Conjugates that enhance total cellular accumulation

Abstract

The present disclosure relates to a conjugated compound antibody covalently linked to an enhancer portion that is composed of a nuclear localization sequence (NLS) covalently linked to a sterol variant, such as cholic acid (ChAc) or its variants.The enhancer portion as encompassed herein can induce the endosomal escape of the compound conjugate by direct membrane destabilization or indirectly by the production of ROS and ceramide, which destabilize endosomal-lysosomal membranes.The conjugated compound can further comprise a payload.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 256,726, filed October 18, 2021, the entire contents of which are incorporated herein by reference.

[0002] Technical Field The present disclosure relates to conjugated compounds that enhance total intracellular accumulation. [Background technology]

[0003] The design of antibody-conjugates (AC) for the delivery of molecules for therapeutic or imaging applications in humans has progressed well, demonstrating clinical efficacy in certain malignancies and reducing the systemic toxicity caused by standard chemotherapy or radiation.

[0004] ACs have been successful in selectively delivering drug payloads to cancer cells for therapeutic or imaging applications in humans. However, AC technology is still expected to evolve into more effective and broadly applicable medicines for improved and broader cancer management, whether in the form of antibodies with radioisotopes or cytotoxic drugs.

[0005] The universal basis for intracellular drug accumulation by antibody-drug conjugates (ADCs) is their reliance on cellular internalization pathways. Once bound to a target antigen, ADCs are internalized, trapped in endosomes, and transported to lysosomes. Lysosomes are membrane-enclosed organelles that contain a series of digestive enzymes and receive proteins transported by endosomes via vesicle membrane fusion, resulting in the release of active drug metabolites. The intracellular accumulation of these metabolites directly correlates with cytotoxic potency. This dependency currently plagues ADCs and prevents them from achieving their full potential. Cancer cells respond to ADCs by increasing the expression of drug efflux pumps and decreasing the expression of target receptors. Receptor recycling pathways and their increased use by cancer cells are also involved in decreasing the intracellular accumulation of internalized ADCs. In essence, the field has long relied on an inefficient process for intracellular accumulation, with no studies directly addressing this issue. Avoiding entrapment in these intracellular pathways is therefore a key area for improving cellular accumulation of the transported drug and maximizing the activity of ADCs.

[0006] Functionalization of monoclonal antibodies (mAbs) with cell-membrane-permeable peptides resulted in a remarkable increase in intracellular accumulation when cells were treated with these types of ACs. However, this advance in cellular accumulation of ACs was primarily to enable mAbs to access and target specific molecules within cells that would otherwise be unavailable for antibodies to target. Of the few reports attempting to utilize ACs equipped with cell-membrane-permeable peptides as therapeutic agents against cancer-specific receptors on the cell surface, all have suffered from high accumulation in non-target cells or tissues, thus limiting their application for targeted delivery.

[0007] Recent advances with ACs functionalized with pH-sensitive polymers have demonstrated superior ability to escape endosomes and enter the cytoplasm while maintaining target cell selectivity. However, it remains to be determined whether increased evasion by these ACs corresponds to increased intracellular accumulation.

[0008] Another recent advance is to have AC achieve multiselective targeting by attaching peptides with compartmental localizing amino acids. In particular, the nuclear localization signal (NLS) sequence from the SV-40 large T antigen was pre-incorporated into a synthetic peptide and conjugated to a protein, demonstrating its ability to direct protein trafficking into the nucleus. The optimized NLS sequence is 25 amino acids long, but mAb 7G3 was also conjugated to a 13-mer peptide (CGYG) with a segment of NLS (underlined) sufficient for nuclear translocation. PKKKRKV The advantage of this short sequence is that it does not penetrate cells, allowing the mAb to maintain cell selectivity. 7G3-NLS was used to conjugate the radioisotope cargo, Indium-111 ( 111 In) was delivered into the nucleus. 111 Molecular damage by In is due to the emission of energetic Auger electrons. They travel only nanometer-micrometer distances and are therefore more effective when delivered into the nucleus. Unfortunately, cytotoxicity is not achieved by standard 111 The evidence, which was less overwhelming compared to In-7G3, suggested that this was due to ineffective nuclear localization caused by entrapment in the endosomal-lysosomal and / or recycling pathways.

