Multivalent multispecific conjugates and related compositions and methods of use

Abstract

A conjugate comprising (a) at least two or more binding motifs, each of which binds to a different cell surface molecule that is overexpressed or selectively expressed in diseased (e.g., cancerous) cells, wherein adjacent binding motifs are separated from each other by a linker, which can be the same as or different from the linkers between other adjacent binding motifs, and (b) an active agent that can be endocytosed by cancerous cells to which the conjugate binds; a composition comprising the conjugate and a pharmaceutically acceptable carrier; a method for selectively targeting cancerous cells in a subject for endocytosis of an anti-cancer agent; and a method for imaging a subject with cancer.

Description

[Technical Field]

[0001] Priority This application is related to and claims priority to U.S. Provisional Patent Application No. 63 / 423,688, filed November 8, 2022. The contents of the aforementioned application are hereby incorporated by reference in their entirety into this disclosure.

[0002] The present disclosure relates to conjugates comprising a binding motif for a cell surface molecule that is overexpressed or selectively expressed in a target cell, a linker, and an active agent, compositions comprising the same, and methods of using the conjugates or compositions comprising the conjugates for targeted delivery of the active agent and / or to treat a subject experiencing a disease state such as cancer, a genetic disorder, etc. [Background technology]

[0003] Many therapeutic agents containing a targeting ligand linked to a drug contain one or more linkers disposed between the targeting ligand and the therapeutically active drug. The targeting ligand can facilitate specific binding to cells (e.g., cancer cells) that express the cell surface molecule (e.g., receptor, transporter, cell-to-cell communication protein, etc.) for the ligand, resulting in delivery of the therapeutically active drug or imaging agent to the target cells. For example, the specific binding of a folate-drug conjugate to a folate receptor (FR) on cancer cells or inflammatory cells can facilitate targeted delivery and specific therapeutic activity directed to cancer cells or sites of inflammation.

[0004] In many, if not all, target-ligand complexes are stabilized by strong intermolecular forces, ultimately leading to a longer residence time at the binding site (higher "on" rate, lower "off" rate). Indeed, high-affinity binding ligands can be effective in binding to cell surface molecules. However, these high-affinity ligands are typically not specific to diseased cells (e.g., cancer cells). For example, FRs are expressed in numerous cancers of epithelial origin, including breast, lung, kidney, and ovarian cancers. However, FRs are also expressed in healthy tissues, such as the brush border membrane of proximal tubule cells in healthy kidneys, making them accessible to drug conjugates in the blood circulation.

[0005] Due to the binding strength of cell surface molecules and high-affinity ligands to cell surface molecules on both diseased and healthy cells, traditional targeted drugs are not accurate in discriminating between targeted and healthy (or otherwise off-target) cells, which can lead to off-target / secondary effects. For example, the therapeutic use of many FR-targeted drugs is problematic due to high FR-mediated renal uptake. This can be particularly problematic for imaging compounds that employ radiation, for example.

[0006] Therefore, there is a need for targeting ligands and compounds with increased selectivity.

[0007] Two important unmet needs in cancer treatment are (i) the development of therapeutic agents that affect cancer cells while sparing normal cells (cancer selectivity), and (ii) the availability of effective strategies to address the variability of cell surface markers between tumors within a single patient and between patients diagnosed with the same cancer (cancer variability). The failure of current strategies to address these aspects of cancer treatment contributes to poor outcomes, tumor resistance, and cancer recurrence.

[0008] In view of the above, it is an object of the present disclosure to provide materials and methods for targeted cell treatment that seek to overcome the shortcomings of current strategies in addressing selectivity generally, and cancer variability in particular. This and other objects and advantages, as well as features of the invention, will be apparent from the detailed description provided herein. Summary of the Invention

[0009] A conjugate is provided, which comprises: (a) at least two or more binding motifs, each of which binds to a different cell surface marker that is overexpressed (or selectively expressed) on target cells (e.g., diseased cells such as cancerous cells), wherein adjacent binding motifs are separated from each other by a linker, and the linker can be the same or different from another linker between other adjacent binding motifs; and (b) an active agent that can induce endocytosis in target cells to which the conjugate binds. Pharmaceutical salts of the conjugate are also provided. As described above, when the conjugate comprises more than one linker, the linkers can be the same or different from each other.

[0010] In some embodiments, the conjugate has Formula I: (chemical 1) BM-(L-BM) n -LA (I) or a pharmaceutically acceptable salt thereof, wherein each BM is one of at least two binding motifs, each L is a linker, n is 1 to 5, and A is an active agent. n can be 5 or more. n can be 1 to 3. n can be 2. n can be 5 to 10. n can be 3. n can be 4.

[0011] The targeted cell can be a pathological cell (eg, a cancerous cell).

[0012] The conjugate can, and preferably does, comprise at least four binding motifs, wherein each of the at least four binding motifs binds to a different cell surface molecule (e.g., a cell surface receptor) that is overexpressed or selectively expressed in the targeted cells. Each of the at least four binding motifs can bind to a different cell surface molecule. The cell surface molecule can be selected from the group consisting of a transporter, a receptor, and a cell-to-cell communication protein. In one embodiment, each cell surface molecule is a cell surface receptor.

[0013] The conjugate can comprise at least four binding motifs, where each of the at least four binding motifs binds to a different cell surface receptor that is overexpressed or selectively expressed on the targeted cell and is selected from the group consisting of fibroblast growth factor receptor 3 (FGFR3), Her2, interleukin-4 receptor alpha (IL-4Rα), and epidermal growth factor receptor (EGFR). In such embodiments, the targeted cell can be or can include a cancerous cell.

[0014] The binding motif for FGFR3 can be VSPPLTLGQLLS (SEQ ID NO: 1) or a functional variant thereof (designated "F"); the binding motif for Her2 can be FCGDGFYACYMDV (SEQ ID NO: 2) or a functional variant thereof (designated "H"); the binding motif for IL-4Rα can be KLAKLAKKLAKLAK (SEQ ID NO: 3) or a functional variant thereof (designated "I"); and the binding motif for EGFR can be YHWYGYTPQNVI (SEQ ID NO: 4) or a functional variant thereof (designated "E").

[0015] When a conjugate includes more than one linker, each linker can be the same or different from each other. Each linker can be approximately 5 nm to 15 nm in length. Each linker can be approximately 7 to 10 nm in length. Each linker can be approximately 7 nm in length. In some embodiments, each linker is approximately 7 nm in length and can be, and preferably is, flexible.

[0016] Each linker can have an amino acid sequence independently selected from GRAQGKAQG (SEQ ID NO: 5), GQAKGQARG (SEQ ID NO: 6), GKQAGRQAG (SEQ ID NO: 7), and GQRAGQKAG (SEQ ID NO: 8). Each linker can have an amino acid sequence independently selected from GRAQGKAQG (SEQ ID NO: 5), GQAKGQARG (SEQ ID NO: 6), GKQAGRQAG (SEQ ID NO: 7), and GQRAGQKAG (SEQ ID NO: 8), and each linker can be approximately 7 nm in length and can be flexible.

[0017] The active agent can be an anti-cancer therapeutic agent or an imaging agent. The active agent can be attached to a nanoparticle or encapsulated in a liposome, where the nanoparticle or liposome is attached to the linker of the conjugate. In some embodiments, the active agent comprises an imaging agent. In some embodiments, the active agent comprises a therapeutic agent.

[0018] The conjugate may comprise SEQ ID NO: 9 or a functional variant thereof.

[0019] The conjugate may comprise SEQ ID NO: 10 or a functional variant thereof.

[0020] The binding motif can be a low affinity binding motif.

[0021] Also provided is a composition comprising the conjugate or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The composition can comprise one of the conjugates. The composition can comprise at least two different conjugates, both of which contain the same four cell surface-binding binding motifs that are overexpressed or selectively expressed in targeted cells, but where the order of the binding motifs differs between the two conjugates. In one embodiment, the composition comprises (i) a first conjugate comprising the sequence YHWYGYTPQNVIGRAQGKAQGKLAKLAKKLAKLAKGQAKGQARGFCGDGFYACYMDVGKQAGRQAGVSPPLTLGQLLSGQRAGQKAG (SEQ ID NO: 9) or a functional variant thereof, and (ii) a second conjugate comprising FCGDGFYACYMDVGRAQGKAQGYHWYGYTPQNVIGQAKGQARGVSPPLTLGQLLSGKQAGRQAGKLAKLAKKLAKLAKGQRAGQKAG (SEQ ID NO: 10) or a functional variant thereof.

[0022] Further provided is a method for selectively targeting target cells (e.g., cancerous or diseased cells) in a subject for endocytosis of a therapeutic agent (e.g., an anti-cancer agent). The method comprises administering to the subject an effective amount of the conjugate or a pharmaceutically acceptable salt thereof, or the composition, wherein the active agent of the conjugate or the pharmaceutically acceptable salt thereof comprises an anti-cancer agent. In some embodiments, when the cancerous cells endocytose the therapeutic agent (e.g., the anti-cancer agent), the endocytosis of the therapeutic agent (e.g., the anti-cancer agent) treats the subject for cancer. The subject can have bladder cancer. The bladder cancer can be non-muscle invasive bladder cancer (NMIBC).

