](OC)[C@@H](C)C(N[C@H](C)[C@@H](O)C%14=CC=CC=C%14)=O)CCC%13)=O)C)=O)=O)=O)C=C%12)=O)CCC%11)=O)=O)=O

[0650] Chemical structure of (S,S)-Tri-ESV6-DOTAGA(11)

[0651]

[0652] - Molecular weight: 2246.2324 Da

[0653] - Exact mass: 2244.8877 Da

[0654] - Chemical formula: C101H122F6N26O27

[0655] - SMILES:

[0656] 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

[0657] ( S Chemical structure of (R)-Tri-ESV6-DOTAGA(12)

[0658]

[0659] -Molecular weight: 2246.2324Da

[0660] - Exact mass: 2244.8877Da

[0661] -Chemical formula: C101H122F6N26O27

[0662] -SMILES:

[0663] 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

[0664] In all experiments described herein, Tri-ESV6-DOTAGA was used as its (S,R)-diastereomer unless otherwise stated.

[0665] Chemical structure of Tri-ESV6-linker-DOTAGA (13)

[0666]

[0667] -Molecular weight: 2581.6294Da

[0668] - Exact mass: 2580.0821Da

[0669] -Chemical formula: C116H151F6N27O34

[0670] -SMILES:

[0671] O=C(NCCNC(CCOCC(COCCC(NCCNC(CCC(NC1=C(N=CC=C2C(NCC(N3CC(F)(F)C[C@H]3C#N)=O)=O)C2=C C=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

[0672] Chemical structure of Tri-ESV6-ValCit-MMAE(14)

[0673]

[0674] -Molecular weight: 3665.9774Da

[0675] - Exact mass: 3663.6397Da

[0676] -Chemical formula: C171H228F6N38O44S

[0677] -SMILES:

[0678] 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

[0679] 14. Preferred conjugates

[0680] For ease of reference, the numbers and structures of some of the compounds of the present invention are summarized in Table 9 and Table 17 below.

[0681] Table 1. Injection dose / g

[0682]

[0683] Table 2. Injection dose / g

[0684]

[0685] Table 3. Injection dose / g

[0686]

[0687] Table 4. Injection dose / g

[0688]

[0689] Table 5. Tumor to organ ratio

[0690]

[0691] Table 6. Tumor to organ ratio

[0692]

[0693] Table 7. Tumor to organ ratio

[0694]

[0695] Table 8. (Tumor to organ ratio)

[0696]

[0697] Table 9.

[0698]

[0699]

[0700]

[0701]

[0702]

[0703]

[0704]

[0705] Table 10. Injection dose / g

[0706]

[0707] Table 11. Injection dose / g

[0708]

[0709] Table 12. Injection dose / g

[0710]

[0711] Table 13. Injection dose / g

[0712]

[0713] Table 14. Tumor to organ ratio

[0714]

[0715] Table 15. (Tumor to Organ Ratio)

[0716]

[0717] Table 16.

[0718]

[0719] Table 17.

[0720]

[0721]

[0722]

[0723]

[0724]

Claims

1. A compound, a single diastereomer thereof, a hydrate thereof, a solvate thereof, a crystalline form thereof, a single tautomer thereof, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following formula I: Each part A is independently represented by the following structure: B is a multifunctional moiety comprising a branch point and covalently links moiety A to moiety C; or wherein B is absent and C comprises a branch point such that three moieties A are attached to C; And C is an atom, molecule or particle and is a therapeutic agent.

2. The compound according to claim 1, represented by the following formula II or III: Among them, L, B S and B L is independently at each occurrence a moiety comprising or consisting of a structural unit selected from the group consisting of alkylene, cycloalkylene, aralkylene, heteroaralkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, aralkenylene, heteroaralkenylene, heteroalkenylene, heterocycloalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, oxyalkylene, dioxyalkylene, aminoalkylene, diaminoalkylene, diacid, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylenealkoxycarbamate, disulfide, 1,2-vinylene, imine, imineamide, phosphoramide, sugar, phosphate, phosphoramide, carbamate, dipeptide, tripeptide and tetrapeptide, each of which is substituted or unsubstituted; J is a moiety comprising a branch point, comprising or consisting of a structural unit, said The structural units are independently selected from the group consisting of alkylene, cycloalkylene, aralkylene, heteroaralkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, aralkenylene, heteroaralkenylene, heteroalkenylene, heterocycloalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, (oxy)alkylene, dioxyalkylene, aminoalkylene, diaminoalkylene, diacid, dialkylsiloxane, amide, sulfide amide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylene alkoxy carbamate, disulfide, 1,2-vinylene, imine, imineamide, phosphoramide, sugar, phosphate, phosphoramide, carbamate, dipeptide, tripeptide, tetrapeptide, each of which is substituted or unsubstituted; each x is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, The integers 17, 18, 19 and 20; Each y is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, The integers 17, 18, 19 and 20; Each z is an integer independently selected from 0, 1, 2, 3, 4, and 5; and Each a is an integer independently selected from 0, 1, 2, 3, 4 and 5.

