Anti-Doppel antibody drug conjugate

Abstract

Described herein are anti-Doppel antibody drug conjugates, compositions comprising them, and related methods of treating Doppel-related diseases and disorders, including cancer. Provided herein are anti-Doppel antibody drug conjugates (ADCs) comprising: (i) a Doppel targeting moiety, (ii) a cleavable linker, and (iii) a therapeutic agent. Also provided herein are methods of treating Doppel-related diseases and disorders, including Doppel-related cancers, using the ADCs described herein, as well as kits comprising the ADCs described herein.

Description

Technical Field

[0001] This article describes anti-Doppel antibody-drug conjugates, compositions comprising them, and related methods for treating Doppel-related diseases and conditions, including cancer. Background Technology

[0002] Chemotherapy is the most commonly used anti-cancer therapy due to its powerful anti-cancer effects. However, its use is often limited by severe side effects and toxicity. To develop selective chemotherapy methods that target tumors rather than healthy tissue, efforts have been made to deliver chemotherapeutic agents to tumor cells, for example, using antibodies or peptides that can recognize and bind to molecules preferentially expressed in tumor cells rather than normal cells. Antibody-drug conjugates (ADCs) may contain a potent cytotoxic drug and a target-specific antibody (e.g., an antibody that selectively binds to a target-specific biomarker, such as a cancer-specific biomarker). An ADC can be described as a multi-stage rocket, consisting of nested components that bring the payload (cytotoxic drug) closer to its target (cancer cells). The primary targeting component of an ADC is the antibody. Once the antibody arrives and binds to its target binding chaperone, effectively binding the ADC to its target binding chaperone, the ADC must be internalized into the cell to exert its intended effect. For optimal efficacy, the cytotoxic drug must possess sufficient cytotoxicity to effectively kill target cells at the dose delivered by the ADC.

[0003] The concept behind ADCs seems simple, but achieving sufficient therapeutic efficacy in vivo has proven extremely difficult. Despite advances in ADC technology, ADC-based therapies still face numerous challenges, such as in vivo toxicity, suboptimal target biomarkers, unpredictable clinical value in combination therapies, and unclear mechanisms of drug resistance.

[0004] In cancer treatment, the identification of tumor-specific biomarkers has transformed our understanding of cancer. Cancer is no longer viewed as a single disease. Because cancer can result from the accumulation of gene mutations, a single cancer can be classified into multiple subtypes, each with its own set of identifying mutations. Conversely, tumors from different parts of the body may also have similar mutations and respond to the same drugs. See, for example, Sun et al., Bioconjugate Chem. (2020) 31(4): 1012-1024. Therefore, in ADC-based chemotherapy, the identification and quantification of tumor-specific antigens as target-specific biomarkers are becoming increasingly important. Despite significant progress in the discovery of tumor-specific and tumor-associated antigens, biomarkers must meet certain requirements for safe and effective use in ADCs, such as high expression on the tumor surface, facilitating endocytosis of ADCs, and limited expression in normal tissues (to prevent off-target toxicity). In this regard, although tumor-associated antigens of solid tumors (such as HER2, TROP2 and nectin 4) have been used to develop monoclonal antibodies for therapeutic and chemotherapeutic purposes, these tumor-associated antigens are also highly expressed in normal tissues, which may lead to non-specific targeting of healthy tissues (off-target toxicity). Summary of the Invention

[0005] Technical problem

[0006] Therefore, there is still a need for safe and effective ADCs that can selectively bind to targets (e.g., tumor cells) without harming non-target cells (e.g., normal tissues) while being effective against target cells (e.g., tumor cells).

[0007] Solution to the problem

[0008] This article provides an anti-Doppel antibody-drug conjugate (ADC) comprising: (i) a Doppel-targeting moiety, (ii) a cleavable linker, and (iii) a therapeutic agent. This article also provides methods for treating Doppel-related diseases and conditions, including Doppel-related cancers, using the ADC described herein, as well as kits containing the ADC described herein.

[0009] Advantages of the invention

[0010] In some respects, this article provides an anti-Doppel ADC comprising: (i) a Doppel-targeting portion connected directly or via a connector to (ii) a cleavable connector connected directly or via a connector to (iii) a therapeutic agent.

[0011] In some respects, the Doppel targeting portion is selected from Doppel-binding monoclonal antibodies, Doppel-binding polyclonal antibodies, Doppel-binding single-chain antibodies, Doppel-binding chimeric antibodies, Doppel-binding humanized antibodies, Doppel-binding veneered antibodies, and any Doppel-binding fragments thereof. In some respects, the Doppel targeting portion is a Doppel-binding antibody selected from the human monoclonal antibody A12 disclosed herein; the human monoclonal antibody B2 disclosed herein; the human monoclonal antibody E9 disclosed herein; the human monoclonal antibody 3D5 disclosed herein; the human monoclonal antibody 3D1 disclosed herein; the human monoclonal antibody 4D1 disclosed herein; the human monoclonal antibody 3H9 disclosed herein; and any Doppel-binding fragments thereof.

[0012] In some respects, cleavable linkers can be cleaved by intracellular proteases. In some respects, cleavable linkers are selected from cleavable dipeptide linkers, such as valine-citrulline, valine-alanine, and phenylalanine-lysine; hydrazone linkers that hydrolyze at pH less than 5.5; and disulfide linkers that cleave in a reducing environment.

[0013] In some aspects, the cleavable peptide linker is a caspase-cleavable peptide linker. In some aspects, the four C-terminal amino acid residues of the caspase-cleavable peptide linker are selected from Asp-Xaa-Xaa-Asp, Leu-Xaa-Xaa-Asp, and Val-Xaa-Xaa-Asp, where Xaa represents any amino acid residue. In some aspects, the four C-terminal amino acid residues of the caspase-cleavable peptide linker are selected from Asp-Glu-Val-Asp (SEQ ID NO: 4), Asp-Leu-Val-Asp (SEQ ID NO: 5), Asp-Glu-Ile-Asp (SEQ ID NO: 6), and Leu-Glu-His-Asp (SEQ ID NO: 7). In some aspects, the six C-terminal amino acid residues of the caspase-cleavable peptide linker are composed of Lys-Gly-Asp-Glu-Val-Asp (SEQ ID NO: 8).

[0014] In some respects, the treatment agent includes chemotherapeutic agents. In some respects, the treatment agent includes chemotherapeutic agents that induce apoptosis in tumor cells. In some respects, the treatment agent is selected from 5-FU, afatinib, aprilidine, azaribin, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, briostatin-1, busulfan, carlicicillin, ethacryne, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatin, COX- 2. Inhibitors, Irinotecan, SN-38, Cladribine, Crizotinib, Cyclophosphamide, Cytarabine, Dacarbazine, Dasatinib, Denaciline, Docetaxel, Actinomycin D, Daunorubicin, Doxorubicin, Podophyllotoxin, Erlotinib, Entenolol, Estrogen Receptor Binding Agents, Etoposide, Exemestane, Fingolimod, Fluuridine, Fludarabine, Flutamide, Flurapirib, Fositatinib, Ganetinib, GDC-08 34. GS-1101, Gefitinib, Gemcitabine, Hydroxyurea, Ibrutinib, Idarubicin, Edraliximab, Ifosfamide, Imatinib, L-Asparaginase, Lapatinib, Lenalidomide, Leucovorin, LFM-A13, Lomustine, Nitrogen Mustard, Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone, Spectinomycin, Mitomycin, Mitotan, Naveline, Neratinib, Nilotinib, Nitrosourea, Olaparib The chemotherapeutic agents are selected from anthracyclines, antibiotics, alkylating agents, platinum-based drugs, antimetabolites, topoisomerase inhibitors, and mitotic inhibitors. In some aspects, the chemotherapeutic agents are selected from doxorubicin, daunorubicin, epirubicin, idarubicin, vararubicin, SU11248, sunitinib, tamoxifen, temozolomide, transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vatalani, vinorelbine, vinblastine, vincristine, vinblastine alkaloids, and ZD183. In some aspects, the chemotherapeutic agents are selected from anthracyclines, antibiotics, alkylating agents, platinum-based drugs, antimetabolites, topoisomerase inhibitors, and mitotic inhibitors. In some aspects, the chemotherapeutic agents are selected from doxorubicin, daunorubicin, epirubicin, idarubicin, vararubicin, and their derivatives. In other respects, the chemotherapeutic agents are selected from actinomycin D, bleomycin, mitomycin C, calichiomycin, and their derivatives.In other aspects, the chemotherapy agents are selected from cyclophosphamide, nitrogen mustard, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozotocin, busulfan, dacarbazine, temozolomide, thiotepa, atratamine, ducamycin, cisplatin, carboplatin, nedaplatin, oxaliplatin, satroplatin, triplatinum tetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cytarabine, fluorouracil, fludarabine, and others. Gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, ezetidine, camptothecin, topotecan, irinotecan, etoposide, teniposide, mitoxantrone, paclitaxel, docetaxel, ixaprone, vincristine, vinblastine, vinorelbine, estradiol, maytansine, DM1 (mettansine), DM4, doralastatin, iorestatin E, iorestatin F, monomethylorestatin E (MMAE), and their derivatives. In some respects, the chemotherapeutic agents are selected from monomethylorestatin E (MMAE) and their derivatives.