[0023] Also provided is a method for imaging or diagnosing a subject having a disease state. The method includes (a) administering to a subject an effective amount of the conjugate or a pharmaceutically acceptable salt thereof, or the composition, wherein the active agent of the conjugate or the pharmaceutically acceptable salt thereof comprises an imaging agent, and (b) imaging the subject or allowing the subject to be imaged. The disease state can be cancer. The disease state can be bladder cancer. The disease state can be NMIBC. The disease state can be a genetic disease. The disease state can be fibrosis. The disease state can be an inflammatory disorder or disease state.

[0024] Imaging the subject can include performing radiological imaging, positron emission tomography (PET) imaging, single-photon emission computed tomography (SPECT) imaging, or magnetic resonance imaging. In some embodiments, imaging the subject further includes imaging an area of ​​the subject affected by the disease state. The imaging agent can include a metal or isotope suitable for radiological imaging, PET imaging, SPECT imaging, or magnetic resonance imaging, or a fluorescent contrast agent, a photodynamic contrast agent, or an optical contrast agent.

[0025] In one embodiment, there is provided a use of the conjugate (or a pharmaceutically acceptable salt thereof) or the composition in the preparation of a medicament for treating a disease condition in a subject. The disease condition can be cancer. The disease condition can be bladder cancer. The disease condition can be NMIBC. [Brief explanation of the drawings]

[0026] Other features, advantages, and aspects of the disclosed embodiments and those contained herein, and the accomplishments thereof, will become apparent in light of the following detailed description of various exemplary embodiments of the present disclosure, which will be better understood when considered in conjunction with the accompanying drawings.

[0027] [Figure 1]Figure 1 is a schematic diagram of the structure of the bladder. GAGs (glycosaminoglycans) can form layers.

[0028] [Figure 2] Figures 2A-2C show receptor microclustering. Figure 2A shows that multivalent agents such as polyclonal antibodies (PAbs), nanoparticles (NPs), oligomerization proteins (OPr), and multivalent peptides (MVs) can induce receptor endocytosis through microclustering (μC) (gray, vertically elongated ovals). Figure 2B shows that multivalent and multispecific (MV-MS) conjugates can microcluster different receptors. Jack et al., Int J Cancer 146(2):449-460 (2020); Coon et al., Fibronectin attachment protein from bacillus Calmette-Guerin as a targeting agent for bladder tumor cells, Int'l J Cancer, 131(3):591-600 (2012).

[0029] Figure 2C shows examples of cargo internalized by μC (the brighter the endosome, the higher the concentration inside). FAP: fibronectin adhesion protein. PDL1: programmed death ligand 1. EGF-PA: epidermal growth factor-protective antigen.

[0030] [Figure 3] Figures 3A-3I illustrate the MV-MS targeting strategy.

[0031] Figure 3A shows the average incidence of overexpression of specific receptors in non-muscle-invasive bladder cancer (NMIBC), where EGFR is epidermal growth factor receptor, ILR4a is interleukin-4 receptor alpha, FGFR3 is fibroblast growth factor receptor 3, and HER2 is human epidermal growth factor receptor 2. Joshi et al., Cancer Medicine 3(6):1615-28(2014), Hashizume et al., Oncotarget 9(75):34066-34078(2018), Rotterud et al., BJU Int'l 95(9):1344-1350(2005).

[0032] Figure 3B (left): Nanoparticles (NPs) decorated with low density conjugates do not favor μC formation within MV-MS conjugates, but rather between them. Figure 3B (right): The conjugates are designed to target EGFR (E 30 ), interleukin-4 receptor alpha (IL-4Rα, I 31 ), Her2(H 32 ) and FGFR3 (F 33 The peptides were generated by combining a binding peptide validated against the nucleotide sequence YPYDVPDYAG (SEQ ID NO: 11) or YPYDVPDYAGYPYDVPDYAGYPYDVPDYA (SEQ ID NO: 12) with a flexible linker (each approximately 7 nm in length) positioned between them and a human influenza hemagglutinin (HA) tag for immunodetection. Chen et al., Advanced Drug Delivery Review 65(10):1357-69(2013); Ching et al., Separation Science & Technology 24:7-8,581-597(1989).

[0033] Figure 3C shows purified P1 and P2 peptides detected by Western blotting with anti-HA antibody.

[0034] Figure 3D shows that the HA-positive cells were significantly different in human (T24), mouse (MB49) and guinea pigs, as revealed by anti-HA immunofluorescence.LE Cells of human (WT: wild-type, which upregulates only IL4R and FGFR3 receptors, but also expresses EGFR) and canine (isolated and immortalized from a sporadic canine tumor) origin bound and internalized the P1 / P2 peptide. Arrows and arrowheads indicate examples of internalized peptides in endosomes and late compartments, respectively. The inset in the middle panel shows the P1 / P2 peptide in Rab5-positive endosomes.

[0035] Figure 3E shows that MB49 cells were synchronized and induced to bind and internalize the peptide. After 5 min, the internalized peptide localized to peripheral early endosomes (arrowheads) and after 45 min, accumulated in late endosomes / lysosomes (arrows).

[0036] Figures 3F-3G show the amount of peptide bound to cells as a function of time estimated by quantitative microscopy (Figure 3F) and as a function of dose (Figure 3G) by quantitative Western blot using an anti-HA antibody (upper panel) followed by band densitometry in triplicates (lower panel).

[0037] Figure 3H shows an ELISA-based study of MV-MS binding to different extracellular domain (ECD) densities to mimic "normal" cells and "cancer" cells displaying one or two overexpressed (OE) receptors.

[0038] Figure 3I shows GFP-Rab5 cells incubated with nanomolar amounts of fluorescent nanoparticles decorated with MV-MS (MVMS-NPs). Q79L The internalized MVMS-NPs were detected as signals inside endosomes (arrows). Scale bar: 10 μm.

[0039] [Figure 4] Figures 4A and 4B show images of immunostaining with anti-HA antibody of normal cells (Figure 4A) and MB49 wild-type cancer cells (Figure 4B) incubated with the highest concentration of P2 conjugate used.

[0040] FIG. 4C shows a graph of quantification of cell-associated P2 per cell (measured as total fluorescence intensity) versus P2 dose.

[0041] While the present disclosure is susceptible to various modifications and alternative forms, exemplary embodiments thereof have been shown by way of example in the drawings and are herein described in detail.

[0042] Sequence Listing The sequences herein (SEQ ID NOS: 1-12) are also provided in computer-readable form encoded in a file submitted herewith and are incorporated herein by reference. The information recorded in computer-readable form is identical to the set forth sequence listing provided herein in accordance with 37 C.F.R. § 1.821(f).

[0043] Detailed Description The present disclosure is based, at least in part, on the discovery that multivalent conjugates containing two or more low-affinity binding motifs that simultaneously target two or more markers present on targeted cells can significantly increase the effective binding affinity (i.e., avidity) of the conjugate for the targeted cells. Furthermore, individual low-affinity binding motifs can be linked by the intercalation of flexible linkers that provide optimal separation between the peptides. This linker-imposed separation can induce microclustering of cell surface markers bound by the peptide units. Taking advantage of these findings, multivalent, multispecific (MV-MS) conjugates are provided.

[0044] The MV-MS conjugates can be used for multiple applications in which selective targeting of cells is necessary and / or beneficial, such as in connection with the administration of a therapeutic to the targeted cells and / or targeted cell removal. For example, the conjugates can be used to selectively deliver a therapeutic to specific kidney cells in connection with the treatment of a genetic condition, or for the selective delivery of a cytotoxic payload to cancer cells (e.g., glioma, medulloblastoma, bladder tumor, etc.). Specific examples are provided herein to facilitate understanding of the concepts described herein, but it will be understood that the conjugates, compositions, and methods can be used in connection with / for any application in which selective targeting of cells may be beneficial or desirable.

[0045] In one embodiment, a conjugate (or a pharmaceutically acceptable salt thereof) is provided, comprising at least two or more binding motifs (BM), each of which binds to a different cell surface molecule (e.g., a transporter, a receptor, a cell-to-cell communication protein, etc.) that is overexpressed or selectively expressed on a target cell, and an active agent (A). Adjacent binding motifs can be separated from each other by a linker (L). For example, when the conjugate comprises multiple linkers (e.g., a first linker located between a first binding motif and a second binding motif, and a second linker located between a second binding motif and a third binding motif), each linker can be the same or different from each other.

[0046] In some embodiments, the compound of Formula I: (Case 2) BM-(L-BM) n -LA (I) or a pharmaceutically acceptable salt thereof, wherein each BM is a binding motif, each L is a linker, n is an integer equal to or greater than 1, and A is an active agent.

[0047] In some embodiments, n is 1 to 5. In some embodiments, n is 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. As noted above, each binding motif can be different from the others, such that each binds to a different cell surface receptor, and each linker can be the same as or different from the other linkers in the conjugate.