[0009] Recently, Leyton JV and Beaudoin S (WO2017 / 156630) showed that the addition of a ChAc molecule to the amine of the N-terminal cysteine ​​of the SV40 large T antigen NLS (SEQ ID NO: 1) by chemical reaction allows the peptide to acquire a new intracellular and nuclear enhancer delivery function by inducing escape from endosomal-lysosomal trapping and its nuclear localization and accumulation. However, it was also observed that the addition of ChAc-SV40 large T antigen to an antibody induces modifications of the solubility, stability, biodistribution and pharmacokinetics of the antibody. The only combination that has been demonstrated there is the combination of SV40 large T antigen (shown in SEQ ID NO: 1) and cholic acid. It is well known that different bile acids have different activities. The best example showing this differential activity is some of the bile acids classified as pro-inflammatory factors, while other bile acids are known to have anti-inflammatory activity.

[0010] Therefore, there remains a need to provide an AC intracellular enhancer delivery agent that is effective in avoiding entrapment in the endosomal-lysosomal and / or recycling pathways. Summary of the Invention

[0011] Provided herein is a conjugate compound comprising an antibody covalently linked to a nuclear localization sequence (NLS), wherein the NLS is covalently linked to a sterol variant, and the antibody is not linked to an SV40 large T antigen NLS linked to cholic acid (ChAc). In one embodiment, the sterol variant is cholic acid (ChAc) or a variant thereof.

[0012] In another embodiment, the NLS is an SV40 large T antigen NLS in combination with a sterol variant other than ChAc.

[0013] In one embodiment, the sterol variant may be located at the C-terminal or N-terminal portion of the conjugated compound as provided herein.

[0014] In another embodiment, the NLS is an NLS other than the SV40 NLS.

[0015] In further embodiments, the NLS is a monopartite or bipartite NLS.

[0016] In another embodiment, the NLS is a non-classical NLS.

[0017] In one embodiment, the non-classical NLS is a hydrophobic PY-NLS or a basic PY-NLS.

[0018] In one embodiment, the antibody is a monoclonal or polyclonal antibody.

[0019] In one embodiment, the antibody is a monospecific, bispecific or multispecific antibody.

[0020] In further embodiments, the antibody is a murine antibody, a goat antibody, a human antibody or a rabbit antibody.

[0021] In one embodiment, the antibody is a humanized antibody.

[0022] In another embodiment, the antibody comprises an epitope-binding fragment selected from the group consisting of Fv, F(ab') and F(ab')2.

[0023] In further embodiments, the nuclear localization sequence is that shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.

[0024] In another embodiment, the cholic acid variant is deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, or glycoursodeoxycholic acid.

[0025] In another embodiment, the antibody is a trastuzumab antibody. In one embodiment, the antibody is conjugated to a deruxtecan (a topoisomerase inhibitor) or a DM1 (a microtubule inhibitor) drug.

[0026] In a further embodiment, the conjugated compound further comprises a payload covalently linked to the antibody.

[0027] In another embodiment, the payload is an imaging molecule.

[0028] In one embodiment, the payload is a fluorescent molecule, an IRM imaging agent or a radionuclide.

[0029] In one embodiment, the fluorescent molecule is 4,4-difluoro-8-(4-carboxyphenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY).

[0030] In another embodiment, the radionuclide is an imaging and / or therapeutic radionuclide.

[0031] In one embodiment, the radionuclide is 47 Sc, 51 Cr, 52 mMn, 55 Co, 58 Co, 52 Fe, 56 Ni, 57 Ni, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 68 Ga, 67 Ga, 72 As, 77 As, 89 Zr, 90 Y, 94 mTc, 99 mTc, 97 Ru, 105 Rh, 109 Pd, 111 Ag,110 In, 111 In, 113 mIn, 114 mIn, 117 mSn, 121 Sn, 127 Te, 142 Pr, 143 Pr, 149 Pm, 151 Pm, 149 Tb, 153 Sm, 157 Gd, 161 Tb, 166 Ho, 165 Dy, 169 Er, 169 Yb, 175 Yb, 172 Tm, 177 Lu, 186 Re, 188 Re, 191 Pt, 197 Hg, 198 Au, 199 Au, 201 Tl, 203 Pb, 211 At, 212 Bi, 213 Bi, 11 C. 75 Br, 76 Br, 77 Br, 82 Br, 18 F, 120 I, 123 I, 124 I, 125 I, 131 I, 89 Sr and 225 At least one of Ac.

[0032] In another embodiment, the payload is a small molecule toxin.