[0015] In some respects, therapeutic agents include immunomodulators. In some respects, immunomodulators are selected from cytokines, lymphokines, monokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony-stimulating factor (CSF), interferon (IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, pro-relaxanthin, follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), liver growth factor, prostaglandins, fibroblast growth factor, prolactin, placental prolactin, OB protein, transforming growth factor (TGF), TGF-α, TGF-β, and insulin. Inorganic growth factor (IGF), erythropoietin, thrombopoietin, tumor necrosis factor (TNF), TNF-α, TNF-β, Müllerian duct inhibitor, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrin, interleukin (IL), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-α, interferon-β, interferon-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7 IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, IL-25, LIF, kit ligand, FLT-3, angiostatin, thromboretin, endostatin, Toll-like receptor (TLR) agonists (e.g., CU-T12-9, Pam3CSK4, FSL-1, Pam2CSK4, and CL429), Poly(A:U), Poly(I:C) Lipopolysaccharide (LPS), MPLA-SM, CRX-527, flagellin, thiazoquinoline derivatives, imidazoquinoline derivatives (e.g., CL097, gardiquimod, imiquimod, and resiquimod), adenine analogs, guanosine analogs, thymidine analogs, benzodiazepine analogs, and CpG oligodeoxynucleotides (ODNs) (e.g., ODN1585, ODN2216, ODN2336, ODN1668, ODN1826, ODN2006, ODN2007, ODN BW006, ODND-SL01, ODN2395, ODNM362, and ODND-SL03).

[0016] In some respects, the therapeutic agent contains a toxin. In some respects, the toxin is selected from ricin, abrinogen, ribonuclease (RNase), DNase 1, Staphylococcus aureus enterotoxin A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

[0017] In some respects, the therapeutic agent contains a radionuclide. In some respects, the radionuclide is selected from... 11 C 13 N、 15 O、 32 P, 33 P, 47 Sc、 51 Cr 57 Co、 58 Co、 59 Fe、 62 Cu、 67 Cu、 67 Ga、 75 Br、 75 Se、 76 Br、 77 As、 77 Br、 80m Br、 89 Sr、 90 Y、 95 Ru、 97 Ru、 99 Mo、 99m Tc, 103m Rh、 103 Ru、 105 Rh、 105 Ru、 107 Hg, 109 Pd, 109 Pt, 111 Ag、 111 In、 113m In、 119 Sb, 121m Te、 122m Te、 125 I, 125m Te、 126 I, 131 I, 133 I, 142 Pr, 143 Pr, 149 Pm, 152 Dy、 153 Sm、 161 Ho、 161 Tb, 165 Tm、 166 Dy、 166 Ho、 167 Tm、 168 TM、 169 Er、 169 Yb、 177 Lu、 186 Re、 188 Re、 189m Os、189 Re、 198 Ir、 194 Ir、 197 Pt, 198 Au、 199 Au、 203 Hg, 211 At、 211 Bi、 211 Pb, 212 Bi、 212 Pb, 213 Bi、 215 Po、 217 At、 219 Rn、 221 Fr、 223 Ra、 225 Ac、 227 Th and 255 Fm.

[0018] In some respects, the therapeutic agent is a DNA cross-linking agent, such as one or more selected from indobenzobenzodiazepine dimer (IGN), pyrrolobenzodiazepine (PBD), and their derivatives.

[0019] In one specific implementation, the Doppel targeting portion is a Doppel targeting antibody, the linker is a caspase-cleavable peptide linker with the amino acid sequence Lys-Gly-Asp-Glu-Val-Asp (SEQ ID NO: 8), and the chemotherapeutic agent is MMAE.

[0020] In one specific implementation, the Doppel targeting portion is human monoclonal antibody 3H9, the linker is a caspase-cleavable peptide linker with the amino acid sequence Lys-Gly-Asp-Glu-Val-Asp (SEQ ID NO: 8), and the chemotherapeutic agent is MMAE.

[0021] This document also provides compositions comprising the ADC described herein and a pharmaceutically acceptable carrier. In some respects, the compositions are formulated for intravenous administration.

[0022] This article also provides methods for treating subjects with Doppel-related diseases or conditions, including administering the ADCs described herein to subjects in need.

[0023] In some respects, Doppel-related diseases or conditions are selected from asthma, tuberculosis, atherosclerosis, and pulmonary hypertension (PAH).

[0024] In some respects, doppel-related diseases or conditions are cancer. In some respects, cancer cells express doppel.

[0025] This article also provides methods for treating subjects with Doppel-related cancer, including administering the ADC described herein to subjects in need.

[0026] This document also provides a kit containing the ADC described herein in a suitable container, and optionally further includes instructions for use.

[0027] The above overview is merely illustrative and is not intended to limit the invention in any way. In addition to the exemplary aspects, embodiments, and features described above, other aspects, embodiments, and features will become apparent from the detailed descriptions and examples below. Attached Figure Description

[0028] Figure 1 The cytotoxicity of the anti-Doppel ADC (name = SEQ ID NO: 65; structure = SEQ ID NO: 66) described herein against HCT116 cells is demonstrated.

[0029] Figure 2 This shows the internalization of 3H9 monoclonal antibody (mAb) into cells expressing Doppel (HCTEC) as observed by confocal microscopy.

[0030] Figure 3 The image shows the colocalization of 3H9 mAb with lysosomes in Doppel-expressing cells (HCTEC) as observed by confocal microscopy.

[0031] Figure 4 The in vivo efficacy of the anti-Doppel ADCs (3D1-vc-MMAE; 3D5-vc-MMAE; 3H9-vc-MMAE; and 4D1-vc-MMAE) described in this article was demonstrated.

[0032] Figure 5 The dose dependence of the anti-Doppel ADCs (3D1-vc-MMAE; 3D5-vc-MMAE; and 3H9-vc-MMAE) described herein was demonstrated.

[0033] Figure 6 The in vivo efficacy of the anti-Doppel ADCs (3H9-KGDEVD-MMAE (name=SEQ ID NO: 65; structure=SEQ ID NO: 66); 3H9-DEVD-MMAE (name=SEQ ID NO: 73; structure=SEQ ID NO: 74) and 3H9-vc-MMAE) with different connectors described herein is demonstrated.

[0034] Figure 7The in vivo efficacy of the anti-Doppel ADCs described herein (3D1-KGDEVD-MMAE (name=SEQ ID NO: 67; structure=SEQ ID NO: 68); 3D5-KGDEVD-MMAE (name=SEQ ID NO: 69; structure=SEQ ID NO: 70); 3H9-KGDEVD-MMAE (name=SEQ ID NO: 65; structure=SEQ ID NO: 66); and 4D1-KGDEVD-MMAE (name=SEQ ID NO: 71; structure=SEQ ID NO: 72)) is demonstrated. Detailed Implementation

[0035] Unless otherwise defined, the technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Various methods known to one of ordinary skill in the art are referenced herein. Publications and other materials illustrating such known methods are incorporated herein by reference in their entirety. One of ordinary skill in the art can practice this invention using any suitable materials and / or methods based on the guidance provided herein. However, the specific materials and methods described herein are for illustrative purposes only. Unless otherwise stated, the materials, reagents, etc., mentioned in the following descriptions and examples are commercially available.

[0036] The singular forms “a,” “an,” and “the” used in this document refer to both the singular and plural forms unless explicitly stated otherwise.

[0037] The term "about" indicates that the included numerical value is not limited to the exact value stated herein, but rather refers to an approximate range around that value without departing from the scope of the invention. The term "about" as used herein will be understood by those skilled in the art, and its meaning may vary depending on the specific context. If those skilled in the art cannot understand the meaning of the term based on the specific context, "about" refers to a value within plus or minus 10%.

[0038] The term "tumor cell" as used in this article refers to any type of tumor tissue cell, including benign or malignant tumor cells.

[0039] As used in this article, the term "cancer" refers to any cancer originating in any part of the body or of any cell type. This includes, but is not limited to, epithelial carcinoma, sarcoma, lymphoma, germ cell tumors, and embryonal tumors. Cancer may be associated with a specific part of the body or a specific disease. The terms "cancer" and "tumor" are often used interchangeably.

[0040] Unless the context otherwise requires, the term “cell” as used herein includes cells in an animal body (e.g., in vivo cells) and cultured cells.

[0041] Unless otherwise stated, as used herein, “Doppel” refers to any Doppel protein, including mouse or human Doppel proteins, including glycosylated or non-glycosylated Doppel proteins, and monomeric or dimer forms of Doppel. In this document, the terms “Doppel,” “PRND,” and “Doppel protein” are generally used interchangeably.

[0042] As used herein, the term "subject" means any animal that is required to receive treatment using any of the methods or approaches described herein, including humans and other mammals such as dogs, cats, rabbits, horses, and cattle. For example, a subject may have or be at risk of developing a Doppel-related disease or condition, including Doppel-related cancers. In some specific respects, a subject is a human diagnosed with a Doppel-related tumor or Doppel-related cancer.

[0043] As used in this article, the term "Doppel-related disease or condition" (or "Doppel-related disease or condition") refers to a disease or condition in which the subject's endothelial cells express Doppel, including asthma, tuberculosis, atherosclerosis, pulmonary hypertension (PAH), and neoplasms and neoplasm-related conditions, including Doppel-related cancers and Doppel-related tumors. As used in this article, the terms "Doppel-related tumor" and "Doppel-related cancer" refer to tumors or cancers in which the cells express Doppel.

[0044] Anti-Doppel antibody-drug conjugate

[0045] This document describes anti-Doppel antibody-drug conjugates (ADCs) comprising: (i) a Doppel-targeting moiety, (ii) a cleavable linker, and (iii) a therapeutic agent. This document also describes methods of treating Doppel-related diseases and conditions, including Doppel-related cancers, using the ADCs described herein. The nomenclature used herein, such as [antibody name]-cleavable linker-[therapeutic agent name], is used to refer to ADCs comprising the antibody, cleavable linker, and therapeutic agent. It should be understood that other components may be present, such as one or more other chemical linkers, as discussed in more detail below and illustrated in the examples.