3. The compound according to claim 1 or 2, wherein the distance (d1+d2) between each portion A and the closest branch point is ≤60, ≤51, ≤50, ≤44 or or wherein the number of bonds separating the two corresponding groups, counted along the shortest path connecting the two corresponding groups, is ≤50, ≤42, ≤41, ≤36, or ≤30.

4. A compound according to any one of the preceding claims, wherein the distance (d1+d2) between each portion A and the closest branch point is ≤30, ≤28, ≤27, ≤26 or Preferably ≤24, ≤22, ≤21, ≤19 or More preferably ≤17, ≤15, ≤13, ≤12, ≤11, ≤10, ≤8, ≤7, ≤6, ≤5, ≤4, ≤2 or or wherein the number of bonds separating two corresponding groups, counted along the shortest path connecting the two corresponding groups, is ≤25, ≤24, ≤23, ≤22 or ≤21; preferably ≤20, ≤19, ≤18, ≤17, ≤16 or ≤15; more preferably ≤14, ≤13, ≤12, ≤11, ≤10, ≤9, ≤8, ≤7, ≤6, ≤5, ≤4, ≤3, ≤2 or 1.

5. The compound according to claim 1 or 2, wherein the distance (d1+d2) between each portion A and the closest branch point is or smaller, preferably or less, more preferably or smaller.

6. A compound according to any one of the preceding claims, wherein between each portion A and the closest branch point, the distance (d1+d2) is or less; or the number of bonds separating the two corresponding groups, counted along the shortest path connecting the two corresponding groups, is 14 or less.

7. A compound according to any one of the preceding claims, wherein the distance (d1) between each portion A and the closest portion J is ≤55, ≤45, ≤38 or or wherein the number of bonds separating two corresponding groups, counted along the shortest path connecting the two corresponding groups, is ≤45, ≤37, ≤31 or ≤25.

8. A compound according to any one of the preceding claims, wherein the distance (d1) between each portion A and the closest portion J is ≤24, ≤23, ≤22, ≤21, ≤20 or Preferably ≤18, ≤17, ≤16, ≤15, ≤13 or More preferably ≤11, ≤10, ≤8, ≤7, ≤6, ≤5, ≤4, ≤2 or or wherein the number of bonds separating the two corresponding groups, counted along the shortest path between the two corresponding groups, is ≤20, ≤19, ≤18, ≤17 or ≤16; preferably ≤15, ≤14, ≤13, ≤12, ≤11 or ≤10; more preferably ≤9, ≤8, ≤7, ≤6, ≤5, ≤4, ≤3, ≤2 or 1.

9. A compound according to any one of the preceding claims, wherein the distance (d1) between each portion A and J is or smaller, preferably or less, more preferably or smaller.

10. A compound according to any one of the preceding claims, wherein the distance (d1) between each portion A and the closest portion J is or less; or the number of bonds separating the two corresponding groups, counted along the shortest path connecting the two corresponding groups, is 9 or less.

11. A compound according to any one of the preceding claims, wherein each moiety (L) a The distance (d2) to the closest branch point is ≤19, ≤17, ≤16, ≤15, or Preferably ≤12, ≤11, ≤10, ≤8 or More preferably ≤6, ≤5, ≤4, ≤2 or or wherein the number of bonds separating two corresponding groups, counted along the shortest path connecting the two corresponding groups, is ≤16, ≤14, ≤13, ≤12, ≤11; preferably ≤10, ≤9, ≤8, ≤7 or ≤6; more preferably ≤5, ≤4, ≤3, ≤2 or 1.

12. A compound according to any one of the preceding claims, wherein the distance (d2) between each portion L and the closest branch point is or smaller, preferably or less, more preferably or smaller.