[0033] In one embodiment, the small molecule toxin is a chemotherapeutic agent.

[0034] In another embodiment, the small molecule toxin is a microtubule disrupting agent, a DNA targeting agent as an RNA polymerase inhibitor, or a topoisomerase inhibitor.

[0035] In further embodiments, the small molecule toxin is vinblastine, emtansine, monomethylauristatin E, or deruxtecan.

[0036] In additional embodiments, the conjugated compounds described herein are for detecting prostate cancer, breast cancer, liver cancer, gastric cancer, colon cancer, pancreatic cancer, ovarian cancer, lung cancer, kidney cancer, brain cancer, testicular cancer, glioblastoma, sarcoma, bone cancer, head and neck cancer, skin cancer, lymphoma, leukemia, colorectal cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or Langerhans cell histiocytosis.

[0037] In one embodiment, the conjugated compounds described herein are for treating prostate cancer, breast cancer, liver cancer, gastric cancer, colon cancer, pancreatic cancer, ovarian cancer, lung cancer, kidney cancer, brain cancer, testicular cancer, glioblastoma, sarcoma, bone cancer, head and neck cancer, skin cancer, lymphoma, leukemia, colorectal cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or Langerhans cell histiocytosis.

[0038] Also provided herein is the use of a conjugated compound as described herein to treat prostate cancer, breast cancer, liver cancer, gastric cancer, colon cancer, pancreatic cancer, ovarian cancer, lung cancer, kidney cancer, brain cancer, testicular cancer, glioblastoma, sarcoma, bone cancer, head and neck cancer, skin cancer, lymphoma, leukemia, colorectal cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or Langerhans cell histiocytosis.

[0039] Further provided herein is the use of a conjugate compound as described herein in the manufacture of a medicament for treating prostate cancer, breast cancer, liver cancer, gastric cancer, colon cancer, pancreatic cancer, ovarian cancer, lung cancer, kidney cancer, brain cancer, testicular cancer, glioblastoma, sarcoma, bone cancer, head and neck cancer, skin cancer, lymphoma, leukemia, colorectal cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or Langerhans cell histiocytosis.

[0040] Additionally provided herein is the use of a conjugated compound as described herein for detecting prostate cancer, breast cancer, liver cancer, gastric cancer, colon cancer, pancreatic cancer, ovarian cancer, lung cancer, kidney cancer, brain cancer, testicular cancer, glioblastoma, sarcoma, bone cancer, head and neck cancer, skin cancer, lymphoma, leukemia, colorectal cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or Langerhans cell histiocytosis.

[0041] Also provided herein are methods of treating and / or detecting prostate cancer, breast cancer, liver cancer, gastric cancer, colon cancer, pancreatic cancer, ovarian cancer, lung cancer, kidney cancer, brain cancer, testicular cancer, glioblastoma, sarcoma, bone cancer, head and neck cancer, skin cancer, lymphoma, leukemia, colorectal cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or Langerhans cell histiocytosis in a subject comprising administering to the subject a conjugate compound as described herein.

[0042] In one embodiment, the subject is a human or an animal.

[0043] Also provided are compositions comprising a conjugate compound as defined herein and a payload.

[0044] In one embodiment, the composition further comprises a carrier.

[0045] Reference is now made to the accompanying drawings, in which: [Brief description of the drawings]

[0046] [Figure 1] A schematic diagram of the conjugate compounds encompassed herein is shown in which an antibody (ligand) is the central component to an enhancer comprised of a nuclear localization signal (NLS) chemically linked to a sterol derivative, to which a payload is covalently linked.

[0047] [Diagram 2] Graph showing cytotoxicity assay of Accum-TDM1 variants.

[0048] [Diagram 3] 1 shows a graph of the cytotoxicity assay of Accum-T-deruxtecan variant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Provided is a novel design of compound conjugate specific to fast internalizing receptors to link endosomal escape and enhanced cellular uptake.More specifically, provided is a conjugate compound, comprising an antibody covalently linked to an enhancer portion that is composed of a nuclear localization sequence (NLS) covalently linked to a sterol variant, such as cholic acid (ChAc) or its variants.Enhancer portions as encompassed herein can induce the endosomal escape of compound conjugates by direct membrane destabilization or indirectly by the production of ROS and ceramide, which destabilize endosomal-lysosomal membranes.