[0048] A "pharmaceutically acceptable salt" refers to a salt of a compound that retains the biological activity of the parent compound and is biologically or otherwise undesirable. Acid and / or base salts can be formed, for example, by reaction with amino and / or carboxyl groups. Pharmaceutically acceptable salt addition salts may be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

[0049] In application, the conjugates herein can simultaneously target multiple upregulated or selectively expressed cell surface molecules (e.g., on cancer cells or other target cells), providing enhanced selectivity for the target cells through endocytosis modulation, and can be highly effective even against target cells with variable expression patterns (e.g., tumor cells), for example, by inducing self-adjustment of affinity (i.e., having low to normal binding affinity but resulting in high binding activity for the target cells) through simultaneous detection of upregulated / selectively expressed molecules. Thus, the target cells can be pathological cells. The target cells can be cancerous cells. The target cells can be any cells with overexpressed and / or selectively expressed cell surface molecule(s) that can be matched with the binding motif of the conjugate.

[0050] Low-affinity binding motif

[0051] As described above, the conjugate comprises at least two or more binding motifs. Each binding motif comprises a peptide or other moiety that corresponds to / binds to a cell surface molecule. As used herein, the terms "protein," "polypeptide," and "peptide" refer to a compound comprising amino acids joined via peptide bonds and are used interchangeably. The binding motifs of the conjugate can be selected based on the type of cell to be targeted, i.e., to correspond to the upregulation or selective expression of two or more cell surface targets in a particular cell. As used herein, "upregulated," "overexpressed," and derivatives thereof (e.g., "upregulated") are used interchangeably and refer to an increase in the level of a marker, such as a receptor, protein, or polypeptide, compared to normal and / or healthy cells. The same consideration applies to "selectively expressed" cell surface molecules.

[0052] At least two of the binding motifs of the conjugate bind to different cell surface molecules. The conjugate can contain at least three binding motifs, at least two of which bind to different cell surface receptors (i.e., in such cases, at least two of the binding motifs are the same). In some embodiments, the conjugate contains at least three binding motifs, each of which binds to a different cell surface receptor that is overexpressed or selectively expressed on the targeted cells. The conjugate can contain four or more binding motifs. In some embodiments, the conjugate contains at least four binding motifs, at least four of which bind to a different cell surface receptor that is overexpressed or selectively expressed on the targeted cells. In some embodiments, the conjugate contains at least four binding motifs, each of which binds to a different cell surface receptor that is overexpressed or selectively expressed on the targeted cells.

[0053] The binding motif can be a low affinity peptide. As used herein, a "low affinity binding motif" or "low affinity peptide" refers to a peptide that, when complexed with a corresponding cell surface molecule or marker, results in only a weak interaction at the protein level. The complex resulting from the interaction between the low affinity binding motif and the cell surface marker has a dissociation constant (K) in the high micromolar range (e.g., ≧0.1 μM). D For example, a complex of a low affinity binding motif and a cell surface marker may have a K of from about 0.1 μM, or from about 1 μM, or from about 100 μM, to about 1000 μM, or to about 500 μM, or to about 250 μM, or to about 100 μM, or to about 10 μM. D Thus, the affinity can be in the range of about 0.1 μM to about 1000 μM (or more), or in the range of about 0.2 μM to about 900 μM, or in the range of about 0.3 μM to about 0.8 μM, or in the range of about 0.5 μM to about 700 μM, or in the range of about 10 μM to about 600 μM, or in the range of about 50 μM to about 500 μM, or in the range of about 100 μM to about 400 μM, or in the range of about 400 μM to about 500 μM, or in the range of about 1 μM to about 200 μM, or in the range of about 450 μM to about 480 μM, or in the range of about 10 μM to about 800 μM, or in the range of about 50 μM to about 100 μM, as measured, for example, by Scatchard analysis, surface plasmon resonance technology (e.g., using BIACORE®), or equivalent technology. The ranges set forth in this paragraph include the defined endpoints and all 0.1 μM increments therein.

[0054] In some embodiments, the binding motif can have a binding affinity to a cell surface molecule of less than about 500 μM, such as 459 μM. In some embodiments, the binding motif can have a binding affinity to a cell surface molecule of less than about 0.5 μM, such as 0.3 μM. In some embodiments, the binding motif can have a binding affinity to a cell surface molecule of less than about 60 μM, such as 55.9 μM.

[0055] Cell surface molecules can include transporters, receptors, cell-to-cell communication proteins, etc. that are selectively expressed on the surface of targeted cells. These molecules can be naturally endogenous to such cells or can appear as a result of mutations, such as in cancer cells. Cell surface molecules can be any molecule known in the literature to be upregulated or selectively expressed in cells of interest.

[0056] In some embodiments, the cell surface molecule comprises a cell surface receptor that is overexpressed on the targeted cell. In some embodiments, the cell surface receptor comprises fibroblast growth factor receptor 3 (FGFR3), Her2, interleukin-4 receptor alpha (IL-4Rα), and epidermal growth factor receptor (EGFR), and optionally, the targeted cell is a cancerous cell. In some embodiments, the cell surface molecule can comprise EGFRviii (e.g., an EGFR variant present in multiple gliomas, prostate cancer, gastric cancer, and other cancers). In some embodiments, the cell surface molecule can comprise PDGFRα and / or PDGFRβ. In some embodiments, the cell surface molecule can comprise megalin (e.g., an endocytic receptor associated with kidney disease).

[0057] Specific cell types can express a specific combination of molecules that are upregulated or selectively expressed on their cell surface.For example, certain cancer cells are known to overexpress FGFR3, Her2, IL-4Rα, and EGFR, each of which is a receptor.When a specific cell type is known to upregulate or selectively express a specific combination of two or more cell surface molecules (for example, three or four different molecules or markers), the low affinity binding motif of the conjugate can be strategically selected to associate with this specific combination of molecules to facilitate targeted delivery and / or binding to such cell types.

[0058] Because the binding motif of the conjugate is a low-affinity peptide, the presence of only one type of corresponding cell surface molecule or a combination of cell surface molecules at a low density on the cell will result in weak or no binding. The synergistic combination of two or more different low-affinity binding motifs of the MV-MS conjugate and the high density or selective expression of molecules on the targeted cell will result in a differentially strong interaction. This therefore allows the MV-MS conjugate to exhibit low affinity to normal or non-targeted cells (i.e., those that do not overexpress the targeted combination of cell surface molecules).

[0059] The combination of binding motifs in the conjugates described herein creates a synergistic effect, allowing the conjugates to not only bind specifically to targeted cells (cells expressing a combination of cell surface molecules that correlates with the combination of low-affinity binding motifs in the conjugates), but also ultimately result in high binding activity when the low-affinity binding motifs in the conjugates simultaneously bind to microclusters of molecules present on the surface of the targeted cells. This affinity switch can occur because certain cells (e.g., cancer cells) upregulate or selectively express two or more cell surface molecules to which the conjugates simultaneously bind, resulting in high binding activity. The simultaneous detection targeting described herein is distinct from traditional multispecific approaches (e.g., bi / trispecific antibodies) because it dynamically enhances the effective affinity of the conjugate only when it is facing the targeted cell. In this way, the MV-MS conjugates can achieve selective delivery of the active agent (A) to targeted cells, thereby reducing off-target toxicity and other side effects associated with nonspecific or less specific delivery techniques.

[0060] As used herein, "specificity" refers to interacting partners that recognize only each other. For example, a binding motif binds to a receptor containing a given receptor sequence of amino acid sequence, and "specifically" binds to the receptor containing that portion, but does not bind to other receptors or proteins that lack that portion of the targeting sequence. A binding motif, or a conjugate containing a binding motif, binds to a receptor (or a variant or mutein thereof) "with specificity" when it binds to that receptor (or a variant or mutein thereof) with an ability that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, at least 15-fold, at least 20-fold, or at least 100-fold higher than its ability to bind to any other receptor tested.

[0061] Additionally, this conjugate can induce endocytosis through receptor microclustering (μC) and delivery of the conjugate's active agent (e.g., a therapeutic agent) to targeted cells via receptor microclustering (μC) (Figures 2A-2C). (See Coon et al., 2012, supra.) Multivalent binding induces the formation of high local cargo density, which can increase the number of endocytic sites and promote the initiation and maturation of endocytic vesicles. (Liu et al., J Cell Biology 191(7):1381-93(2010); Pedersen et al., J Cell Biology 219(11):e202002160(2020)). Microcluster-mediated uptake is unaffected by several types of receptor mutations that affect dimerization and classical endocytosis, as well as other scenarios that impair internalization (e.g., the presence of Her2 instead of EGFR).

[0062] Furthermore, this conjugate can address challenges arising from tumor variability due to the multiple specificities of the conjugate binding motifs.

[0063] Specific examples of binding motifs include, but are not limited to, a binding motif for FGFR3 that is or comprises VSPPLTLGQLLS (SEQ ID NO: 1) or a functional variant thereof (designated "F"), a binding motif for Her2 that is or comprises FCGDGFYACYMDV (SEQ ID NO: 2) or a functional variant thereof (designated "H"), a binding motif for IL-4Rα that is or comprises KLAKLAKKLAKLAK (SEQ ID NO: 3) or a functional variant thereof (designated "I"), and a binding motif for EGFR that is or comprises YHWYGYTPQNVI (SEQ ID NO: 4) or a functional variant thereof (designated "E").