13. A compound according to any one of the preceding claims, wherein each moiety (L) a The distance (d2) from the closest branch point is or less; or the number of bonds separating the two corresponding groups, counted along the shortest path connecting the two corresponding groups, is 5 or less.

14. The compound according to claim 2, wherein the distance or spacing between each portion A and the closest branch point is as defined in claim 6.

15. The compound according to claim 14, wherein the distance or spacing between each portion A and the closest portion J is as defined in claim 10.

16. A compound according to any one of the preceding claims, wherein L, B S and / or B L Each independently selected from: (a) a group comprising or consisting of structural units independently selected from the group consisting of: Each of R, R 1 , R 2 and R 3 independently selected from H, OH, SH, NH2, halogen, cyano, carboxyl, alkyl, cycloalkyl, aryl and heteroaryl, each of which is substituted or unsubstituted; R 4 and R 5 Each of is independently selected from alkyl, cycloalkyl, aryl and heteroaryl, each of which is substituted or unsubstituted; R a , R b and R c Each of which is independently selected from the side chain residues of proteinogenic amino acids or non-proteinogenic amino acids, each of which can be further substituted; Each X is independently selected from NH, NR, S, O and CH2, preferably NH; Each of n and m is independently an integer from 0 to 100, preferably from 0 to 50, more preferably from 0 to 30, still more preferably an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20; and where each * indicates that the shortest path to section A contains fewer attachment points for atoms than the shortest path to section A contains; and each · indicates that the shortest path to section C contains fewer attachment points for atoms than the shortest path to section C contains; (c) one or more of the following structural units: wherein in each of the above structures, n is 1, 2, 3 or 4; and Each * indicates that the shortest path to part A contains fewer atoms than the shortest path to part A; and each · indicates that the shortest path to part C contains fewer atoms than the shortest path to part C, provided that when n>1 and the corresponding attachment point is marked in R a , R b and R c When it is on any one of the attachment points, it can be present independently in one or more of the peptide monomer units, preferably in the one peptide monomer unit farthest from the other attachment point indicated in the corresponding structure; (d)B L and B S One or more of are independently selected from the following structures: *–Val-Ala–·; *–Val-Lys–·; *–Val-Arg–·, where each * indicates that the shortest path to section A contains fewer attachment points for atoms than the shortest path to section A contains; and each · indicates that the shortest path to section C contains fewer attachment points for atoms than the shortest path to section C contains; and / or (e) y is 1, 2 or 3; and / or at least one B L Also comprises a cleavable linker group independently selected from the following structures: Each * indicates that the shortest path to section A contains fewer attachment points than the shortest path to section A contains; and each · indicates that the shortest path to section C contains fewer attachment points than the shortest path to section C contains; or (f) Where ((B S ) x (B L ) y ) z Represented by the following structure: Where B' s and B" s Each independently selected from the group consisting of: Each B L Independently selected from the group consisting of: Each n is 0, 1, 2, 3, 4, or 5; Each m is 0, 1, 2, 3, 4, or 5; Each x' is 0, 1, or 2; Each x” is 0, 1, or 2; Each y is 0, 1, or 2; and z is 1 or 2, Among them, R 1 , R 2 , R 3 , R a , R b , R c , X, * and · as defined in any one of the preceding claims.

17. A compound according to any one of claims 2 to 16, wherein J has a structure represented by J-1, J-2, J-3, J-4 or J-5: wherein each n1 is independently 0, 1, 2, 3, 4, 5 or 6, preferably 4; Each n2 is independently 0, 1, 2, 3 or 4, preferably 2; Each n6 is independently 0 or 1, preferably 1; Each Y is independently O, NR, CR2, preferably O; Each R p Independently N + or C, preferably C; Each R p ' is independently N or CR; Wherein each R is independently H or selected from H, SH, NH2, halogen, cyano, carboxyl, C 1-6 -alkyl, O(C 1-6 Alkyl), S(C 1-6 -alkyl), C 2-6 Alkenyl, C 2-6 Alkynyl, 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.

18. A compound according to any one of claims 2 to 16, wherein J has a structure selected from the following: wherein each q is 0, 1, 2 or 3; each r is 0, 1, 2 or 3; q+r=3; and all other groups and variables are as defined in claim 6.