[0050] Thus, conjugate compounds are provided that comprise an antibody covalently linked to a nuclear localization sequence (NLS) that is covalently linked to a sterol variant, excluding the ChAc-SV40 large T antigen NLS (SEQ ID NO:1).

[0051] It is known that each bile acid may have different effects on drug formulations and may not result in increased bioavailability and delivery to certain tissues. Furthermore, the SV40 large T antigen sequence is not predictive of any other NLS sequence, so it is unclear whether other combinations will achieve the desired intracellular and nuclear delivery activity.

[0052] In one embodiment, the conjugated compound further comprises a payload covalently linked to the antibody.

[0053] In one embodiment, the sterol variant may be located at the C-terminal or N-terminal portion of the conjugated compound as provided herein.

[0054] Thus, provided are compositions comprising a conjugate compound as described herein, a payload, and a carrier.

[0055] For example, conjugated compounds are provided that escape trapping within the endosomal-lysosomal system and subsequently translocate to the nucleus.

[0056] Examples of conjugate compounds include, but are not limited to, antibodies, oligonucleotides, antisense, drugs, or siRNA molecules, and do not exclude any small molecules and biological entities with intracellular targets that cannot permeate mammalian membranes.

[0057] In one embodiment, the antibody is a monoclonal or polyclonal antibody.

[0058] In one embodiment, the antibody is a monospecific, bispecific or multispecific antibody.

[0059] In another embodiment, the antibody is a murine antibody, a goat antibody, a human antibody or a rabbit antibody, or a humanized antibody.

[0060] As encompassed herein, antibodies can also include epitope-binding fragments, such as, for example, Fv, F(ab') and / or F(ab')2.

[0061] In one embodiment, the attachment of sterol variants is included. The term "variant" for sterol variants is well known. The bile acid core is based on a sterol molecule. Cholic acid is the primary bile acid synthesized in liver cells, which is further processed to produce secondary bile acids or variants of cholic acid, such as deoxycholic acid, chenodeoxycholic acid, or lithocholic acid. The cholic acid variants as included herein can be deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, and / or glycoursodeoxycholic acid.

[0062] It is the conjugation of NLS and ChAC (ChAcNLS) that allows efficient endosomal escape and nuclear localization of antibodies. Bile acids trigger the enzyme acid sphingomyelinase, which cleaves sphingomyelin, which is abundant in the inner leaflet of endosomes. An increase in the amount of ceramide destabilizes the membrane by forming a channel or lipid flip-flop that is sufficient for proteins to pass through, and thus, the linkage of ChAc to NLS allows mAbs to efficiently localize to the nucleus, as demonstrated in this application by increasing the potency of antibody conjugates and further explored in encapsulation. Thus, the efficiency of conjugated compounds to localize to the nucleus depends not only on the sequence of the NLS, but also on the conjugation to bile acids such as ChAc to induce escape from endosomal / lysosomal capture.

[0063] Thus, a conjugate compound is disclosed comprising an antibody covalently linked to a nuclear localization sequence (NLS), said NLS being covalently linked to cholic acid (ChAc) or a variant thereof, said NLS being a classical nuclear localization sequence (mono-binal type), excluding ChAc-SV40NLS, a hydrophobic PY-NLS or a basic PY-NLS, a nucleolar localization signal.

[0064] As encompassed herein, the NLS to be conjugated can be a classical nuclear localization sequence. Alternatively, hydrophobic PY-NLS and basic PY-NLS are also encompassed. In one embodiment, the NLS is PQBP1 NLS (SEQ ID NO:2), hnRNPA1 (SEQ ID NO:3), GWG-SV40 NLS (SEQ ID NO:4), NLS2 RPS1 (SEQ ID NO:5), NLS1 RPS17 (SEQ ID NO:6), NLS3 RPS17 (SEQ ID NO:7), nucleoplasmin NLS (SEQ ID NO:8), cMyc NLS (SEQ ID NO:9), TUS NLS (SEQ ID NO:10), hnRNP D NLS (SEQ ID NO:11), hnRNP M NLS (SEQ ID NO:12), HuR NLS (SEQ ID NO:13), and / or NLS2-RG RPS17 (SEQ ID NO:14).

[0065] As further encompassed, the NLS can be SV40NLS conjugated to a sterol variant other than ChAc, such as deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, or glycoursodeoxycholic acid.