[0064] As used herein, a "functional variant" of an amino acid sequence or peptide is an amino acid sequence or peptide that can provide the same biological function as the reference sequence or peptide. In some embodiments, the variant has fewer than 20, 11, 9, 8, 7, 6, 5, 4, 3, or fewer than 1 amino acid substitution compared to the reference sequence or peptide.

[0065] Desirably, the sequences maintain about 90% to about 100% identity (e.g., 90% identity to about 100% identity, about 90% identity to 100% identity, or 90% identity to 100% identity), about 95% identity to about 96% identity (e.g., 95% identity to about 96% identity, about 95% identity to 96% identity, or 95% identity to 96% identity), etc., or about 92.5% identity to about 97.5% identity (e.g., 92.5% identity to about 97.5% identity, about 92.5% identity to 97.5% identity, or 92.5% identity to 97.5% identity), etc. The ranges set forth in this paragraph include the defined endpoints and each 1% increment subsumed therein.

[0066] The term "identity," when referring to amino acid or polypeptide sequences, is defined as the percentage of amino acid or nucleic acid residues in a candidate sequence that are identical to those in a reference sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100 to obtain a percentage. Thus, two copies of the exact same sequence will have 100% identity, while two sequences that have relative amino acid deletions, insertions, or substitutions will have a lower degree of identity. Alignment for purposes of determining percent sequence identity can be accomplished by a variety of methods within the skill of the art, for example, using publicly available computer software. For example, determining the percent identity or similarity between sequences can be performed, for example, by using the GAP program (Genetics Computer Group, software, now available through Accelrys Online), and alignment can be performed, for example, using the ClustalW algorithm (VNTI software, InforMax Inc.). Furthermore, the nucleic acid or amino acid sequence of interest can be used to search sequence databases. Algorithms for database searches are typically based on BLAST software (Altschul et al., 1990), but those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm needed to obtain maximum alignment across the full length of the sequences being compared.

[0067] Linker

[0068] Adjacent binding motifs can be connected by a linker (L in Formula I). ​​In some embodiments, the binding motif and the active agent are connected by a linker. When the conjugate includes multiple linkers, the linkers can be the same or different (or any combination thereof).

[0069] Each linker can be any suitable linker. The linker can contain atoms selected from C, N, O, S, Si, and P; C, N, O, S, and P; or C, N, O, and S. The linker can have a backbone ranging in length from as few as two atoms to as many as 100 or more consecutive atoms in the linker backbone. The "backbone" of a linker is the shortest chain of consecutive atoms that forms a covalent link between adjacent binding motifs and / or between a binding motif and an active agent. In some embodiments, the polyvalent linker has a branched backbone, with each branch acting as a section of the backbone linker, all the way to the end.

[0070] Linkers can be non-releasable, i.e., non-labile, however, in some embodiments, it may be desirable for one or more linkers in a conjugate to be releasable, i.e., labile, such as, for example, photocleavable, acid-labile, base-labile, or enzyme-cleavable.

[0071] The length of each linker can be selected to optimize the separation imposed by the linker on the molecules at the surface of the targeted cell, which in turn can induce microclustering of cell surface molecules bound by the binding motif (e.g., when the conjugate is administered). The linker can have a chain length of at least about 5 nm. In some embodiments, each linker is approximately 5 nm to 15 nm in length. In some embodiments, the linker is at least about 7 nm in length. In some embodiments, each linker is approximately 7 nm in length and flexible. In some embodiments, each linker is approximately 7 to 10 nm in length. In some embodiments, the linker is at least about 14 nm in length. In some embodiments, the linker is approximately 15 nm in length. In some embodiments, the linker is about 7 nm to about 31 nm in length (e.g., about 7 to 31, 7 to about 31, or 7 to 31), about 7 nm to about 24 nm in length (e.g., about 7 to 24, 7 to about 24, or 7 to 24), or about 7 nm to about 20 nm in length (e.g., about 7 to 20, 7 to about 20, or 7 to 20). In some embodiments, the linker is about 14 nm to about 31 nm in length (e.g., about 14 to 31, 14 to about 31, or 14 to 31), about 14 nm to about 24 nm in length (e.g., about 14 to 24, 14 to about 24, or 14 to 24), or about 14 nm to about 20 nm in length (e.g., about 14 to 20, 14 to about 20, or 14 to 20). In some embodiments, the linkers have a chain length of at least 7 nm, at least 14 nm, at least 20 nm, at least 25 nm, at least 30 nm, or at least 40 nm, or 5 nm to 15 nm, 5 nm to 10 nm, 7 nm to 10 nm, 5 nm to 20 nm, 10 nm to 40 nm, or 25 nm to 100 nm. In certain embodiments, the length of each linker is selected to provide a separation of 7 to 10 nm or about 7 to 10 nm (e.g., about 7 nm to about 10 nm, 7 nm to about 10 nm, about 7 nm to 10 nm, or 7 nm to 10 nm) between them to promote microclustering of the bound molecules on the cell surface. The ranges specified in this paragraph include the specified endpoints and all 1 nm increments subsumed within the specified range.

[0072] The linker can include at least one carbon-carbon bond and / or at least one amide bond. The linker can include one or more natural or unnatural amino acids in the L or D configuration, or any combination of the foregoing.

[0073] In some embodiments, a linker is a group comprising one or more covalently linked structural units.

[0074] In some embodiments, the linker group is an optionally substituted polyethylene glycol (PEG) having between 1 and about 100 ethylene glycol units. In some embodiments, the linker is substituted with an aryl, phenyl, benzyl, alkyl, alkylene, or heterocyclic group. In some embodiments, the linker is asymmetric. In some embodiments, the linker is symmetric.

[0075] Alternatively, or in addition to chain length, in some embodiments, the linker can have suitable substituents that affect its hydrophobicity or hydrophilicity. Thus, for example, the linker can have a hydrophobic side group, such as an alkyl, cycloalkyl, aryl, arylalkyl, or similar group, each of which is optionally substituted. When the linker comprises one or more amino acids, the linker can contain hydrophobic amino acid side chains, such as those derived from Phe and Tyr, including substituted variants thereof, and analogs and derivatives of such side chains.

[0076] The linker can include a spacer (e.g., conjugated to a spacer and / or include a spacer). The spacer can be any suitable spacer. The spacer of the linker can include hydrophilic, hydrophobic, amphipathic, non-peptidic, peptidic, and / or aromatic monomers. The length of the spacer can range from 1 to 30 (e.g., PEG having 1 to 30 carbon atoms, 1 to 30 units, etc.). Examples of hydrophilic spacers include, but are not limited to, PEG polymers and derivatives thereof. Examples of hydrophobic spacers include, but are not limited to, pure or mixed branched hydrocarbon, fluorocarbon, alkane, alkene, and / or alkyne polymers. Examples of amphipathic spacers include, but are not limited to, pure or mixed phospholipids and / or derivatives thereof. Examples of peptidic spacers include, but are not limited to, pure or mixed single, branched, L- or D-configuration, essential, non-essential, natural, and unnatural amino acids, and derivatives thereof. Examples of aromatic spacers include, but are not limited to, pure and mixed repeating quinoids.

[0077] In some embodiments, the linker is formed via click chemistry / click chemistry-derived synthetic methods. Those skilled in the art will appreciate that the terms "click chemistry" and "click chemistry-derived" generally refer to a class of small molecule reactions commonly used in conjugation, which allow selected substrates to be conjugated with specific molecules. While click chemistry is not a single specific reaction, it describes a method for generating products that follows natural examples, and it also generates substances by joining small modular units. In many applications, click reactions join biomolecules and reporter molecules. Click chemistry is not limited to biological contexts; the "click" reaction concept has been used in pharmacological and various biomimetic applications. However, it has been found to be particularly useful in the detection, localization, and qualification of biomolecules.

[0078] Click reactions can occur in one pot, are typically unhindered by water, produce minimal by-products, and are "spring-loaded"—characterized by a high thermodynamic driving force that rapidly and irreversibly drives high yields of a single reaction product with high reaction specificity (in some cases, both regio- and stereospecificity). These properties make click reactions well-suited to the problem of isolating and targeting molecules in complex biological environments. In such environments, therefore, products must be physiologically stable, and any by-products must be non-toxic (e.g., for in vivo systems).

[0079] In some embodiments, each linker has an amino acid sequence independently selected from GRAQGKAQG (SEQ ID NO: 5), GQAKGQARG (SEQ ID NO: 6), GKQAGRQAG (SEQ ID NO: 7), and GQRAGQKAG (SEQ ID NO: 8). In some embodiments, each linker has an amino acid sequence independently selected from GRAQGKAQG (SEQ ID NO: 5), GQAKGQARG (SEQ ID NO: 6), GKQAGRQAG (SEQ ID NO: 7), and GQRAGQKAG (SEQ ID NO: 8), is approximately 7 nm to 10 nm in length (e.g., 7 nm, 8 nm, 9 nm, or 10 nm), and is flexible.

[0080] Active Agent

[0081] The active agent can be attached to one or the other end of the alternating motif and linker chain, or to the side chain of an amino acid, such as an amino acid that includes the linker. The active agent can be attached directly to the alternating motif and linker chain, such as by covalent bonding or cross-linking. Alternatively, the active agent can be attached to a nanoparticle or encapsulated in a liposome, where the nanoparticle or liposome is attached to the alternating motif and linker chain.