19. The compound according to any one of claims 2 to 17, wherein J has a structure represented by J-1a, J-1b, J-1c, ​​J-1d, J-2a, J-2b, J2-c or J2-d:

20. A compound according to any one of claims 2 to 16 and 18, wherein J has a structure selected from the following:

21. A compound according to any one of claims 2 to 20, wherein each L independently comprises or is represented by a structure selected from the following:

22. A compound according to any one of claims 2 to 21, wherein each (L) a It is represented by: wherein each of n3, n4, n7 and n8 is independently 0, 1, 2, 3, 4, 5 or 6, preferably 2; Each n5 is independently 0 or 1, preferably 1: Each m1, m2 and m3 is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, preferably 1; Each l1 and l2 is independently 0 or 1, preferably 1; Each l3 is independently 0 or 1, preferably 0; Each R is independently H or selected from H, SH, NH2, halogen, cyano, carboxyl, C 1-6 -alkyl, O(C 1-6 Alkyl), S(C 1-6 -alkyl), C 2-6 Alkenyl, C 2-6 Alkynyl, 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; and Each W is independently selected from NR and O.

23. A compound according to any one of claims 2 to 22, wherein the fragment ((L) a )3J is represented by the following structure K-1, K-2, K-3 or K-4, wherein each n1 is independently 0, 1, 2, 3, 4, 5 or 6, preferably 4; Each n2 is independently 0, 1, 2, 3 or 4, preferably 2; wherein each of n3, n4, n7 and n8 is independently 0, 1, 2, 3, 4, 5 or 6, preferably 2; Each n5 and n6 is independently 0 or 1, preferably 1; Each Y is independently O, NR, CR2, preferably O; Each R p Independently N + or C, preferably C; Each R p ' is independently N or CR; Each m1, m2 and m3 is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, preferably 1; Each l1 and l2 is independently 0 or 1, preferably 1; Each l3 is independently 0 or 1, preferably 0; Wherein each R is independently H or selected from H, SH, NH2, halogen, cyano, carboxyl, C 1-6 -alkyl, O(C 1-6 Alkyl), S(C 1-6 -alkyl), C 2-6 Alkenyl, C 2-6 Alkynyl, 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; and Each W is independently selected from NR and O.

24. A compound according to any one of claims 2 to 23, wherein the fragment ((L) a )3J is selected from the following structures:

25. A compound according to any one of claims 2 to 23, wherein the fragment ((L) a )3J is selected from the following structures: wherein each m3 is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, preferably 0, 1, 2, 3 or 4; and Each l3 is independently 0 or 1, preferably 0.

26. A compound according to any one of the preceding claims, wherein the moiety C is selected from: (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 and a chelator; (d) a cytotoxic agent and / or a cytostatic agent; (f) an immunomodulator; or (g) a protein.

27. A compound according to any one of the preceding claims, wherein the moiety C is a chelator group suitable for radiolabeling with a therapeutic nuclide selected from compounds having a structure according to any one of the following formulae: in: n is 0, 1, 2, 3, 4 or 5; preferably 1; R 1e is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH; R 2e is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH; Each R 3e is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH; R 4e is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH; R 1f is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH; R 2f is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH; R 3f is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH; and X is O, NH or S; preferably O.

28. A compound according to any one of the preceding claims, wherein the moiety C is a chelator group suitable for radiolabeling with a therapeutic nuclide selected from the group consisting of 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), 29. A compound according to any one of the preceding claims, wherein the moiety C is a therapeutic radioactive group comprising a therapeutic radioisotope selected from 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 Lu; it is not intended for use in diagnostic applications.

30. A compound according to any one of claims 26 to 29, wherein the chelate of the therapeutic radioisotope is a chelate of an isotope listed in claim 29 and / or a chelate with a chelator listed in claim 27 or 28.

31. A compound according to any one of the preceding claims, wherein moiety C is a radioactive group selected from any one of the following structures: Wherein M is a therapeutic radioisotope, preferably selected from the list described in claim 29.

32. A compound according to any one of the preceding claims, wherein moiety C is selected from 33. A compound according to any one of claims 1 to 26, wherein moiety C is a cytotoxic and / or cytostatic therapeutic 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, anti-vascular agents, and combinations of two or more thereof.

34. A compound according to any one of claims 1 to 26 and 33, wherein moiety C is an auristatin having a structure according to the following formula: in: R 1d Independently H or C1-C6 alkyl; preferably H or CH3; R 2d Independently C1-C6 alkyl; preferably CH3 or iPr; R 3d 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 Independently for C3-C 10 Aryl or C3-C 10 Heteroaryl; preferably optionally substituted phenyl or pyridinyl.