[0066] A 13-mer peptide (CGYG) that is non-cell-permeable and has a segment of the classical NLS (underlined) derived from the SV-40 large T antigen. PKKKRKV GG; SEQ ID NO:1) has been previously conjugated to an anti-CD123 (IL-3Rα) antibody (7G3) and its chimeric version (CSL360) (Leyton et al., 2011, J Nucl Med, 52:1465-1473). 111 The ability of NLS-7G3 and NLS-CSL360, which transport In (In), to the nucleus was evaluated. Despite the presence of the NLS sequence, the percentage of radioactivity delivered to the nucleus was very low, with the majority of the radioactivity remaining on the cell surface or in the cytoplasm (Zerashkian et al., 2014, Nucl Med Biol, 41:377-383). Thus, 111 In-NLSCSL360 remained trapped within endosomes.

[0067] WO2017 / 156630, the entire contents of which are incorporated herein, discloses the coupling of cholic acid to the peptide CGYGPKKKRKVGG (SEQ ID NO: 1), which contains a segment of the nuclear localization sequence (NLS) from the SV40 large T antigen.

[0068] Conjugate ("Accum") peptide variants as listed in Table 1, synthesized according to the method described in WO2017 / 156630, as provided herein. At least 2 mg of TDM1 was pooled for conjugation of each Accum peptide variant in a single tube. For example, when preparing conjugation reactions of 10 Accum peptide variants simultaneously, 20 mg of TDM1 was dispensed into a single tube. A 10-fold excess of SM(PEG)4 crosslinker was added and incubated at room temperature for 1 hour. After the first 1 hour step reaction, at least 2 mg of TDM-PEG4-maleimide was dispensed into a different tube for the second step reaction, addition of Accum peptide variants. A 10-fold excess of selected Accum peptide variants was added to each tube reaction at room temperature for 1 hour or O / N at 4°C. Unreacted crosslinker and Accum peptide variants were removed by G25 Sephadex and 100 kDa cutoff filter. The Accum-TDM1 variant low DAR construct was passed through a 0.22um filter to remove any potential aggregates or contaminants. [Table 1-1] [Table 1-2]

[0069] Following synthesis of the Accum constructs above, cytotoxicity assays were performed. On day 0, 5000 JIMT-1 cells per well were plated in a 96-well plate. On day 1, cells were treated with TDM1 or each Accum-TDM1 variant at concentrations ranging from 0 to 100ug / ml. Cells were incubated at 37C for 72 hours. To determine cell viability, Prestoblue assay was used according to the manufacturer's protocol. [Table 2] [Table 3] [Table 4]

[0070] As can be seen in Figure 2 and Table 2, each Accum variant construct increases the cytotoxicity of TDM1. At concentrations of 0.1ug / ml and 1ug / ml of TDM1, only 5% and 34% cell death was observed, respectively, but with Accum-TDM1 constructs, cytotoxicity increases by 11-34% at 0.1ug / ml and 36%-88% cell death (Table 2). In Figure 2, it is clearly observed that higher concentrations of TDM1 are required to induce cell death with the same efficacy as Accum-TDM1 constructs. In summary, each Accum variant low DAR construct (1-3 Accum moieties per antibody) increases the cytotoxicity of TDM1 by 2-10 fold. [Table 5]

[0071] As can be seen in Figure 3 and Table 3, each Accum variant construct increases the cytotoxicity of T-deruxtecan. At concentrations of 0.001ug / ml and 0.1ug / ml T-deruxtecan, only 87% and 54% cell death was observed, respectively, whereas with Accum-T-deruxtecan constructs, cytotoxicity increases by 63-84% and cell death increases by 43-51%, respectively (Table 3). In Figure 3, it is clearly observed that higher concentrations of T-deruxtecan are required to induce cell death with the same efficacy as Accum-T-deruxtecan constructs. In summary, each Accum variant low DAR construct (1-3 Accum moieties per antibody) increases the cytotoxicity of T-deruxtecan by 2-10 fold.

[0072] Thus, conjugated antibodies, such as, but not limited to, Trastuzamab in breast cancer systems, or small molecules as described herein are provided.