[0046] As discussed in more detail below, the anti-Doppel ADCs and methods described herein are based on the inventors' findings that Doppel is a tumor endothelial cell (TEC) surface marker that plays a role in pathological angiogenesis. Inhibiting the angiogenic activity of Doppel, for example by inhibiting the interaction of Doppel with tyrosine kinase receptors (e.g., VEGFR2), or by binding to Doppel, can selectively inhibit pathological angiogenesis, including tumor angiogenesis and pathological angiogenesis associated with other diseases (e.g., asthma, tuberculosis, atherosclerosis, pulmonary hypertension (PAH), tumors, and tumor-related diseases) related to Doppel-expressing endothelial cells. For example, during pathological angiogenesis, the expression level of Doppel on endothelial cells may be increased compared to the expression level under normal or physiological angiogenesis conditions. Furthermore, Doppel expression on TECs may be associated with pathological tumor-associated angiogenesis or tumorigenesis.

[0047] Doppel

[0048] Doppel is a prion-like protein encoded by the gene PRND, located near the PRNP (prion protein-coding gene) locus. See, for example, Golaniska et al., Folia Neuropathol, 42(Supplement A) 47-54 (2004). Doppel expression is conserved across human to mouse evolution, suggesting that Doppel expression may play an important role under certain physiological conditions. See, for example, Behrens et al., EMBO J. 21:3652 (2002). Full-length human Doppel is a 179-amino acid protein (UniProtKB_Q9UKY0; NCBI Ref._NP_036541.2) with a molecular weight of 14 kDa in its unglycosylated form. Doppel undergoes C-terminal glycosylphosphatidylinositol (GPI) modification and is expressed on the cell surface via GPI anchoring on lipid rafts.

[0049] Doppel is transiently expressed in neonatal brain endothelial cells, but in adults, it is expressed only in testicular cells (Li et al., Am. J. Pathol. (2000) 157(5): 1447-1452). According to the human protein atlas, PRND is almost absent in tissues outside the gonads in normal adults. Since Doppel is lacking on most normal cells, the anti-Doppel antibody-drug conjugate (ADC) described herein provides a highly targeted and specific ADC-targeting approach that is not possible with other tumor biomarkers. Therefore, the method described herein will have limited off-target toxicity and represents a significant improvement over existing methods.

[0050] On the other hand, doppel is associated with a variety of diseases and conditions for which safe and effective treatments are currently lacking. For example, doppel is associated with neurodegenerative diseases and angiogenesis. Importantly for this invention, doppel is highly expressed in tumors. Comincini et al. (Anticancer Research, 2004, 24: 1507-1518) conducted the most extensive study on the relationship between malignancy and doppel expression in astrocytomas. They investigated PRND expression in glioblastoma cell lines and non-glial tumor samples, finding a direct correlation between PRND expression and tumor malignancy. The authors reported that immunohistochemical analysis showed diffuse cytoplasmic distribution of doppel in various astrocytomas, infiltrating lymphocytes, and vascular endothelial cells. The authors also reported high levels of PRND detected in non-glial malignant tumor samples such as gastric adenocarcinoma and anaplastic meningioma, suggesting that doppel may serve as a biomarker for other types of cancer besides glioblastoma. Al-Hilal et al. (J. Clin. Invest. 2016, 126(4): 1251-66) reported a study on Doppel as a potential therapeutic target for tumor angiogenesis, finding that Doppel is expressed in tumor endothelial cells (TECs) but not in normal endothelial cells (ECs), and pointed out that blocking Doppel can selectively inhibit tumor angiogenesis.

[0051] For the purposes of this invention, Doppel is located on the surface of TEC, making it a superior ADC target than the tumor itself, for the following reasons.

[0052] First, the TEC is located within the blood vessel and is the first layer of cells encountered by intravenously injected drugs. Therefore, in some respects, anti-Doppel ADCs are actually vascular-targeting ADCs (VT-ADCs). One advantage of VT-ADCs compared to ADCs targeting other biomarkers is the proximity of the target to the blood vessel. In conventional chemotherapy targeting other biomarkers, chemotherapeutic agents must overcome multiple barriers to reach the target. For example, even before reaching the cells, it must cross the tumor's extracellular matrix (ECM), which constitutes multiple barriers to chemotherapeutic agents. For instance, the tumor ECM itself is under high pressure due to vascular leakage and lymph node obstruction. This interstitial high pressure inhibits the extravasation of macromolecules and intercellular transport. Furthermore, the dense collagen fiber network in the ECM and the increased distance between tumor cells further complicate target localization. In this respect, the relative volume of the interstitial space is 3–5 times larger than that of normal cells, thus increasing the distance that chemotherapeutic agents need to travel (Kratz, F. et al., Drug delivery inoncology, (2011) John Wiley & Sons, Ltd. pp. 40–44). Furthermore, even if chemotherapeutic agents reach the tumor center, their efficacy may be limited by their design characteristics. Most chemotherapeutic agents target the rapid proliferation of tumor cells. However, malignant cells far from blood vessels are quiescent due to unfavorable environments and may therefore not respond to chemotherapeutic agents (Kratz, F. et al., Drug delivery in oncology, (2011) John Wiley & Sons, Ltd., p. 15). In contrast, the anti-Doppel ADC targeting Doppel on the surface of TEC described in this paper does not need to cross the intervascular space, thus avoiding the aforementioned problems. This not only makes the ADC more effective at the target but also limits off-target toxicity and provides a more predictable PK / PD relationship. Moreover, all types of tumors, if they wish to grow to greater than 1 cm, can be targeted. 3 All of these depend on angiogenesis. Therefore, since the chemotherapeutic agents carried by the anti-Doppel ADCs described in this article cause damage to vascular structures, the anti-Doppel ADCs described in this article can effectively combat most tumor masses (e.g., including tumor cells far from blood vessels).

[0053] Given that doppel is located on the surface of TEC, another advantage of targeting doppel relates to tumor heterogeneity. Even within the same tumor, cells may express different biomarkers, and mutations occur frequently. This may be because the hypoxic environment within the tumor leads to further changes in the proteome and genome, thereby driving tumor progression toward malignancy, including the development of chemotherapy resistance (Kratz, F. et al., Drug delivery in oncology, (2011) JohnWiley & Sons, Ltd., p. 49). In contrast, TEC originates from normal hematopoietic cells and is less likely to undergo somatic mutations.

[0054] As mentioned above, Doppel is selectively expressed not only on tumor cells but also on endothelial cells under pathological conditions such as atherosclerosis, tuberculosis, asthma, and pulmonary hypertension (PAH). Although the role of Doppel in the development of these diseases is not fully elucidated, the Doppel protein has been identified as a potential therapeutic target. Since Doppel is actively involved in angiogenesis signaling, targeting Doppel could be an effective way to selectively inhibit pathological angiogenesis.

[0055] Doppel targeting moiety

[0056] As described above, the anti-Doppel ADC described herein includes a Doppel-targeting moiety. In some aspects, the Doppel-targeting moiety is an antibody that binds to Doppel or a related type of anti-Doppel antibody, such as a Doppel-binding antibody fragment, including but not limited to antibody fragments or peptides that bind Doppel. In some aspects, this binding inhibits the interaction of Doppel with tyrosine kinase receptors, such as one or more of VEGFR2, VEGFR1, VEGFR3, bFGFR, and PDGFR. In some aspects, the Doppel-targeting moiety binds to Doppel, thereby inhibiting the interaction of Doppel with VEGFR2. In some embodiments, the anti-Doppel antibody targets the extracellular domain (ECD) of Doppel.

[0057] The term “antibody” or “anti-Doppel antibody” as used in this article includes monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies (e.g., see Jones et al., Nature 321 (1986), 522-525; Riechmann et al., Nature 332 (1988), 323-329; Presta, Curr. Op. Struct. Biol. 2 (1992), 593-596), chimeric antibodies (e.g., see Morrison et al., Proc. Natl. Acad. Sci. USA 81 (1984), 6851-6855), human antibodies, fully humanized antibodies (e.g., see Tomizuka et al., Nature Genetics (1997) 16: 133-143; Kuroiwa et al., Nucl. Acids Res. (1998) 26: 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects Vol. 10, 69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.) (Kluwer Academic Publishers, 1999); Tomizuka et al., Proc. Natl. Acad. Sci. USA (2000) 97: 722-727, WO 2007 / 077028), multispecific antibodies (e.g., bispecific antibodies) formed from at least two antibodies, or antibody fragments of any of the above antibodies.

[0058] The term “Doppel-binding antibody fragment” as used in this article refers to any portion of the antibody of the above types that can bind to Doppel, typically including the antigen-binding region or variable region. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, biantibodies (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA (1993) 90: 6444-6448), single-chain antibody molecules (see, e.g., Pluckthun, in: The Pharmacology of Monoclonal Antibodies 113 (edited by Rosenburg and Moore) (Springer Verlag, NY (1994), 269-315), and other fragments capable of binding to Doppel. Therefore, antibody fragments that can be used as the Doppel-targeting portion of the ADC described herein can contain a portion of a full-length antibody, such as its antigen-binding domain or variable region. Examples of suitable antibody fragments include Fab, F(ab')2, Fv, and single-chain Fv (scFv) constructs (where the Fv fragments of the heavy and light chains are linked by suitable linkers) (see, e.g., Huston et al., Proc. Natl. Acad. Sci. USA). (1988) 85: 5879-5883); biantibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0059] As used in this article, the term "dual antibody" refers to a small antibody fragment having two antigen-binding sites, which contains a heavy chain variable domain (V) linked to the same polypeptide chain via a linker. H ) and light chain variable domain (V L (V) H -V L The linker is too short to allow two domains on the same chain to pair, so these domains are forced to pair with complementary domains on another chain, thus forming two antigen-binding sites. Detailed descriptions of biantibodies can be found, for example, EP404097; WO93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA (1993) 90:6444-6448.