35. A compound according to any one of claims 1 to 26 and 33, wherein moiety C is a topoisomerase inhibitor selected from camptothecin (CPT) and derivatives thereof derived by replacement of a hydrogen atom from topotecan, irinotecan, silatecan, cosinotecan, exatecan, lortotecan, gimatecan, belotecan, rubitecan, derutecan and DXd.

36. A compound according to any one of claims 1-26 and 33, wherein moiety C is a cytotoxic and / or cytostatic therapeutic selected from the following structures: Where each n is 0, 1, 2, 3, 4, 5 or 6; 37. according to the compound of any one of claims 1 to 26, wherein part C is an immunomodulator, which is selected from molecules known to be able to modulate the immune system, such as CD3, CD25, TLR, STING, 4-1BBL, 4-1BB, PD-1, Ligands of mTor, PDL-1, NKG-2D IMiD, wherein the ligand can be an agonist and / or antagonist.

38. A compound according to any one of claims 1 to 26 or 37, wherein moiety C is a therapeutic protein or a therapeutic antibody, the therapeutic protein being selected from a cytokine, such as IL2, IL10, IL12, IL15, TNF, interferon gamma.

39. A compound according to any one of the preceding claims, wherein the fragment ((B S ) x (B L ) y ) z C is represented by one of the following structures: wherein each of AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8 and AA9 represents a proteinogenic amino acid or a non-proteinogenic amino acid, or is absent, and in, Unless otherwise stated, all groups and variables are as defined in any of the preceding claims.

40. The compound of claim 39, wherein AA5 is an amino acid with a charged side chain and AA8 is an amino acid with an aliphatic side chain.

41. The compound according to claim 39 or 40, wherein: AA1 is selected from Asp and Glu, or is absent; AA2 is selected from Asp and Glu, or is absent; AA3 is Lys; AA4 is selected from Asp and Glu; AA5 is selected from Lys and Arg; AA6 is selected from Asp and Glu; AA7 is selected from Cys; and AA8 is selected from Gly, Ala and Val; and AA9 is selected from Pro and citrulline (Cit).

42. A compound according to any one of the preceding claims, represented by the formula: wherein K represents a fragment as defined in any one of the preceding claims ((L) a )3J; (B S ) x Independently by 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(CH2CH2O) 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)-, preferably -C(O)(CH2) n C(O)- or -(CH2) n C(O)-denotes; Each n and m is independently an integer, preferably selected from 0, 1, 2, 3, 4, 5 and 6; Each of AA3, AA4, AA5, AA6, AA7 and AA8 independently represents a proteinogenic amino acid or a non-proteinogenic amino acid, or is absent; and C is a cytotoxic and / or cytostatic agent.

43. The compound of claim 42, wherein AA5 is an amino acid with a charged side chain and AA7 is an amino acid with an aliphatic side chain.

44. A compound according to claim 42 or 43, wherein AA3 is absent; AA4 is selected from Asp and Glu; AA5 is selected from Lys and Arg; AA6 is selected from Asp and Glu; AA7 is selected from Gly, Ala and Val; and AA8 is selected from Pro and citrulline (Cit).

45. A compound according to any one of claims 42 to 44, wherein C is a topoisomerase inhibitor as defined in claim 35.

46. ​​A compound according to any one of claims 42 to 44, wherein C is an auristatin as defined in claim 34.

47. A compound according to any of the preceding items, which is represented by one of the following formulae: Each of the S ) x ,(B L ) y or ((B S ) x -(B L ) y ) independently by 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(CH2CH2O) 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)-, preferably -C(O)(CH2) n O(CH2CH2O) m (CH2) n NH- means; Each n and m is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, preferably selected from 0, 1, 2, 3, 4, 5 and 6; and Each z is independently an integer selected from 0, 1, 2, 3, 4 and 5, preferably 1; Each m3 is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, preferably 1; and Each l3 is independently 0 or 1, preferably 0.

48. A compound according to any one of the preceding formulae, which is represented by the following formula: wherein each m3 is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, preferably 1; Each l3 is independently 0 or 1, preferably 0; z is an integer independently selected from 0, 1, 2, 3, 4 and 5, preferably 1; and Each m is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, preferably selected from 0, 1, 2, 3, 4, 5 and 6.

49. A compound according to claim 47 or 48, wherein moiety C is a chelator group suitable for radiolabelling with a therapeutic nuclide.