[0082] "Liposome" refers to small, spherical vesicles composed of lipids, specifically vesicle-forming lipids that can spontaneously assemble into lipid bilayer structures in water, with a hydrophobic portion, which is the hydrophobic region of the bilayer membrane that contacts the interior, and a head group, which is the polar surface of the membrane that faces the exterior. Vesicle-forming lipids typically have two hydrocarbon chains, particularly acyl chains, and either a polar or nonpolar head group. Vesicle-forming lipids are composed of either naturally occurring or synthetic lipids, including phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, and sphingomyelin, where the two hydrocarbon chains are typically about 14-22 carbon atoms in length and have varying degrees of unsaturation. Such lipids and phospholipids can be obtained commercially or prepared according to published methods. Other suitable lipids include glycolipids and sterols, such as cholesterol and its various analogs. Cationic lipids can also be used in liposomes, typically having a hydrophilic moiety such as a sterol, acyl, or diacyl chain, where the lipid has an overall net positive charge. The head group of the lipid typically carries a positive charge. The cationic vesicle-forming lipid may be a neutral lipid or an amphiphilic lipid derivatized with a cationic lipid. The liposome may contain a vesicle-forming lipid derivatized with a hydrophilic polymer to form a surface coating of hydrophilic polymer chains on the surface of the liposome. The polymer may be a block / random polymer. For the preparation of vesicle-forming lipids derivatized with hydrophilic polymers, see, for example, US Pat. No. 5,395,619, US Pat. No. 5,013,556, US Pat. No. 5,631,018, and International Patent Application Publication No. WO 98 / 07409.

[0083] The active agent can be or can include a therapeutic (or prophylactic) agent, an imaging agent, a diagnostic agent, a compound comprising a drug moiety, etc., and / or a pharmaceutically acceptable salt of such a compound or drug moiety.

[0084] In some embodiments, the active agent is or includes an imaging agent (e.g., covalently or non-covalently attached directly or indirectly (e.g., via a nanoparticle) to a binding moiety via a linker), such as a radioimaging agent, radiosensitizing agent, radioprotective agent, or radiotherapeutic agent.

[0085] Examples of imaging agents include, but are not limited to, metals or isotopes suitable for radiological imaging, positron emission tomography (PET) imaging, single photon emission computed tomography (SPECT) imaging, or magnetic resonance imaging, fluorescent contrast agents, photodynamic contrast agents, or optical contrast agents. Metals include, but are not limited to, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or a derivative thereof, TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid) or a derivative thereof, SarAr (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexazabicyclo[6.6.6]-eicosane-1,8-diamine or a derivative thereof, NOTA (1,4,7-triazacyclopentadiene-1,8-diamine or a derivative thereof), CITA (CITA-1,4,7,10-triazabicyclopentadiene-1,8-diamine or a derivative thereof), CITA-1,4,7,10-triazabicyclopentadiene-1,8-diamine or a derivative thereof, ... nonan-1,4,7-triacetic acid) or a derivative thereof, NETA (4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl)acetic acid or a derivative thereof, TRAP (1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid) or a derivative thereof, HBED (N,N0-bis(2-hydroxybenzyl)-ethylenediamine-N,N0-diacetic acid) or a derivative thereof, ) or derivatives thereof, 2,3-HOPO (3-hydroxypyridin-2-one) or derivatives thereof, PCTA (3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-3,6,9-triacetic acid) or derivatives thereof, DFO (desferrioxamine) or derivatives thereof, DTPA (diethylenetriaminepentaacetic acid) or derivatives thereof, OCTAPA (N,N0-bis(6-carboxy-2-pyridylmethyl)methyl) H2-MACROPA (N,N'-bis[(6-carboxy-2-pyridipmethyl)]-4,13-diaza-18-crown-6) or its derivatives, H2-DEDPA (1,2-[[carboxy)-pyridin-2-yl]-methylamino]ethane or its derivatives, and EC containing β-l-diaminopropionic acid, aspartic acid, and cysteine. 20 The metal chelating group can be chelated with a chelating group selected from the group consisting of: 11W, 13 W, 13 N 15 THE, 18 F 32 P 44 Sc 47 Sc 52 Mn、 55 Co 60 Co 64 Ass, 67 Ass, 67 Ga 68 Ga 86 Y 89 Mr. 89 Zr 90 Y 99m Tc 111 In 114m In 117m Sn、 123 I 124 I 125 I 131 I 149 Also, 153 Sm、 152 Also, 155 Also, 161 Also, 169 Er, 177 Lu 186 Re, 188 Re, 211 At 212 Pb 212 Bi, 213 Bi, 223 Frog, 224 Frog, 225 Ab、 225 Ac、または 227Fluorescent imaging agents include carbocyanines, indocarbocyanines, oxacarbocyanines, thiacarbocyanines and merocyanines, polymethines, coumarins, rhodamines, xanthenes, fluoresceins, borondipyrromethane (BODIPY), CyS, CyS.S, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S7S0, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor7S0, AlexaFluor790, Dy677, Dy676, Dy682, Dy7S2, Dy780, DyLightS47, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 7S0, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, ADS832WS and S0456.

[0086] The active agent can be or can include a therapeutic agent. The term "therapeutic agent" is intended in its broadest sense to include a compound, chemical, microorganism, or any agent capable of producing an effect in a subject or in biological tissues or cells when administered. Thus, the term includes both prophylactic and therapeutic agents, as well as diagnostic agents and any other category of agent capable of having a desired effect. Therapeutic agents include, but are not limited to, pharmaceuticals and vaccines, nucleic acid sequences (such as supercoiled, relaxed, and linear DNA and fragments thereof, antisense constructs, artificial chromosomes, RNA and fragments thereof, and any other nucleic acid-based therapy), cytokines, small molecule drugs, proteins, peptides and polypeptides, oligonucleotides, oligopeptides, fluorescent molecules (e.g., fluorophores) and other imaging agents, hormones, chemotherapy, and combinations of interleukins, lectins, and other stimulatory agents.

[0087] In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound (e.g., a compound capable of disrupting cellular mechanisms important for cell survival and / or proliferation and / or causing apoptosis). In some embodiments, the therapeutic agent comprises a therapeutic drug selected from the group consisting of an anti-cancer agent, a chemotherapeutic agent, a cytotoxic drug, an immunomodulator, an immunosuppressant, an immunostimulator, and / or an agonist.

[0088] Examples of therapeutic agents include, but are not limited to, erdafitinib, cisplatin, doxorubicin, monomethyl auristatin E, erdafitinib, mitomycin, pemigatinib, SN-38, thiotepa, barbican, a combination of cisplatin and gemcitabine, and MVAC (methotrexate, vinblastine sulfate, doxorubicin hydrochloride, and cisplatin).

[0089] The immunomodulatory agent can be any suitable immunotherapeutic drug. Examples of suitable immunotherapeutic agents include, but are not limited to, transforming growth factor beta (TGF-β) inhibitors, such as R268712.

[0090] The anti-cancer agent can be any suitable anti-cancer drug. Examples of suitable anti-cancer drugs include, but are not limited to, kinase inhibitors such as dasatinib.

[0091] The chemotherapeutic agent can be any suitable chemotherapy drug. Examples of suitable chemotherapy drugs include, but are not limited to, an anthracycline such as doxorubicin, a taxane such as docetaxel, a cyclophosphamide such as cytoxin, or 5-fluorouracil.

[0092] The conjugate can comprise SEQ ID NO: 9 or a functional variant thereof. The conjugate can comprise SEQ ID NO: 10 or a functional variant thereof. Such sequences can tolerate some modifications as discussed above.

[0093] When targeting cells other than cancerous cells (e.g., diseased or genetically altered cells), the active agent can be any type of therapeutic agent effective against the specific condition or cells being targeted.

[0094] The conjugates can be synthesized according to methods known in the art. Solid-phase synthesis, solution-phase synthesis, or a combination of both can be used. The peptide chain can be assembled on the solid phase, and cyclization or other modifications can be performed on the resin or in solution (see, e.g., Chan and White, Fmoc Solid Phase Peptide Synthesis, A Practical Approach, Oxford University Press (2000), and references cited therein). The conjugates are easily purified with high yields. See, e.g., Figure 3C.

[0095] composition

[0096] In view of the above, a composition comprising the above-mentioned conjugate and a pharmaceutically acceptable carrier is also provided. "Pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as, but not limited to, buffers, preservatives, anesthetics, solubilizers, isotonicity agents, wetting agents, and stabilizers. The term also encompasses any drug approved by a regulatory agency, such as the U.S. Food and Drug Administration, or listed in the U.S. Pharmacopoeia for use in animals (e.g., mammals, such as humans). The carrier can be a phosphate-buffered saline solution, water, or an emulsion, such as an oil / water or water / oil emulsion.

[0097] The composition can include a pharmaceutically acceptable salt, hydrate, or solvate of the conjugate.