[0060] Furthermore, the term “antibody” or “anti-Doppel antibody” as used herein may include antibody-like molecules containing engineered antibody subdomains or naturally occurring antibody variants. These antibody-like molecules may be single-domain antibodies, such as antibodies containing only the VH or only the VL domains, derived from natural sources (e.g., camels) (see, e.g., Muyldermans et al., J. Biotech. (2001) 74: 277-302) or obtained by in vitro display of libraries from humans, camels, or other species (see, e.g., Holt et al., Trends Biotechnol., (2003) 21:484-90).

[0061] As used in this article, the term "monoclonal antibody" refers to an antibody obtained from a single B lymphocyte clone or from cells transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are highly specific, targeting only a single antigenic site. Furthermore, unlike polyclonal antibody formulations, which typically contain multiple antibodies targeting different determinants (epitopes), each monoclonal antibody targets only a single determinant (epitope) on the antigen.

[0062] Therefore, the Doppel targeting portion of the ADC described herein can be any antibody or antibody-like molecule, including but not limited to polyclonal or monoclonal antibodies, or antibody derivatives such as single-chain antibodies, chimeric antibodies, humanized antibodies (or other modified species-specific antibodies for other target species), surface-modified antibodies, etc. The antibody can be glycosylated or non-glycosylated, or have a modified glycosylation pattern.

[0063] The Doppel targeting portion applicable to the ADCs and methods described herein includes the Doppel targeting molecule described in U.S. Patent Application 17 / 350,763, filed June 17, 2021, the entire contents of which are incorporated herein by reference.

[0064] The general structure of antibodies is well known in the art and will only be briefly outlined here. Immunoglobulin monomers consist of two heavy chains and two light chains linked by disulfide bonds. Each heavy chain pairs with one light chain and is directly linked by a disulfide bond. Each heavy chain contains a constant region (whose sequence depends on the antibody isotype) and a variable region. The variable region contains three hypervariable regions (or complementarity-determining regions), named CDRH1, CDRH2, and CDRH3, which are located within the frame region. Each light chain also contains a constant region and a variable region; its variable region contains three hypervariable regions (named CDRL1, CDRL2, and CDRL3), which are located within the frame region in a similar manner to the heavy chain variable region.

[0065] The hypervariable regions of each pair of heavy and light chains cooperate to form antigen-binding sites capable of binding to the target antigen. The binding specificity of a pair of heavy and light chains is determined by the sequence of their respective CDRs. Therefore, once a set of CDR sequences (i.e., the sequences of the three CDRs of the heavy and light chains) is determined, resulting in specific binding specificity, this set of CDR sequences can, in principle, be inserted into appropriate positions in any other antibody framework region and linked to any antibody constant region to obtain different antibodies with the same antigen-binding specificity.

[0066] In some embodiments, the Doppel targeting portion is an anti-Doppel antibody. In a particular embodiment, the Doppel targeting portion is a human monoclonal antibody selected from human monoclonal antibody A12, human monoclonal antibody B2, human monoclonal antibody E9, human monoclonal antibody 3D5, human monoclonal antibody 3D1, human monoclonal antibody 4D1, human monoclonal antibody 3H9, and any Doppel-binding fragment thereof. In a particular embodiment, the amino acid sequence of the Doppel targeting portion has at least 85%, 90%, 95%, or 99% identity with the amino acid sequence of monoclonal antibody A12, human monoclonal antibody B2, human monoclonal antibody E9, human monoclonal antibody 3D5, human monoclonal antibody 3D1, human monoclonal antibody 4D1, or human monoclonal antibody 3H9.

[0067] In some implementations, the Doppel targeting portion is an anti-Doppel antibody whose CDR sequence has at least 85%, 90%, 95%, 99%, or 100% identity with the CDR sequences of human monoclonal antibodies A12, B2, E9, 3D5, 3D1, 4D1, or 3H9.

[0068] In some embodiments, the Doppel targeting portion is an anti-Doppel antibody whose heavy chain sequence has at least 85%, 90%, 95%, 99%, or 100% identity with the heavy chain sequence of human monoclonal antibody A12 (SEQ ID NO: 9), human monoclonal antibody B2 (SEQ ID NO: 10), human monoclonal antibody E9 (SEQ ID NO: 11), human monoclonal antibody 3D5 (SEQ ID NO: 12), human monoclonal antibody 3D1 (SEQ ID NO: 13), human monoclonal antibody 4D1 (SEQ ID NO: 14), or human monoclonal antibody 3H9 (SEQ ID NO: 15). In some implementations, the Doppel targeting portion is an anti-Doppel antibody whose heavy chain variable region sequence has at least 85%, 90%, 95%, 99%, or 100% identity with the heavy chain variable region sequence of human monoclonal antibody A12, human monoclonal antibody B2, human monoclonal antibody E9, human monoclonal antibody 3D5, human monoclonal antibody 3D1, human monoclonal antibody 4D1, or human monoclonal antibody 3H9. In a particular embodiment, the CDRH1 amino acid sequence contained in the heavy chain variable region has at least 85%, 90%, 95%, 99%, or 100% identity with any one of the following sequences: the CDRH1 sequence of human monoclonal antibody A12 (SEQ ID NO: 16), the CDRH1 sequence of human monoclonal antibody B2 (SEQ ID NO: 17), the CDRH1 sequence of human monoclonal antibody E9 (SEQ ID NO: 18), the CDRH1 sequence of human monoclonal antibody 3D5 (SEQ ID NO: 19), the CDRH1 sequence of human monoclonal antibody 3D1 (SEQ ID NO: 20), the CDRH1 sequence of human monoclonal antibody 4D1 (SEQ ID NO: 21), or the CDRH1 sequence of human monoclonal antibody 3H9 (SEQ ID NO: 22). In a particular embodiment, the CDRH2 amino acid sequence contained in the heavy chain variable region has at least 85%, 90%, 95%, 99%, or 100% identity with the CDRH2 sequence of any one of the following: human monoclonal antibody A12 (SEQ ID NO: 23), human monoclonal antibody B2 (SEQ ID NO: 24), human monoclonal antibody E9 (SEQ ID NO: 25), human monoclonal antibody 3D5 (SEQ ID NO: 26), human monoclonal antibody 3D1 (SEQ ID NO: 27), human monoclonal antibody 4D1 (SEQ ID NO: 28), or human monoclonal antibody 3H9 (SEQ ID NO: 29).In a particular embodiment, the CDRH3 amino acid sequence contained in the heavy chain variable region has at least 85%, 90%, 95%, 99%, or 100% identity with the CDRH3 sequence of any one of the following: human monoclonal antibody A12 (SEQ ID NO: 30), human monoclonal antibody B2 (SEQ ID NO: 31), human monoclonal antibody E9 (SEQ ID NO: 32), human monoclonal antibody 3D5 (SEQ ID NO: 33), human monoclonal antibody 3D1 (SEQ ID NO: 34), human monoclonal antibody 4D1 (SEQ ID NO: 35), or human monoclonal antibody 3H9 (SEQ ID NO: 36).

[0069] In some embodiments, the Doppel targeting portion is an anti-Doppel antibody whose light chain sequence has at least 85%, 90%, 95%, 99%, or 100% identity with the light chain sequence of human monoclonal antibody A12 (SEQ ID NO: 37), human monoclonal antibody B2 (SEQ ID NO: 38), human monoclonal antibody E9 (SEQ ID NO: 39), human monoclonal antibody 3D5 (SEQ ID NO: 40), human monoclonal antibody 3D1 (SEQ ID NO: 41), human monoclonal antibody 4D1 (SEQ ID NO: 42), or human monoclonal antibody 3H9 (SEQ ID NO: 43). In some implementations, the Doppel targeting portion is an anti-Doppel antibody whose light chain variable region sequence has at least 85%, 90%, 95%, 99%, or 100% identity with the light chain variable region sequence of human monoclonal antibody A12, human monoclonal antibody B2, human monoclonal antibody E9, human monoclonal antibody 3D5, human monoclonal antibody 3D1, human monoclonal antibody 4D1, or human monoclonal antibody 3H9. In a particular embodiment, the CDRL1 amino acid sequence contained in the light chain variable region has at least 85%, 90%, 95%, 99%, or 100% identity with the CDRL1 sequence of any one of the following: human monoclonal antibody A12 (SEQ ID NO: 44), human monoclonal antibody B2 (SEQ ID NO: 45), human monoclonal antibody E9 (SEQ ID NO: 46), human monoclonal antibody 3D5 (SEQ ID NO: 47), human monoclonal antibody 3D1 (SEQ ID NO: 48), human monoclonal antibody 4D1 (SEQ ID NO: 49), or human monoclonal antibody 3H9 (SEQ ID NO: 50). In a particular embodiment, the CDRL2 amino acid sequence contained in the light chain variable region has at least 85%, 90%, 95%, 99%, or 100% identity with the CDRL2 sequence of any one of the following: human monoclonal antibody A12 (SEQ ID NO: 51), human monoclonal antibody B2 (SEQ ID NO: 52), human monoclonal antibody E9 (SEQ ID NO: 53), human monoclonal antibody 3D5 (SEQ ID NO: 54), human monoclonal antibody 3D1 (SEQ ID NO: 55), human monoclonal antibody 4D1 (SEQ ID NO: 56), or human monoclonal antibody 3H9 (SEQ ID NO: 57).In a particular embodiment, the CDRL3 amino acid sequence contained in the light chain variable region has at least 85%, 90%, 95%, 99%, or 100% identity with any one of the following CDRL3 sequences: human monoclonal antibody A12 (SEQ ID NO: 58), human monoclonal antibody B2 (SEQ ID NO: 59), human monoclonal antibody E9 (SEQ ID NO: 60), human monoclonal antibody 3D5 (SEQ ID NO: 61), human monoclonal antibody 3D1 (SEQ ID NO: 62), human monoclonal antibody 4D1 (SEQ ID NO: 63), or human monoclonal antibody 3H9 (SEQ ID NO: 64).