50. The compound according to claim 47 or 48, wherein moiety C is as defined in any one of claims 27 to 32.

51. A compound having a structure selected from the compounds in Table 9, an individual diastereomer thereof, a hydrate thereof, a solvate thereof, a crystalline form thereof, an individual tautomer thereof, or a pharmaceutically acceptable salt thereof.

52. A compound according to any one of the preceding claims having one of the following structures:

53. A compound according to any one of claims 1 to 51 having one of the following structures:

54. A pharmaceutical composition comprising a compound according to any one of the preceding claims and a pharmaceutically acceptable excipient.

55. A pharmaceutical combination comprising (i) a compound according to any one of claims 1 to 53 or a pharmaceutical composition according to claim 54 and (ii) an immunocytokine.

56. The pharmaceutical combination according to claim 55, wherein the immunocytokine specifically binds to the alternatively spliced ​​ED-B domain of fibronectin isoform B-FN, optionally binding to the epitope sequence represented by SEQ ID NO:

40.

57. A pharmaceutical combination according to any one of claims 55 or 56, wherein the immunocytokine comprises a sequence having IL2 activity.

58. according to the drug combination described in any one of claims 55 to 57, wherein the immunocytokine comprises one or more of the CDRs of any one of SEQ ID NOs: 13-18, optionally all of SEQ ID NOs: 13-18, further optionally wherein the immunocytokine comprises a VH sequence of SEQ ID NO: 19 and / or a VL sequence of SEQ ID NO:

20.

59. The pharmaceutical combination according to any one of claims 55 to 58, wherein the immunocytokine comprises a scFv sequence, optionally a human monoclonal scFv sequence.

60. The pharmaceutical combination according to any one of claims 55 to 59, wherein the immunocytokine comprises the sequence of SEQ ID NO:

12.

61. A compound according to any one of claims 1 to 53, a pharmaceutical composition according to claim 54, or a pharmaceutical combination according to any one of claims 55 to 60, for use in the following methods: (a) a method of surgical or therapeutic treatment of the human or animal body; or (b) a method of treating or preventing a subject having, or at risk of, a disease or condition; or (c) Methods for targeted delivery of a therapeutic agent to a subject having a disease or condition or at risk of a disease or condition.

62. A compound according to any one of claims 1 to 53, a pharmaceutical composition according to claim 54, or a pharmaceutical combination according to any one of claims 55-60 for use as defined in claim 61, wherein the disease or condition is independently selected from cancer, inflammation, atherosclerosis, fibrosis, tissue remodeling and keloid conditions, preferably, wherein 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, bile duct cancer, clear cell renal carcinoma, neuroendocrine tumors, carcinogenic osteomalacia, sarcoma, CUP (carcinoma of unknown primary), thymic carcinoma, desmoid tumor, glioma, astrocytoma, cervical cancer, skin cancer, kidney cancer and prostate cancer; and / or wherein the compound has prolonged retention at the disease site at therapeutically relevant levels, e.g., for at least 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 24 hours, 48 ​​hours, 72 hours, or 96 hours after injection, preferably for more than 1 hour, more preferably for more than 6 hours, even more preferably for more than 24 hours.

63. A compound according to any one of claims 1 to 53 or a pharmaceutical composition according to claim 54 for use according to claim 61 or 62, further comprising administering an immunocytokine as defined in any one of claims 55 to 60.

64. An immunocytokine as defined in any one of claims 55 to 60 for use as defined in claim 61 or 62, said method further comprising administering a compound according to any one of claims 1 to 53 or a pharmaceutical composition according to claim 54.

65. A compound according to any one of claims 1 to 53 or a pharmaceutical composition according to claim 54 for use according to any one of claims 61 to 64, wherein the compound is 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, most preferably 90-125 nmol / kg expressed as a mouse dose, or the corresponding human equivalent dose.

66. A compound according to any one of claims 1 to 53 or a pharmaceutical composition according to claim 54 for use according to any one of claims 61 to 64, wherein the compound is 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, most preferably 7-10 nmol / kg.

67. A compound according to any one of claims 1 to 53 or a pharmaceutical composition according to claim 54 for use according to any one of claims 61 to 64, wherein the compound is administered to a human subject at a dose of 7 nmol / kg or higher, preferably 7-20 nmol / kg.

68. A compound according to any one of claims 1 to 53 or a pharmaceutical composition according to claim 54 for use according to any one of claims 61 to 64, wherein the compound is administered to a human subject at a dose of 1 mg or more, preferably 1 to 3 mg.

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