[0098] The composition can include at least two different conjugates, both of which contain binding motifs that bind to the same four cell surface molecules that are overexpressed or selectively expressed on targeted cells (e.g., cancerous cells), but where the order of the binding motifs differs between the two conjugates. The composition can include (i) a first conjugate containing SEQ ID NO: 9 or a functional variant thereof and (ii) a second conjugate containing SEQ ID NO: 10 or a functional variant thereof. Such sequences and functional variants can tolerate some modifications as discussed above.

[0099] Uses and Methods

[0100] Further provided is a method for selectively targeting cells (e.g., diseased cells, cancer cells, or another type of cell) in a subject for endocytosis of an active agent. The method can include administering to the subject an effective amount of any of the conjugates or compositions described herein. As used herein, "subject" refers to any warm-blooded animal, vertebrate, domesticated animal (e.g., livestock, horses, cats, and dogs), and humans.

[0101] An "effective" amount is the amount of active agent that provides a desired effect, such as an anti-cancer effect or the ability to image cancerous cells, in a subject. The amount that is "effective" will vary from subject to subject, depending on the age and general condition of the individual, the mode of administration, etc. Therefore, it is not always possible to specify an exact "effective amount." However, an appropriate "effective amount" can be determined by one of ordinary skill in the art using routine experimentation.

[0102] In some embodiments, the method includes selectively targeting cancerous cells in a subject for endocytosis of an active agent, comprising administering an effective amount of (a) a conjugate or a pharmaceutically acceptable salt thereof, or (b) this composition, wherein the active agent of the conjugate or pharmaceutically acceptable salt comprises an anticancer agent. The cancerous cells can endocytose the active agent (e.g., an anticancer agent), and the endocytosis of the active agent treats the subject for a disease state such as cancer. The subject can have bladder cancer (e.g., transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and combinations thereof). In some embodiments, the bladder cancer is NMIBC. When the cancerous cells endocytose the anticancer agent, the endocytosis of the anticancer agent can treat the subject for cancer.

[0103] It will be understood that the disease state can include any disease state in which the targeted cells overexpress or selectively express two or more cell surface molecules that can be targeted by the binding motifs of the conjugate.

[0104] In one embodiment, a method comprises selectively targeting diseased cells in a subject for endocytosis of a therapeutic agent, comprising administering an effective amount of (a) this conjugate or (b) this composition, wherein the active agent of the conjugate comprises a therapeutic agent, and the diseased cells are able to endocytose the therapeutic agent, wherein endocytosis of the therapeutic agent treats the subject for the disease.

[0105] The composition can be administered by any suitable route known in the art.For administration to a subject with bladder cancer, the composition can be directly administered to the bladder lumen, such as by injection or catheterization.In some embodiments, when a subject suffers from a genetic condition associated with specific kidney cells, the composition can be administered (and formulated to be administered) directly to the kidney and / or via injection into the bloodstream.

[0106] "Treat," "treating," "treated," and "treatment" are used to refer to the prevention, alleviation, or elimination of symptoms and signs associated with a specific disease, disorder, or condition (e.g., cancer). "Treating" cancer can include, for example, maintaining, reducing, or eliminating detectable cancer from a subject, such as a patient, and / or inhibiting or preventing its metastasis.

[0107] Also provided is a method for imaging or diagnosing a subject having a disease state. In one embodiment, the subject has cancer. The method can include (a) administering to the subject an effective amount of the conjugate, a pharmaceutical salt thereof, or a composition described herein, wherein the active agent of the conjugate or its pharmaceutically acceptable salt comprises an imaging agent, and (b) imaging the subject or allowing the subject to be imaged. In one embodiment, the disease state is cancer.

[0108] Imaging the subject can include performing radiographic imaging, PET imaging, SPECT imaging, magnetic resonance imaging, or using any other imaging technique known in the art. Imaging the subject can further include imaging an area of ​​the subject affected by a disease state (e.g., the location of a tumor, a kidney where the subject is experiencing a genetic condition affecting the kidney, etc.). In some embodiments, the imaging agent includes a metal or isotope suitable for radiographic imaging, PET imaging, SPECT imaging, or magnetic resonance imaging, or a fluorescent contrast agent, a photodynamic contrast agent, or an optical contrast agent.

[0109] It will be understood that the selective targeting of this conjugate and composition can be used to identify the location of targeted cells in a subject using imaging techniques.In addition or alternatively, this conjugate and composition can be used to diagnose disease states.For example, when this conjugate is administered to a subject, where this conjugate targets specific targeted cells, imaging the subject after administering the conjugate will provide a positive indication of the presence of targeted cells, which can support that the subject has targeted cells.In such an example, the method can further include treating the subject for a disease state associated with targeted cells.

[0110] Also provided is the use of the conjugate or composition in the preparation of a medicament for treating a disease state in a subject. In some embodiments, the disease state is cancer. In some embodiments, the disease state is bladder cancer. In some embodiments, the disease state is NMIBC.

[0111] example The present disclosure will be better understood with reference to the following examples, which are provided as illustrative examples and not by way of limitation.

[0112] Example 1 Production and purification of conjugates for the treatment of bladder cancer The advantages of this conjugate can be illustrated by their application to cancer cells. Given the ready availability of reagents and cell lines, this disclosure is described in the context of bladder cancer, but this approach is applicable to any form of cancer.

[0113] Invasive bladder cancer is treated by radical cystectomy, the removal of the bladder. Non-muscle-invasive bladder cancer (NMIBC) is currently treated with therapeutic agents that have very limited efficacy and poor cancer selectivity. Current anti-bladder cancer drugs do not adapt to tumor cell variability, which narrows the range of patients who can be treated with some success and therefore leads to cancer progression, recurrence, tumor resistance, and severe side effects. In addition, the global shortage of Bacillus Calmette-Guerin (BCG), the adjuvant therapy of choice for NMIBC, highlights the need for other treatment options. Collectively, these factors result in bladder cancer having the highest postoperative recurrence rate (>75%) of any malignant tumor, with 25% of cases progressing to invasive bladder cancer leading to radical cystectomy. The resulting economic burden on the U.S. healthcare system currently exceeds $4 billion per year.

[0114] A common practice in NMIBC treatment is the application of therapeutic agents into the bladder lumen to take advantage of the differential exposure of tumor cells and normal cells shielded by a glycosaminoglycan (GAG) layer, as shown in Figure 1. This treatment approach allows access to tumor cells, but diffusion of the therapeutic agent through loose tumor-cell junctions allows the anti-cancer therapeutic agent to contact normal (e.g., healthy) cells / tissues as well as blood vessels. Even if the administration of the therapeutic agent is limited and tumor regression, even if transient, increases the likelihood that the agent will come into contact with normal cells / tissues.

[0115] Potential side effects on normal cells / tissues can be reduced by utilizing agents that are selective for cancer cells, which is particularly important when cell surface receptor expression can vary within tumors, between tumors in a single patient, and between tumors in different patients with the same diagnosis.

[0116] In consideration of the above, we provide an MV-MS conjugate that includes a low-affinity binding motif for interacting with cell surface receptors on cancer cells to achieve high binding activity. In contrast, the low density of such cell surface receptors on normal cells does not allow binding driven by avidity, allowing the MV-MS conjugate to interact with cell surface receptors on normal cells with low affinity, i.e., very weak and transient, if any. In other words, the MV-MS conjugate exhibits low affinity to normal cells while differentially exhibiting strong and persistent interaction with cancer cells.

[0117] The binding motif is selected based on the upregulation of two or more cell surface receptor targets, selected from a strategically selected group of receptors that can be simultaneously bound by the MV-MS conjugate. As described above, receptor microcluster binding can induce endocytosis of the conjugate and, consequently, intracellular delivery of the active agent contained in the conjugate. Multivalent binding induces the formation of high local cargo density, which increases the number of endocytic sites and promotes the initiation and maturation of endocytic vesicles.

[0118] The MV-MS conjugates were used to detect FGFR3 (Jin et al., FGFR3 signaling and reverses the lethal phenotype of mice mimicking human thanatophoric dysplasia, Human Molecular Genetics 21(26):5443-55(2012)), Her2 (Florczak et al., Cellular uptake, intracellular distribution and degradation of Her2-targeting silk nanospheres 2019:6855-6865(2019)), IL-4Rα (Permpoon et al., Inhibition of tumor growth against chemoresistant cholangiocarcinoma by a proapoptotic peptide targeting interleukin-4 receptor Molecular Pharmaceutics 17(11):4077-4088(2020)), and EGFR (Li et al., Identification and characterization of a novel peptide ligand of It was prepared by combining validated low-affinity binding peptides to epidermal growth factor receptor (FASEB J 19(14):1933-2088(2005)).

[0119] Each of the at least four binding motifs included VSPPLTLGQLLS (SEQ ID NO: 1) (binding motif for FGFR3, designated "F"), FCGDGFYACYMDV (SEQ ID NO: 2) (binding motif for Her2, designated "H"), KLAKLAKKLAKLAK (SEQ ID NO: 3) (binding motif for IL-4Rα, designated "I"), and YHWYGYTPQNVI (SEQ ID NO: 4) (binding motif for EGFR, designated "E"). Peptide "E" had a binding affinity for EGFR of less than about 500 μM, such as 459 μM. Peptide "H" had a binding affinity for Her2 of less than about 0.5 μM, such as 0.3 μM. Peptide "F" had a binding affinity for FGFR3 of about 1 μM, such as 1 μM. Peptide "I" had a binding affinity for IL-4Rα of less than about 60 μM, such as 55.9 μM.