[0070] In some implementations, the Doppel targeting portion is an anti-Doppel antibody whose frame region sequence has at least 85%, 90%, 95%, 99%, or 100% identity with the frame sequence of human monoclonal antibody A12, human monoclonal antibody B2, human monoclonal antibody E9, human monoclonal antibody 3D5, human monoclonal antibody 3D1, human monoclonal antibody 4D1, or human monoclonal antibody 3H9.

[0071] In some implementations, the Doppel targeting portion is an anti-Doppel antibody whose heavy chain constant region sequence has at least 85%, 90%, 95%, 99%, or 100% identity with the heavy chain constant region sequence of human monoclonal antibody A12, human monoclonal antibody B2, human monoclonal antibody E9, human monoclonal antibody 3D5, human monoclonal antibody 3D1, human monoclonal antibody 4D1, or human monoclonal antibody 3H9.

[0072] In some implementations, the Doppel targeting portion is an anti-Doppel antibody whose light chain constant region sequence has at least 85%, 90%, 95%, 99%, or 100% identity with the light chain constant region sequence of human monoclonal antibody A12, human monoclonal antibody B2, human monoclonal antibody E9, human monoclonal antibody 3D5, human monoclonal antibody 3D1, human monoclonal antibody 4D1, or human monoclonal antibody 3H9.

[0073] Table 1

[0074]

[0075]

[0076]

[0077] Table 2

[0078]

[0079] Table 3

[0080]

[0081]

[0082] Table 4

[0083]

[0084] As described above, due to the localization of Doppel on tumor cells, anti-Doppel antibodies suitable for the ADCs and methods described herein can target tumor cells and thus exhibit one or more properties, such as the ability to recognize tumor cells, the ability to bind to tumor cells, the ability to be endocytosed by tumor cells, and the ability to kill tumor cells.

[0085] As described above, due to the localization of Doppel on tumor endothelial cells (TECs), anti-Doppel antibodies suitable for the ADCs and methods described herein can target tumor endothelial cells (TECs) and thus exhibit one or more properties, such as the ability to recognize TECs, the ability to bind to TECs, the ability to be internalized by TECs, and the ability to kill TECs.

[0086] Since ADCs can contain individual chemotherapeutic agents, the Doppel-targeting antibody itself does not necessarily need to have antitumor activity. Nevertheless, in some implementations, the Doppel-targeting antibody itself has antitumor activity. As mentioned above, the advantage of the Doppel-targeting antibody is its ability to be internalized by tumor cells, thereby enhancing the cytotoxic effects of chemotherapeutic compounds in a manner that specifically and selectively targets tumor cells.

[0087] Flow cytometry can be used to confirm the binding activity of antibodies to tumor cells. The process of antibody internalization into tumor cells can be confirmed by: (1) observing intracellular antibodies under a fluorescence microscope using a fluorescently labeled secondary antibody bound to a Doppel-targeting antibody (see, for example, Adams et al., Cell Death and Differentiation (2008) 15: 751-761); (2) measuring intracellular fluorescence using a fluorescently labeled secondary antibody bound to a Doppel-targeting antibody (Austin et al., Mol. Biol. of the Cell, (2004) 15:5268-5282); or (3) using an immunotoxin bound to a Doppel-targeting antibody for Mab-ZAP detection, in which the toxin is released after entering the cell, thereby inhibiting cell growth (see, for example, Kohls et al., BioTechniques, (2000) 28:162-165). For example, a recombinant complex protein of diphtheria toxin catalytic domain and protein G can be used as an immunotoxin.

[0088] Alternatively, conventional techniques can be used to screen for Doppel binding activity of antibodies and antibody fragments. For example, Doppel binding activity can be measured using absorbance measurements, enzyme-linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), radioimmunoassays (RIA), Western blotting, and / or immunofluorescence. For example, in ELISA, a known anti-Doppel-targeting antibody can be immobilized on a plate, Doppel can be added to the plate, and then a sample containing the antibody to be tested, such as culture supernatant from antibody-producing cells or purified antibody, can be added. Then, a secondary antibody capable of recognizing the primary antibody and labeled with an enzyme (e.g., alkaline phosphatase) is added, and the plate is incubated. Next, after washing, an enzyme substrate (e.g., nitrophenyl phosphate) is added to the plate, and absorbance is measured to assess the antigen-binding activity of the sample. C-terminal or N-terminal fragments of the Doppel protein can be used as antigens. In another example, surface plasmon resonance analysis can be used to assess the activity of the antibodies according to the invention.

[0089] In some embodiments, the anti-Doppel antibody binds to one or more forms of Doppel, such as one or more of the monomeric, dimeric, glycosylated, and non-glycosylated forms described above. In some embodiments, the anti-Doppel antibody binds to one or more forms of human Doppel, such as one or more of the monomeric, dimeric, glycosylated, and non-glycosylated forms described above. In some embodiments, the anti-Doppel antibody binds to one or more non-human Doppel, such as one or more of the monomeric, dimeric, glycosylated, and non-glycosylated forms described above.

[0090] In some embodiments, the anti-Doppel antibody preferentially binds to one or more forms of Doppel, for example, preferentially binding to one or more forms of Doppel compared to other forms. In some embodiments, the anti-Doppel antibody preferentially binds to one or more forms of human Doppel. In some embodiments, the anti-Doppel antibody preferentially binds to one or more forms of non-human Doppel.

[0091] In some embodiments, the anti-Doppel antibody preferentially binds to one form of Doppel, for example, preferentially binding to one form of Doppel compared to other forms of Doppel. In some embodiments, the anti-Doppel antibody preferentially binds to one form of human Doppel. In some embodiments, the anti-Doppel antibody preferentially binds to one form of non-human Doppel.

[0092] The antitumor activity of antibodies can be confirmed in vitro by measuring their inhibitory activity on cell growth. For example, cancer cell lines overexpressing the antibody target protein can be cultured, and different concentrations of the antibody can be added to the culture system to determine its inhibitory activity on lesion formation, colony formation, and globular growth. Antitumor activity can also be confirmed in vivo, for example, by administering the antibody to nude mice transplanted with tumor cell lines that highly express the target protein and measuring changes in the cancer cells (tumors).

[0093] The Doppel targeting portion suitable for the ADCs and methods described herein can be prepared using methods known in the art. Anti-Doppel antibodies suitable for the ADCs and methods described herein can be derived from (e.g., generated therein) any species. Typical species include human, rat, mouse, and rabbit. Anti-Doppel antibodies derived from non-human species can be chimeric or humanized antibodies. Anti-Doppel antibodies suitable for the ADCs and methods described herein can be polyclonal or monoclonal antibodies.

[0094] For example, antibodies can be prepared in a host (e.g., a mammalian host) using an antigen containing the Doppel protein or fragments thereof (e.g., its N-terminal or globular domain), and these antibodies can be screened for their ability to bind to Doppel and (optionally) their ability to inhibit interaction with tyrosine kinase receptors (e.g., VEGFR2, etc.). For example, polyclonal antibodies against Doppel can be prepared by collecting blood from a mammal immunized with Doppel and isolating the desired antibody from the serum. Methods for isolating serum are known in the art. Serum containing polyclonal antibodies and / or components containing polyclonal antibodies can be isolated and purified.

[0095] Monoclonal antibodies used in the ADCs and methods described herein can be produced by methods known to those skilled in the art. For example, immune cells can be collected from non-human mammals immunized with an antigen (whose serum contains the desired antibody) and fused. Immune cells used for cell fusion are typically taken from the spleen. Other suitable parental cells that can be fused with immune cells include, for example, mammalian myeloma cells, such as mammalian myeloma cells with the acquired property of drug-selective fusion. Immune cells and myeloma cells can be fused according to known methods. See, for example, Milstein et al., Methods Enzymol., (1981) 73: 3-46. Hybridomas obtained by cell fusion can be selected by culturing in a standard selection medium, such as HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). Cell culture is typically carried out in HAT medium for several days to several weeks, for example, enough time for all other cells except the desired hybridoma (non-fused cells) to die. Standard limiting dilutions can then be performed to screen and clone hybridoma cells that produce the desired antibody.

[0096] Humanized forms of nonhuman (e.g., murine) antibodies can be obtained as chimeric antibodies containing a very small amount of nonhuman immunoglobulin sequence. Typically, humanized antibodies contain at least one or two variable regions, where the variable region sequence is derived from a nonhuman immunoglobulin, and the frame region (FR) sequence corresponds to a human immunoglobulin sequence. Thus, in some embodiments, the anti-Doppel antibody used herein contains a human antibody frame region. Such antibodies can be prepared using known techniques. Humanized antibodies may optionally contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. See, for example, Jones et al., Nature (1986) 321: 522-525; Reichmann et al., Nature, (1988) 332: 323-329; Presta, Curr. Op. Struct. Biol., (1992) 2: 593-596.