[0073] Also included herein, but not limited to, payloads are radionuclides conjugated to the compounds described herein, which include: 47 Sc, 51 Cr, 52 mMn, 55 Co, 58 Co, 52 Fe, 56 Ni, 57 Ni, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 68 Ga, 67 Ga, 72 As, 77 As, 89 Zr, 90 Y, 94 mTc, 99 mTc, 97 Ru, 105 Rh, 109 Pd, 111 Ag, 110 In,111 In, 113 mIn, 114 mIn, 117 mSn, 121 Sn, 127 Te, 142 Pr, 143 Pr, 149 Pm, 151 Pm, 149 Tb, 153 Sm, 157 Gd, 161 Tb, 166 Ho, 165 Dy, 169 Er, 169 Yb, 175 Yb, 172 Tm, 177 Lu, 186 Re, 188 Re, 191 Pt, 197 Hg, 198 Au, 199 Au, 201 Tl, 203 Pb, 211 At, 212 Bi, 213 Bi, 11 C. 75 Br, 76 Br, 77 Br, 82 Br, 18 F, 120 I, 123 I, 124 I, 125 I, 131 I, 89 Sr and 225 Ac. Additionally, conjugated chemotherapeutic agents as described herein are included, such as, but not limited to, DM1 (mertansine) and deruxtecan.

[0074] Thus, in one embodiment, a novel cholic acid (ChAc)-NLS fusion peptide (ChAcNLS) conjugated to an antibody is provided as shown in Figure 1, which functionalizes the complex to escape endosomal trapping, delivers it to the nucleus, and utilizes the nucleus as a reservoir for enhanced intracellular accumulation. ChAcNLS is conjugated to a mAb and the complex is coupled to copper-64 ( 64 When radiolabeled with 1000 ng / cm2 (Cu) and injected in vivo, the amount of radioactivity delivered to the tumor is superior to, for example, versions unable to escape endosomal trapping. Not only has the ability of altered conjugate compounds to enhance intracellular accumulation of antibodies been clearly demonstrated, but the conjugate compounds provided herein do not affect the affinity and selectivity of the antibodies.

[0075] In general, the attachment of ChAcNLS to a mAb can be controlled.

[0076] 64 The conjugate compound further linked to a payload such as Cu 64 It has also been described that ChAcNLS has the ability to enhance the cellular uptake of Cu. Thus, ChAcNLS should not disrupt the pharmacokinetics of antibody in vivo. The conjugate compounds provided herein may result in more effective radiation toxicity or more sensitive detection of cancer cells, because of the increased radioactivity retention observed in tumors as shown herein.

[0077] Thus, by targeting the receptor that is rapidly internalized, it provides a method that can be widely applied to other molecules or antibodies.Many interleukin receptors involved in various cancers undergo rapid endocytosis upon ligand binding.The use of conjugates as described herein can increase the accumulation of actual chemotherapy molecules in targeted cells, for example.

[0078] In one embodiment, an antibody-drug conjugate (ADC) as described herein is composed of three components - a monoclonal antibody (mAb), a crosslinker, and a cytotoxin (e.g., a small molecule chemotherapeutic drug). For example, the cytotoxin is conjugated to the mAb via a crosslinker.

[0079] Antibody-drug conjugates (ADCs) as described herein are encompassed herein that include a payload, such as, for example, but not limited to, a small molecule toxin, such as a microtubule disrupting agent (e.g., vinblastine, monomethylauristatin E or MMAE, DM1) and / or a DNA targeting agent (deruxtecan, a topoisomerase inhibitor).

[0080] For example, antibodies conjugated with ChAcNLS together with an attached chemotherapeutic molecule (e.g., 4,4-difluoro-8-(4-carboxyphenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (abbreviated BODIPY), which is a cytotoxic molecule used in photodynamic therapy applications in cancer) result in increased cytoplasmic accumulation of the antibody and chemotherapeutic molecule, providing faster blood clearance and concomitantly better tumor uptake, as ChAcNLS does not interfere with tumor targeting.

[0081] Not only do conjugates such as those described herein provide a means to enhance the delivery of antibodies, but ChAcNLS can also enhance the delivery of attached molecular payloads, not just antibodies, and ChAcNLS can deliver increased amounts of molecular payloads to the nucleus.

[0082] Further encompassed herein is the possibility of conjugating the antibody with additional drugs, such as vinblastine, which is used in combination with other chemotherapeutic agents to treat Hodgkin's lymphoma (Hodgkin's disease) and non-Hodgkin's lymphoma, as well as cancer of the testes, and is also used to treat Langerhans cell histiocytosis.

[0083] Thus, the conjugated compounds described herein may be used to detect or treat prostate cancer, breast cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, ovarian cancer, lung cancer, kidney cancer, brain cancer, testicular cancer, glioblastoma, sarcoma, bone cancer, head and neck cancer, skin cancer, lymphoma, leukemia or colorectal cancer.