[0120] A linker was positioned between each adjacent binding motif, each linker was approximately 7 nm in length and flexible, and the conjugate further contained an HA-tag for immunodetection. Each linker had an amino acid sequence independently selected from GRAQGKAQG (SEQ ID NO: 5), GQAKGQARG (SEQ ID NO: 6), GKQAGRQAG (SEQ ID NO: 7), and GQRAGQKAG (SEQ ID NO: 8).

[0121] Two conjugates were prepared, purified, and tested (Figure 3C), the first containing SEQ ID NO:9 (designated "P1:EIHF") and the second containing SEQ ID NO:10 (designated "P2:HEFI"). Peptides "P1" and "P2" covered all receptor microclustering possibilities for NMIBC, achieving multiple specificities and addressing patient and tumor variability (see, e.g., Figure 3B).

[0122] The tested conjugates targeted and internalized bladder cancer cells. For a description of the methodology, see Coon et al. (2012) supra. See, e.g., Figures 3D and 3E. Given the receptor conservation across species, the conjugates targeted cells of human, mouse, and canine origin (Figure 3D) and were internalized into GFP-Rab5-positive endosomes in a time-dependent manner (Figure 3E) (see, e.g., Figure 3D, middle panel set). In contrast to nonspecific interactions, preliminary results showed a time-dependent binding of the ligand to MB49 cells (see, e.g., Figure 3F).

[0123] Example 2 Cell-free binding assay To determine the binding of a nanoparticle-containing conjugate (MV-MS-NP) to bladder cancer cells compared with control cells, different concentrations of fluorescent nanoparticles, either decorated with the conjugate or not, were incubated with a test panel of cells at 4°C (to avoid uptake) for 45 minutes (see Jack et al. (2020)). After washing, binding was measured by flow cytometry, quantitative microscopy, and quantitative Western blotting (Figure 3G). To provide an additional and independent method for determining binding, this assay used membranes isolated from bladder cancer cells, thus lacking the confounding effect of uptake on binding.

[0124] To measure the rate and extent of MV-MS-NP uptake by bladder cancer cells compared with control cells, different concentrations of fluorescent nanoparticles decorated with this conjugate or not were incubated with a test panel of cells for different times (0–30 min) at 37 °C. After an acid wash to remove non-internalized nanoparticles, the internalized fraction was assessed by flow cytometry, quantitative microscopy, and quantitative Western blotting.

[0125] To determine the extent of MV-MS-NP-mediated delivery of a cytotoxic load to bladder cancer cells versus control cells, MV-MS-NPs loaded with or without MMC were used, with a 30-minute fixed exposure time followed by washing and incubation in complete medium, as described above. After 48 hours, viability was measured by MTT assay. (Jack et al., 2020).

[0126] Example 3 Simultaneous detection of receptor concentration and receptor upon conjugate binding in cancer cells To test the ability of the ligands to self-regulate affinity, an ELISA assay was designed (Figure 3H). Purified EGFR and / or Her2 extracellular domains (ECDs) produced by bladder cancer cells, His6-tagged, and engineered to be soluble, were immobilized on anti-His6 plates. Normal cells were simulated at low ECD density, while cancer cells displaying upregulated levels of one or two receptors were simulated at high density with one ECD (amount of a single EGFR ECD = amount of a single Her2 ECD) or two ECDs (EGFR ECD + Her2 ECD = amount of either single ECD) (Figure 3H).

[0127] Although rebinding caused by a single ECD at high density led to conjugate retention higher than normal cell levels, simultaneous detection of two ECDs revealed substantial avidity effects that led to superior binding of the MV-MS conjugate. Nonlinear regression of these preliminary results generated estimates for the observed affinities. Specifically, for low ECD densities, the peptide bound with low affinity (>6 μM), whereas rebinding effects due to high ECD densities led to an effective affinity of ∼0.1 μM, and simultaneous detection revealed high avidity (<0.5 nM).

[0128] Example 4 MV-MS-mediated delivery of nanoparticles to bladder cancer cells Nanomolar concentrations of the MV-MS conjugate were highly efficient in binding and delivering nanoparticles to bladder cancer cells (Figure 3I). The conjugate was stable and required only small amounts of the drug to induce substantial uptake by cancer cells.

[0129] Normal (labeled A) and MB49 wild-type cancer (labeled B) cells isolated from bladder urothelium were disaggregated and cultured in vitro. Cultured cells were incubated with different concentrations of P2 MV-MS conjugate, and the presence of cell-associated peptide was examined by immunostaining with an anti-HA antibody using the methodology described in Example 1.

[0130] The P2 conjugate exhibited remarkable selectivity for cancer cells, with no detectable binding or uptake into normal urothelium (Figures 4A-4C).

[0131] Example 5 In vitro binding studies The in vitro ability of the conjugates to bind, internalize, and deliver a cytotoxic payload was evaluated in different cohorts of cells: dogs bearing spontaneous bladder tumors, mouse orthotopic models of bladder cancer bearing tumors with engineered expression profiles (i.e., lacking upregulation of several receptors and / or expressing different combinations of FGFR3, EGFR, Her2, and IL4Rα), and immortalized normal urothelium and unmodified MB49 cells as controls. LE These include cell lines and disaggregated cells obtained from resected spontaneous bladder cancer tumors from dogs and human subjects with different expression profiles.

[0132] To measure the uptake rate of conjugates by bladder cancer versus control cells, serum-starved cells were incubated with 100 μM of the selected conjugate for 45 min at 4°C. After removal of unbound conjugate by ice-cold washing, 37°C-warmed medium was added and the cells were incubated at 37°C for different periods (ranging from 0 to 30 min) to allow uptake. During this incubation period, Binds but does not internalizeThe drug is stripped using an acid wash at 4°C.

[0133] The internalized fraction of the conjugate is assessed by quantitative Western blotting, flow cytometry, and quantitative microscopy. For the latter, after removal of non-internalized conjugate, samples are fixed and the sample and control GFP-Rab5 Q79L Uptake is measured by determining cell-associated fluorescence intensity inside labeled endosomes (Image J). Ligands can be detected based on the presence of an HA tag at the C-terminus of the conjugate. Cell density is determined by counting DAPI structures ≥ 7 microns in diameter.

[0134] A similar approach is followed for the attachment of conjugate-decorated nanoparticles (NPs): pH-sensitive (PEG-stabilized to deliver encapsulated cytotoxic drugs from acidic compartments to the cytoplasm) nanoliposomes (50 nm diameter, compatible with endocytic vesicles) are selected as NP carriers and prepared according to known protocols.

[0135] Briefly, a chloroform solution of 13 μmol of total lipids (DPPC:cholesterol:DSG-PEG2K-NTA:DSPE-Cy5, 64.46:35:0.04:0.5) was prepared. (This ratio, based on a 50 nm diameter nanocarrier, is estimated to yield NPs with PEG2K-NTA-His6-MV-MS conjugates separated by 25 nm.) The solution was carefully evaporated under a stream of N2 gas to produce a uniform thin film. The film was placed under a 50 μm Hg vacuum for 3 hours to remove traces of solvent impurities, and then hydrated in 4 ml of 15 mM 4-(2-hydroxyethyl)-piperazineethanesulfonic acid (HEPES) (with and without 0.4 mg / ml mitomycin C (MMC), 50% encapsulation efficiency) via 10 freeze-thaw-vortex cycles. The resulting nanocarrier solution was extruded seven times at 50-55 °C through three laminated polycarbonate filters with pore sizes of 800 nm, 200 nm, and 50 nm using an extrusion device charged with N2 pressure of 200-300 psi. 2+ : Tris-NTA content is monitored in each formulation by inductively coupled plasma mass spectrometry (ICP-MS).

[0136] NTA-NPs and His6 ligand (1:20 ratio) were incubated at room temperature for 1 hour, followed by removal of unbound peptides. Retention of the His6 ligand was adjusted as previously described, leading to a loss of ≦10% of the ligand from the NPs over 24 hours of incubation at room temperature. The use of a low NTA density (see above) and a tag with ≦6 histidines can reduce nonspecific binding and increase NP stability.

[0137] Example 6 In vivo binding studies Dogs bearing spontaneous bladder tumors, mouse orthotopic models of bladder cancer bearing tumors with engineered expression profiles (i.e., lacking upregulation of several receptors and / or expressing different combinations of FGFR3, EGFR, Her2, and IL4Rα), and immortalized normal urothelium and unmodified MB49 as controls.LE The in vivo antitumor activity of the conjugates will be evaluated in cell lines. A cohort of mice, MB49, stably expressing the targeted receptor after electrocautery will be used. LE Implantation of the cells into the bladder produces tumors.

[0138] Briefly, using a limited number of animals (n=6 for each test cohort), tumor size in the mouse cohort is determined for each animal after treatment with the conjugate or placebo, and tumor size in the dog cohort is determined for each animal both before and after treatment with the conjugate.

[0139] The extent of penetration for each agent is also determined by immunohistochemistry on post-treatment tumor biopsies.