[0097] As another method for obtaining antibodies that can be used in the ADCs and methods described herein, a transgenic animal carrying a human antibody gene can be immunized with the Doppel protein, cells expressing the Doppel protein, or their lysates. The resulting antibody-producing cells can be collected and fused with myeloma cells to obtain a hybridoma, from which human antibodies against Doppel can then be prepared according to the methods described above. Alternatively, antibody-producing immune cells (e.g., immune lymphocytes) can be immortalized via oncogenes and used to prepare monoclonal antibodies.

[0098] Recombinant genetic engineering techniques can also be used to prepare monoclonal antibodies against Doppel that can be used in the ADCs and methods described herein. See, for example, Borrebaeck et al., Therapeutic Monoclonal Antibodies (MacMillan Publishers Ltd. (1990)). For example, DNA encoding an antibody against Doppel can be cloned from immune cells (e.g., antibody-producing hybridomas or immune lymphocytes), inserted into a suitable vector, and introduced into host cells to prepare recombinant anti-Doppel antibodies.

[0099] As described above, the Doppel-targeting portion of the ADC described herein can be an antibody fragment that binds to Doppel. As mentioned above, the term "antibody fragment that binds to Doppel" as used herein includes any Doppel-binding fragment of an antibody or antibody-like molecule, including but not limited to Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments and smaller fragments, biantibodies, etc. Antibody "fragments" can be prepared from full-length antibodies or synthesized using, for example, recombinant technologies. Doppel-binding antibody fragments can be generated by treating an antibody that binds to Doppel with an enzyme (e.g., papain or pepsin). Alternatively, a gene encoding a Doppel antibody fragment can be constructed, inserted into an expression vector, and expressed in a suitable host cell. See, for example, Co et al., J. Immunol. (1969) 152: 2968-2976; Better and Horwitz, Methods Enzymol. (1989) 178: 476-496; Pluckthun and Skerra, Methods Enzymol. (1989) 178:497-515; Lamoyi, Methods Enzymol. (1986) 121: 652-663; Rousseaux et al., Methods Enzymol. (1986) 121: 663-669; Bird and Walker, Trends Biotechnol. (1991) 9: 132-137.

[0100] Cleavable linker

[0101] As described above, the anti-Doppel ADC described herein contains a cleavable linker. In some respects, linking the anti-Doppel moiety to the therapeutic agent via the cleavable linker can serve a dual purpose: to inactively link the therapeutic agent to the anti-Doppel moiety before reaching the target site (e.g., thereby preventing potential systemic toxicity associated with chemotherapy agents), and to release the therapeutic agent at the target site (thereby maximizing efficacy at the desired site).

[0102] Suitable cleavable linkers for ADCs are known in the art, including peptide linkers and non-peptide linkers. Typically, the cleavable linker for the anti-Doppel ADC conjugate described herein can be cleaved under intracellular or extracellular conditions, or both. In some respects, the linker can be cleaved by intracellular proteases, such as lysosomal proteases or endosomal proteases. In some respects, the linker can be hydrolyzed at pH less than 5.5. In some respects, the linker can be cleaved by caspases, as discussed in more detail below.

[0103] In some respects, the adapter comprises or is composed of a dipeptide adapter. In some respects, the dipeptide adapter is a valine-citrulline (Val-Cit) adapter, a valine-alanine (Val-Ala) adapter, or a phenylalanine-lysine (Phe-Lys) adapter, which can be cleaved by cathepsin B.

[0104] In some respects, the joint is or is contained in disulfide bond joints that break down in a reducing environment.

[0105] In some respects, the connector is or is contained in a hydrazone connector that is hydrolyzed under conditions of pH less than 5.5.

[0106] As described above, in some respects, this linker is a caspase-cleavable peptide linker. Examples of suitable caspase-cleavable peptide linkers are described in U.S. Patent No. 10,357,572, the entire contents of which are incorporated herein by reference.

[0107] As used herein, the term "cystasparase" refers to cysteine-aspartic proteases and cysteine-dependent aspartic-directed proteases that are activated (e.g., expressed) by cells undergoing apoptosis. In specific embodiments, the cystasparases are cystasparase-3, cystasparase-7, and / or cystasparase-9. Cysteines are not tumor cell-specific but are expressed as a result of apoptosis. Therefore, cystasparases must be activated by other factors. This gives drugs with cystasparase-cleavable peptide linkers (e.g., linkers comprising or composed of DEVD peptide sequences (SEQ ID NO: 4)) several unique advantages. First, cleavage of the cystasparase-cleavable peptide linker is independent of the expression of the tumor enzyme, which can vary considerably. Second, the cystasparase-cleavable peptide linker is cleaved only in the presence of cystasparase, for example, in cells undergoing apoptosis. Furthermore, when the chemotherapeutic payload itself is not selective, cleavage of the linker releases the chemotherapeutic agent, thereby killing nearby tumor cells regardless of their heterogeneity (this is known as the bystander killing effect). More notably, cleavage of caspase-cleavable peptide linkers can trigger the cleavage of more linkers, resulting in an amplification effect. For example, cells killed by chemotherapeutic agents express caspase, which in turn cleaves the caspase-cleavable peptide linkers of other ADCs, releasing the chemotherapeutic agents it carries, killing more tumor cells, and so on, effectively enhancing and prolonging the therapeutic effect. (See, for example, Sun et al., Bioconjugate Chem. (2020) 31(4): 1012-1024; Kovtun et al., Cancer Research (2006) 66(6): 3214-21).

[0108] As used herein, the term "caspasparase-cleavable peptide linker" refers to a peptide sequence consisting of two or more amino acid residues that can be cleaved by caspase. In some respects, caspase-cleavable peptide linkers can be cleaved by caspase-3 or caspase-7, for example, peptides containing the sequence Asp-Xaa-Xaa-Asp (where "Xaa" represents any amino acid, and can be in L- or D-isomer form). In other respects, caspase-cleavable peptide linkers can be cleaved by caspase-9, for example, peptides containing the amino acid sequences Leu-Xaa-Xaa-Asp or Val-Xaa-Xaa-Asp (where "Xaa" represents any amino acid, and can be in L- or D-isomer form).

[0109] In certain respects, the caspase-cleavable peptide linker comprises or consists of one of the following sequences:

[0110] Asp-Glu-Val-Asp (SEQ ID NO: 4)

[0111] Asp-Leu-Val-Asp (SEQ ID NO: 5)

[0112] Asp-Glu-Ile-Asp (SEQ ID NO: 6), or

[0113] Leu-Glu-His-Asp (SEQ ID NO: 7).

[0114] In certain respects, the caspase-cleavable peptide linker comprises or consists of the sequence Lys-Gly-Asp-Glu-Val-Asp (SEQ ID NO: 8), also denoted as KGDEVD (SEQ ID NO: 8).

[0115] Generally, the anti-Doppel ADCs described herein are inactive until the linker is cleaved. Therefore, the anti-Doppel ADCs containing chemotherapeutic agents described herein cause minimal damage to healthy cells. Furthermore, the anti-Doppel ADCs with caspase-cleavable peptide linkers described herein are only activated in the presence of caspase, for example, in the presence of cells undergoing apoptosis (e.g., tumor cells undergoing apoptosis). Therefore, the anti-Doppel ADCs with caspase-cleavable peptide linkers described herein achieve target specificity in two ways: (i) target specificity provided by the Doppel targeting portion; and (ii) target specificity provided by the caspase-cleavable peptide linker. As mentioned above, the anti-Doppel ADCs with caspase-cleavable peptide linkers described herein have additional advantages due to the amplification effect. Furthermore, the anti-Doppel ADC described herein, which has a caspase-cleavable peptide linker, wherein the four C-terminal amino acid residues of the caspase-cleavable peptide linker are selected from Asp-Xaa-Xaa-Asp, Leu-Xaa-Xaa-Asp, and Val-Xaa-Xaa-Asp, where Xaa represents any amino acid residue, has the additional advantage that the linker is cleaved at its junction with the therapeutic agent, releasing the unmodified form of the therapeutic agent, which will exert its inherent therapeutic effect (e.g., antitumor or cytotoxic effects).

[0116] Table 5

[0117]

[0118] Therapeutic agent

[0119] As described above, the anti-Doppel ADC described herein contains a therapeutic agent.

[0120] In some respects, the therapeutic agent is or comprises a chemotherapeutic agent. The terms “chemotherapeutic agent” and “cytotoxic agent” are generally used interchangeably herein. Herein, “chemotherapeutic agent” refers to the portion that can be used to treat cancer, such as a small molecule compound used to treat cancer. Similarly, herein, “cytotoxic agent” refers to the portion that can be used to induce cell death (apoptosis), such as a small molecule compound used to induce the death of target cells. In a specific embodiment, the chemotherapeutic agent can induce apoptosis in target cells (e.g., tumor cells and tumor tissue). Any chemotherapeutic agent known in the art may be used as a chemotherapeutic agent in the anti-Doppel ADC described herein. Typically, the chemotherapeutic agent used has or can be prepared to have substituents, structures, or portions that can be coupled to another portion of the ADC (directly or via a linker). Suitable chemotherapeutic agents include the cytotoxic agents described in U.S. Patent No. 10,357,572, the entire contents of which are incorporated herein by reference.