[0084] While the disclosure has been described in relation to specific embodiments thereof, it will be understood that further modifications are possible, and this application is intended to cover any variations, uses, or adaptations thereof as may be applied to the essential features hereinbefore described and including such departures from the present disclosure as come within known or customary practice within the art to which this invention pertains, and as fall within the scope of the appended claims.

Claims

1. A conjugate compound comprising an antibody covalently linked to a nuclear localization sequence (NLS), wherein the NLS is covalently linked to a sterol variant, the sterol variant being a bile acid, and the NLS is not an SV40 large T antigen NLS.

2. 2. The conjugate compound of claim 1, wherein the bile acid is cholic acid (ChAc) or a variant thereof.

3. 2. The conjugate compound of claim 1, wherein the sterol variant is conjugated to the C-terminal or N-terminal portion of the NLS.

4. The conjugate compound according to any one of claims 1 to 3, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

5. The conjugate compound according to any one of claims 1 to 3, wherein the antibody is a humanized antibody.

6. The conjugate compound according to any one of claims 1 to 3, wherein the antibody is a monospecific antibody, a bispecific antibody or a multispecific antibody.

7. The antibody is a Fv, F(ab') and F(ab') ２ 4. The conjugate compound of any one of claims 1 to 3, comprising an epitope-binding fragment selected from the group consisting of:

8. The conjugate compound of any one of claims 1 to 3, wherein the nuclear localization sequence is cMyc NLS (SEQ ID NO: 9), hnRNPA1 (SEQ ID NO: 3), PQBP1 NLS (SEQ ID NO: 2), NLS2 RPS1 (SEQ ID NO: 5), NLS1 RPS17 (SEQ ID NO: 6), NLS3 RPS17 (SEQ ID NO: 7), nucleoplasmin NLS (SEQ ID NO: 8), TUS NLS (SEQ ID NO: 10), hnRNP D NLS (SEQ ID NO: 11), hnRNP M NLS (SEQ ID NO: 12), HuR NLS (SEQ ID NO: 13), or NLS2-RG RPS17 (SEQ ID NO: 14).

9. 4. The conjugate compound of claim 1, wherein the bile acid is chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), or glycoursodeoxycholic acid (GUDCA).

10. The conjugate compound of any one of claims 1 to 3, wherein the antibody is a trastuzumab antibody.

11. The conjugate compound of any one of claims 1 to 3, wherein the antibody is conjugated to deruxtecan or DM1.

12. The conjugate compound of any one of claims 1 to 3, further comprising a payload covalently linked to said antibody.

13. 13. The conjugate compound of claim 12, wherein the payload is a radionuclide or a small molecule toxin.

14. The payload is () ４７ 3、 ５１ 、 ５２ m*、 ５５ o、 ５８ o、 ５２ e、 ５６ 、、 ５７ 、、 ６１ u、 ６２ u、 ６４ u、 ６７ u、 ６６ |、 ６８ |、 ６７ |、 ７２ 1.、 ７７ 1.、 ８９ 、 ９０ 、 ９４ mc、 ９９ mc、 ９７ __、 １０５ (2) １０９ P、 １１１ __、 １１０ .、 １１１ .、 １１３ m.、 １１４ m.、 １１７ mn、 １２１ 、 １２７ ()、 １４２ 0.、 １４３ 0.、 １４９ m、 １５１ m、 １４９ (b、) １５３ 3m、 １５７ 、、 １６１ (b、) １６６ 、 １６５ y、 １６９ ॥、 １６９ b、 １７５ __________、|____、 １８６ 2)、 １８８ 2)、 １９１ 0.、 １９７ 、 １９８ __、 １９９ __、 ２０１ .、 ２０３ pi.、 ２１１ 1.、 ２１２ 、、 ２１３ 、、 １１ 、 ７５ 、 ７６ 、 ７７ 、 ８２ 、 １８ F. １２０ I, １２３ I, １２４ I, １２５ I, １３１ I, ８９ Sr and ２２５ a radionuclide being at least one of Ac, (b) a chemotherapeutic agent; (c) a microtubule-disrupting agent, a DNA-targeting agent, a topoisomerase inhibitor, or a DNA-alkylating agent; (d) a small molecule toxin that is vinblastine, emtansine, monomethyl auristatin E, or deruxtecan; or (e) Imaging molecules 14. The conjugate compound of claim 13, wherein:

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