[0140] general

[0141] All patents, patent application publications, journal articles, textbooks, and other publications mentioned in this specification are indicative of the levels of those skilled in the art to which this disclosure pertains, and all such publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0142] The invention illustratively described herein may suitably be practiced in the absence of any element(s) or limitation(ies) not specifically disclosed herein. Thus, for example, any instances of the terms "comprising," "consisting essentially of," and "consisting of" herein may be replaced with either of the other two terms. Similarly, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more types of methods and / or steps described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0143] The terms "about" and "approximately" are used interchangeably herein when referring to a number or numerical value or range (e.g., including integers, fractions, and percentages), and each means that the referenced number or numerical range is an approximation within experimental variation (or within statistical experimental error), and thus the numerical value or range can vary by up to 10% of the stated number or numerical range (e.g., ±5% to 10% of the recited value), to the extent that one of ordinary skill in the art would consider it equivalent to the recited value (e.g., having the same function or result). The term "about" or "approximately" allows for some variation in the value or range, for example, including the specified endpoints, and within 90%, 95%, 99%, 99.5%, 99.9%, 99.99%, or 99.999% or more of the stated endpoints of the stated value or range. Each value or range of values ​​preceded by the term "about" is also intended to encompass the embodiment of the stated absolute value or absolute range of values.

[0144] The terms and expressions employed are used as explanations of terms and not as limitations of terms. In this regard, if a term is defined and otherwise described or discussed elsewhere in the "Detailed Description," all such definitions, explanations, and discussions are intended to be attributed to such terms. Nor is there any intention in the use of such terms and expressions to exclude any equivalents of the features shown and described or portions thereof. Furthermore, although subheadings may be used in the "Detailed Description," such use is merely for ease of reference and is not intended to limit any disclosure made in one section to only that section; rather, any disclosure made under one subheading is intended to constitute a disclosure under all other subheadings.

[0145] It is recognized that various modifications are possible within the scope of the invention as claimed. Thus, while the invention has been specifically disclosed in the context of preferred embodiments and optional features, those skilled in the art may employ modifications and variations of the concepts disclosed herein. Such modifications and variations are deemed to be within the scope of the invention as claimed herein.

Claims

1. (a) at least two or more binding motifs; wherein each of the at least two or more binding motifs binds to a different cell surface molecule that is overexpressed or selectively expressed in the targeted cell; wherein adjacent binding motifs are separated from each other by a linker, and (b) a conjugate comprising an active agent, wherein the active agent is capable of being endocytosed by a targeted cell to which the conjugate binds; wherein, when comprising more than one linker, said linkers can be the same or different from each other; The conjugate, or a pharmaceutically acceptable salt thereof.

2. Formula I: (Chem.1) BM-(LMM) ｎ -L-A (I) 2. The conjugate of claim 1, comprising the structure: wherein each BM is one of said at least two binding motifs; each L is a linker; n is 1 to 5; A is the active agent; The conjugate, or a pharmaceutically acceptable salt thereof.

3. 3. The conjugate of claim 2, wherein n is 1 to 3.

4. 2. The conjugate of claim 1, wherein the targeted cell is a pathological cell (e.g., a cancerous cell).

5. 3. The conjugate of claim 2, wherein the targeted cell is a pathological cell (e.g., a cancerous cell).

6. 2. The conjugate of claim 1, wherein the conjugate comprises at least four binding motifs, each of which binds to a different cell surface molecule that is overexpressed or selectively expressed on the targeted cell.

7. 7. The conjugate of claim 6, wherein each cell surface molecule is selected from the group consisting of a transporter, a receptor, and a cell-to-cell communication protein.

8. The conjugate of any one of claims 1 to 7, wherein each cell surface molecule is a cell surface receptor.

9. 7. The conjugate of claim 6, wherein each cell surface molecule is a cell surface receptor.

10. wherein the conjugate comprises at least four binding motifs, each of which binds to a different cell surface receptor that is overexpressed or selectively expressed on the targeted cell and is selected from the group consisting of fibroblast growth factor receptor 3 (FGFR3), Her2, interleukin-4 receptor alpha (IL-4Rα), and epidermal growth factor receptor (EGFR); The conjugate of claim 2 , wherein the targeted cell is a cancerous cell.

11. 4. The conjugate of claim 3, wherein the binding motif for FGFR3 is SEQ ID NO: 1 or a functional variant thereof (designated "F"), the binding motif for Her2 is SEQ ID NO: 2 or a functional variant thereof (designated "H"), the binding motif for IL-4Rα is SEQ ID NO: 3 or a functional variant thereof (designated "I"), and the binding motif for EGFR is SEQ ID NO: 4 or a functional variant thereof (designated "E").

12. 12. The conjugate of any one of claims 1 to 7 and 9 to 11, wherein each linker is approximately 5 nm to 15 nm in length.

13. 12. The conjugate of any one of claims 1 to 7 and 9 to 11, wherein each linker is approximately 7 to 10 nm in length.

14. 12. The conjugate of any one of claims 1 to 7 and 9 to 11, wherein each linker is approximately 7 nm in length and flexible.

15. 2. The conjugate of claim 1, wherein each linker has an amino acid sequence independently selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:

8.

16. 16. The conjugate of claim 15, wherein each linker is approximately 7 nm in length and flexible.

17. 17. The conjugate of any one of claims 1 to 7, 9 to 11, 15, and 16, wherein the active agent is an anti-cancer therapeutic agent or an imaging agent.

18. 17. The conjugate of any one of claims 1 to 7, 9 to 11, 15, and 16, wherein the active agent is attached to a nanoparticle or encapsulated in a liposome, and wherein the nanoparticle or liposome is attached to a linker of the conjugate.

19. 10. The conjugate of claim 1, wherein the active agent comprises an imaging agent.

20. 17. The conjugate of any one of claims 1 to 7, 9 to 11, 15 and 16, comprising SEQ ID NO: 9 or a functional variant thereof.

21. 17. The conjugate of any one of claims 1 to 7, 9 to 11, 15 and 16, comprising SEQ ID NO: 10 or a functional variant thereof.

22. 17. The conjugate of any one of claims 1 to 7, 9 to 11, 15 and 16, wherein each binding motif is a low affinity binding motif.

23. A composition comprising the conjugate according to any one of claims 1 to 22 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

24. 24. The composition of claim 23, comprising two conjugates, both of which contain binding motifs that bind to the same four cell surface molecules that are overexpressed or selectively expressed on targeted cells, but wherein the order of the binding motifs differs between the two conjugates.

25. 25. The composition of claim 24, comprising (i) a first conjugate comprising SEQ ID NO: 9 or a functional variant thereof and (ii) a second conjugate comprising SEQ ID NO: 10 or a functional variant thereof.

26. and (b) a composition according to any one of claims 23 to 25, wherein the composition comprises a pharmaceutically acceptable salt thereof or a conjugate according to any one of claims 1 to 22, wherein the composition comprises a pharmaceutically acceptable salt thereof or a conjugate according to any one of claims 1 to 22, wherein the composition comprises a pharmaceutically acceptable salt thereof or a conjugate according to any one of claims 1 to 22, wherein the composition comprises a pharmaceutically acceptable salt thereof or a conjugate according to any one of claims 1 to 22, wherein the composition comprises a pharmaceutically acceptable salt thereof or a conjugate according to any one of claims 1 to 22, wherein the conjugate ... wherein the active agent of the conjugate or a pharmaceutically acceptable salt thereof comprises an anti-cancer agent.

27. 27. The method of claim 26, wherein the cancerous cells endocytose the anti-cancer agent and endocytosis of the anti-cancer agent treats the subject for cancer.

28. 28. The method of claim 27, wherein the subject has bladder cancer.

29. 29. The method of claim 28, wherein the bladder cancer is non-muscle invasive bladder cancer (NMIBC).

30. 10. A method of imaging or diagnosing a subject having a disease state, comprising: (a) administering to the subject an effective amount of (i) the conjugate of any one of claims 1 to 22 or a pharmaceutically acceptable salt thereof, or (ii) the composition of any one of claims 23 to 25, wherein the active agent of the conjugate comprises an imaging agent; and (b) imaging the object or causing the object to be imaged; wherein the disease state is cancer.

31. 31. The method of claim 30, wherein imaging the subject comprises radiological imaging, positron emission tomography (PET) imaging, single photon emission computed tomography (SPECT) imaging, or magnetic resonance imaging.

32. 31. The method of claim 30, wherein imaging the subject further comprises imaging a region of the subject affected by the disease state.

33. 31. The method of claim 30, wherein the imaging agent comprises a metal or isotope suitable for radiological imaging, PET imaging, SPECT imaging, or magnetic resonance imaging, or a fluorescent imaging agent, a photodynamic imaging agent, or an optical imaging agent.

34. wherein the use of a conjugate according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, or a composition according to any one of claims 22 to 24, in the preparation of a medicament for treating a disease condition in a subject.

35. 32. The use of claim 31 , wherein the disease state is cancer.

36. 36. The use of claim 34 or 35, wherein the disease state is bladder cancer.

37. 36. The use of claim 34 or 35, wherein the disease state is non-muscle invasive bladder cancer (NMIBC).

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