[0121] Suitable cytotoxic agents include, for example, oliquistatin, DNA minor groove binders, DNA minor groove alkylating agents, enediyne, reciprocoloxine, ducamycin, taxane, puromycin, dolalastatin, maytansine compounds, and vinca alkaloids. In some respects, cytotoxic agents are selected from oliquistatin phenylalanine diamine (AFP), monomethyloliquistatin F (MMAF), monomethyloliquistatin E (MMAE), oliquistatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino doxorubicin, rhizotoxin, cyanomorpholino doxorubicin, dolalastatin-10, echinomycin, combutatin, carlicycin, maytansine, DM1, neteroprexine, and their derivatives. Other suitable cytotoxic agents include anti-microtubule agents such as oliguria alkaloids, podophyllotoxin, taxanes, baccatin derivatives, cryptofolin, maytansine, combutatin, and dolatastatin. Specifically, anti-microtubule agents include oliguria phenylalanine diamine (AFP), monomethyloliguria alkaloid F (MMAF), monomethyloliguria alkaloid E (MMAE), oliguria alkaloid E, vincristine, vinblastine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epochyam A, epochyam B, nocodazole, colchicine, colchicine, estradiol, simadotine, discosponyl lactone, maytansine, DM1, or eleutherobin. In some embodiments, the anti-microtubule agent is monomethyloliguria alkaloid E (MMAE).

[0122] In some implementations, the chemotherapeutic agents are anthracyclines such as doxorubicin, daunorubicin, epirubicin, idarubicin, vararubicin, or derivatives thereof; antibiotics such as actinomycin D, bleomycin, mitomycin C, carlicimycin, or derivatives thereof; alkylating agents such as cyclophosphamide, nitrogen mustard, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozotocin, busulfan, dacarbazine, temozolomide, thiotepa, atratamine, ducamycin, or derivatives thereof; platinum-based drugs such as cisplatin, carboplatin, nedaplatin, oxaliplatin, satroplatin, triplatin tetranitrate, or derivatives thereof; and antimetabolites such as 5-fluorouracil. The chemotherapeutic agents include pyridine, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cytarabine, fluorouridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, or derivatives thereof; topoisomerase inhibitors, such as ethatecan, camptothecin, topotecan, irinotecan, etoposide, teniposide, mitoxantrone, or derivatives thereof; mitotic inhibitors, such as paclitaxel, docetaxel, ixaprone, vincristine, vinblastine, vinorelbine, estradiol, maytansine, DM1 (metansine), DM4, dolalastatin, oliquistatin E, oliquistatin F, monomethyloliquistatin E, monomethyloliquistatin F, or derivatives thereof. In a specific embodiment, the chemotherapeutic agent is doxorubicin. In a specific embodiment, the chemotherapeutic agent is daunorubicin. In a specific embodiment, the chemotherapeutic agent is ethatecan.

[0123] In one embodiment, the chemotherapeutic agent is selected from 5-FU, afatinib, aprilidine, azaribin, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, briostatin-1, busulfan, carlicicillin, ethacryne, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatin, CO X-2 inhibitors, irinotecan, SN-38, cladribine, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib, denaciline, docetaxel, actinomycin D, daunorubicin, doxorubicin, podophyllotoxin, erlotinib, entenoxate, estrogen receptor binders, etoposide, exemestane, fingolimod, fluorouridine, fludarabine, flutamide, flurapirib, fostatinib, ganetinib, GDC-0 834, GS-1101, Gefitinib, Gemcitabine, Hydroxyurea, Ibrutinib, Idarubicin, Edraliximab, Ifosfamide, Imatinib, L-Asparaginase, Lapatinib, Lenalidomide, Leucovorin, LFM-A13, Lomustine, Nitrogen Mustard, Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone, Spectinomycin, Mitomycin, Mitotan, Naveline, Neratinib, Nilotinib, Nitrosourea, Olaparib Priligy, Procarbazine, Paclitaxel, PCI-32765, Pentostatin, PSI-341, Raloxifene, Semustine, Sorafenib, Levozocin, SU11248, Sunitinib, Tamoxifen, Temozolomide, Transplatinum, Thalidomide, Thioguanine, Thiotepa, Teniposide, Topotecan, Uracil Mustard, Vatalani, Vinorelbine, Vincristine, Vincristine Alkaloids, and ZD183.

[0124] As mentioned above, chemotherapeutic agents that can be used in the anti-Doppel ADC described herein include monomethylolpropionate E (MMAE), the chemical name of which is:

[0125] ((S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butyramido)butyramide

[0126] Its chemical formula is as follows:

[0127]

[0128] As mentioned above, chemotherapeutic agents that can be used in the anti-Doppel ADC described herein include ethanotecan, whose chemical name is:

[0129] (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-10,13-dione

[0130] Its chemical structural formula is as follows:

[0131]

[0132] As mentioned above, other suitable chemotherapeutic agents include doxorubicin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, platinum-based antitumor drugs (cisplatin or its derivatives), paclitaxel and its derivatives, and camptothecin and its derivatives, including ethathecan.

[0133] In some respects, therapeutic agents are or contain immunomodulators. As used herein, “immunomodulator” refers to a drug that helps the body fight off pathogens (such as tumor cells) by modulating the immune response. In specific implementation methods, immunomodulators are selected from cytokines, lymphokines, monokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony-stimulating factor (CSF), interferon (IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, pro-relaxanthin, follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), liver growth factor, prostaglandins, fibroblast growth factor, prolactin, placental prolactin, OB protein, transforming growth factor (TGF), TGF-α, TGF-β, and insulin-like growth factor (IG). F), erythropoietin, thrombopoietin, tumor necrosis factor (TNF), TNF-α, TNF-β, Müllerian duct inhibitor, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrin, interleukin (IL), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-α, interferon-β, interferon-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1. IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, IL-25, LIF, kit ligand, FLT-3, angiostatin, thromboretin, endostatin, Toll-like receptor (TLR) agonists (e.g., CU-T12-9, Pam3CSK4, FSL-1, Pam2CSK4, and CL429), Poly(A:U), Poly(I:C), lipopolysaccharide (LPS), MPLA-SM, CRX-527, flagellin, Thiazolylquinoline derivatives, imidazoquinoline derivatives (e.g., CL097, gardiquimod, imiquimod, and resiquimod), adenine analogs, guanosine analogs, thymidine analogs, benzodiazepine analogs, and CpG oligodeoxynucleotides (ODNs) (e.g., ODN1585, ODN2216, ODN2336, ODN1668, ODN1826, ODN2006, ODN2007, ODNBW006, ODND-SL01, ODN2395, ODNM362, and ODND-SL03).

[0134] In some respects, the therapeutic agent is or contains a toxin. As used herein, the term "toxin" refers to a naturally occurring organic substance produced by the metabolic activity of living cells or organisms. In a particular embodiment, the toxin is selected from ricin, abrinogen, ribonuclease (RNase), DNase 1, Staphylococcus aureus enterotoxin A, pokeweed antiviral protein, gel toxin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

[0135] In some respects, the therapeutic agent is or contains a radionuclide. In this document, the terms “radionuclide,” “radioisotope,” or “radioisotope” are generally used interchangeably. As used herein, the term “radionuclide” refers to a nuclide that possesses excessive nuclear energy, making it unstable. Radionuclides may be naturally occurring or artificially synthesized. Exposure to radionuclides can have harmful effects on living organisms. In certain embodiments, the radionuclide is selected from… 11 C 13 N、 15 O、 32 P, 33 P, 47 Sc、 51 Cr 57 Co、 58 Co、 59 Fe、 62 Cu、 67 Cu、 67 Ga、 75 Br、 75 Se、 76 Br、 77 As、 77 Br、 80m Br、 89 Sr、 90 Y、 95 Ru、 97 Ru、 99 Mo、 99m Tc, 103m Rh、 103 Ru、 105 Rh、 105 Ru、 107 Hg, 109 Pd, 109 Pt, 111 Ag、 111 In、 113m In、 119 Sb, 121m Te、 122m Te、 125 I, 125m Te、 126 I, 131 I, 133 I,142 Pr, 143 Pr, 149 Pm, 152 Dy、 153 Sm、 161 Ho、 161 Tb, 165 Tm、 166 Dy、 166 Ho、 167 Tm、 168 TM、 169 Er、 169 Yb、 177 Lu、 186 Re、 188 Re、 189m Os、 189 Re、 198 Ir、 194 Ir、 197 Pt, 198 Au、 199 Au、 203 Hg, 211 At、 211 Bi、 211 Pb, 212 Bi、 212 Pb, 213 Bi、 215 Po、 217 At、 219 Rn、 221 Fr、 223 Ra、 225 Ac、 227 Th and 255 Fm.

[0136] In some embodiments, the therapeutic agent is or comprises a DNA cross-linking agent. In some embodiments, the DNA cross-linking agent is selected from indobenzobenzodiazepine dimer (IGN), pyrrolobenzodiazepine (PBD), and derivatives thereof.

[0137] Mode of attachment

[0138] In some embodiments of the anti-Doppel ADC described herein, the Doppel targeting portion is connected directly or via a connector to a cleavable connector, and the cleavable connector can be connected directly or via a connector to the therapeutic agent.

[0139] Therefore, in some embodiments, the Doppel targeting moiety is directly coupled to, for example, a caspase-cleavable peptide linker, via a covalent bond between a group on the Doppel targeting moiety and a group on the N-terminus of the peptide linker or a group on the side chain of the peptide linker. Furthermore, in some embodiments, the cleavable linker (e.g., a caspase-cleavable peptide linker) is directly coupled to a therapeutic agent, for example, via a covalent bond between a group on the C-terminus of the peptide linker or a group on the side chain of the peptide linker and a group on the therapeutic agent.

[0140] Alternatively, one or both connections can be made via a connector. Any connector suitable for pharmaceutical compounds can be used for this purpose. Suitable connectors include p-aminobenzylcarbamate (PABC).

[0141] Although the above description assumes that the Doppel targeting portion is attached to the N-terminus of the caspase-cleavable peptide linker and the therapeutic agent is attached to the C-terminus of the caspase-cleavable peptide linker, the possibility that the therapeutic agent is attached to the N-terminus of the caspase-cleavable peptide linker and the Doppel targeting portion is attached to the C-terminus of the caspase-cleavable peptide linker is also considered.

[0142] In some other embodiments, the anti-Doppel ADC described herein comprises a peptide that can be cleaved by caspase, which is directly or via a linker to a therapeutic agent, which in turn is directly or via a linker to the Doppel targeting moiety. For example, daunorubicin exerts its chemotherapeutic effect when conjugated to other moieties at its 14-CH3 site. Therefore, caspase-induced cleavage can produce a chemotherapeutic effect without releasing free daunorubicin. Thus, in some embodiments, the ADC comprises a peptide that can be cleaved by caspase, which is directly or via a linker to daunorubicin, which is directly or via a linker to the Doppel targeting moiety at its 14-CH3 site.

[0143] Anti-Doppel ADC

[0144] The anti-Doppel ADC described herein can be prepared using methods known in the art for preparing conjugates.

[0145] The anti-Doppel ADC described herein can be prepared stepwise, for example, by first preparing a cleavable linker-therapeutic agent conjugate, and then conjugating one or more of these conjugates to the Doppel targeting moiety. For example, a caspase-cleavable peptide linker-therapeutic agent conjugate can be prepared according to the method described in U.S. Patent No. 10,357,572 (the entire contents of which are incorporated herein by reference), and then one or more of these conjugates can be conjugated to the Doppel targeting moiety described herein. Other suitable methods for preparing the ADC described herein are known in the art and are illustrated in the following examples.

[0146] The number of therapeutic agent molecules on each Doppel targeting moiety (e.g., each Doppel targeting antibody) can affect the efficacy and safety of antibody-drug conjugates (ADCs). Therefore, ADCs can be prepared under reaction conditions that control the number of therapeutic agent molecules on each Doppel targeting moiety, such as controlling the relative amounts of reactants, other reagents, and other reaction conditions. However, it is possible to obtain a mixture of ADCs with varying numbers of therapeutic agent molecules conjugated. Therefore, the number of therapeutic agent molecules conjugated to an ADC can be expressed as an average value, representing the average number of therapeutic agent molecules conjugated on each Doppel targeting moiety. Thus, in the following examples, unless otherwise specified (e.g., when describing the number of therapeutic agent molecules conjugated on each Doppel targeting moiety of a particular ADC in a mixture), the number of therapeutic agent molecules conjugated on each Doppel targeting moiety refers to an average value.

[0147] The following embodiments and figures illustrate specific examples of anti-Doppel ADCs, including 3D1-KGDEVD-MMAE (name = SEQ ID NO: 67; structure = SEQ ID NO: 68), 3D5-KGDEVD-MMAE (name = SEQ ID NO: 69; structure = SEQ ID NO: 70), 3H9-KGDEVD-MMAE (name = SEQ ID NO: 65; structure = SEQ ID NO: 66), 3H9-KGDEVD-ethanotecan (name = SEQ ID NO: 75; structure = SEQ ID NO: 76), 4D1-KGDEVD-MMAE (name = SEQ ID NO: 71; structure = SEQ ID NO: 72), 3D1-vc-MMAE, 3D5-vc-MMAE, 3H9-vc-MMAE, and 4D1-vc-MMAE.

[0148] Pharmaceutical composition

[0149] In some respects, anti-Doppel ADCs are provided as pharmaceutical compositions, such as compositions comprising anti-Doppel ADCs and pharmaceutically acceptable carriers, excipients, and / or diluents. Examples of suitable carriers, excipients, and diluents include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, minerals, etc.

[0150] This pharmaceutical composition can be formulated for use in any route of administration, including any parenteral or topical route. In some respects, this pharmaceutical composition is suitable for injection or infusion, such as intravenous injection or infusion, for example, as a sterile composition for injection or infusion. Suitable components and excipients for such compositions are known in the art.

[0151] Examples of other formulations for parenteral administration include sterile aqueous solutions, water-insoluble solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solutions and suspensions may include, for example, propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), or injectable esters (e.g., ethyl oleate). The matrix of suppository formulations may include, for example, Wiptsol, polyethylene glycol (macrogol), Tween 61, cocoa butter, lauryl acetate, glycerin gelatin, etc.

[0152] In a particular embodiment, the conjugate is dissolved in water or other pharmaceutically acceptable aqueous carrier, wherein the conjugate has good solubility and optionally contains or does not contain other pharmaceutically acceptable excipients, preservatives, etc.

[0153] In addition, a kit is provided. The kit may contain one or more ADCs described herein in a suitable container and may optionally include instructions for use with the methods described herein.

[0154] Using anti-Doppel ADC methods

[0155] As described above, the anti-Doppel ADCs presented in this article can be used to treat Doppel-related diseases and conditions, including cancer.

[0156] Anti-Doppel ADCs can be administered via any route of administration. In some embodiments, ADCs are administered intravenously. The dosage of an ADC will vary depending on the subject and the disease condition being treated, and can be determined by those skilled in the art. In some embodiments, the dosage can be between about 1 mg / kg and about 100 mg / kg, including about 5 mg / kg and about 75 mg / kg, for example about 10 mg / kg and about 50 mg / kg, or higher. As mentioned above, ADCs containing chemotherapeutic agents are generally less toxic than chemotherapeutic agents alone, therefore the dosage of an ADC can be higher than the non-toxic dose when chemotherapeutic agents are used alone.

[0157] As described above, in embodiments where the ADC comprises a caspase-cleavable peptide linker and a chemotherapeutic agent, an amplification effect may occur: as described above, the treatment method induces apoptosis, leading to caspase expression. Upon administration of the ADC, caspase cleaves the caspase-cleavable peptide linker, releasing the chemotherapeutic agent. The chemotherapeutic agent induces further apoptosis, leading to further caspase expression, which in turn leads to more ADC being cleaved / activated by caspase, resulting in an amplification effect of apoptosis. This amplification effect makes the method highly efficient and specific in killing target cells (e.g., target tumor cells). Furthermore, this amplification effect can prolong the time interval between apoptosis-inducing treatments and / or between ADC administration doses. Therefore, in some embodiments, this amplification effect can reduce the chemotherapy dose required to treat a certain number of cancer cells.

[0158] As described above, ADCs are inactive until the caspase-cleavable peptide linker is cleaved. Therefore, ADCs are non-toxic to healthy cells (or do not induce apoptosis). In certain embodiments, the methods described herein can reduce damage to normal cells by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more compared to the administration of the same dose of an unconjugated form of chemotherapeutic agent.

[0159] Furthermore, the apoptotic effect of ADCs is selective for cells expressing caspase (e.g., cells undergoing apoptosis). Therefore, once apoptosis is induced in a target cell region (e.g., a target tissue), the method described herein can selectively and effectively induce apoptosis in other target cells, thereby treating, for example, cancer.

[0160] Inventive approach

[0161] Example

[0162] Example 1 - Preparation of an ADC named 3H9-KGDEVD-MMAE (name = SEQ ID NO: 65)

[0163] The cytotoxic drug monomethyl ozretamine E (MMAE) was linked to the anti-Doppel human monoclonal antibody 3H9 via a caspase-cleavable peptide linker composed of Lys-Gly-Asp-Glu-Val-Asp (SEQ ID NO: 8), resulting in an ADC named 3H9-KGDEVD-MMAE (name = SEQ ID NO: 65).

[0164]

[0165] (SEQ ID NO: 66)

[0166] The synthesis method of 3H9-KGDEVD-MMAE (SEQ ID NO: 65) is described below, and its overall structure is shown in the figure above (SEQ ID NO: 66).

[0167] A. Synthesis of maleimide-KGDEVD-MMAE (SEQ ID NO: 77) drug-connector conjugate

[0168] The preparation method of the maleimide-KGDEVD-MMAE (SEQ ID NO: 77) conjugate is as described in US Patent No. 10,357,572 (the entire contents of which are incorporated herein by reference). Specifically, ε-maleimide hexanoate and MMAE are linked to the amino and C-terminus of the lysine side chain of the AcKGDEVD peptide (SEQ ID NO: 1), respectively.

[0169] B. Coupling with mAb 3H9

[0170] To prepare this ADC, mAb 3H9 was partially reduced with tris(2-carboxyethyl)phosphine (TCEP), and the reduced cysteine ​​residues were then reacted with a maleimide-KGDEVD-MMAE (SEQ ID NO: 77) conjugate (e.g., a maleimide-terminated linker-load).

[0171] Specifically, 4.2 molar excess of tris(2-carboxyethyl)phosphine (TCEP) was added to a solution of 100 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid buffer) (pH 7.0) and 1 mM diethylenetriaminepentaacetic acid (DTPA), and the mixture was reacted at 37 °C for 1 hour to partially reduce 3H9 mAb. The concentration of the reduced antibody was determined by the BCA method of the solution, and the thiol concentration was determined by reacting with DTNB and measuring the absorbance at 412 nm, thereby determining the thiol / antibody ratio.

[0172] The reduced mAb was cooled on ice. Maleimide-terminated linker-load was added to the reaction mixture at a linker-load / mAb-thiol molar ratio of 2:1, and the reaction was continued at 4°C for 1 hour. After 1 hour of incubation, the reaction mixture was concentrated by centrifugation and ultrafiltration, and purified by elution with PBS via desalting G25 at 4°C. Then, ADC 3H9-KGDEVD-MMAE (name = SEQ ID NO: 65; structure = SEQ ID NO: 66) was aseptically filtered through a 0.2 μm filter membrane and immediately frozen at -80°C. The ADC was analyzed as follows: (1) concentration, determined by BCA protein assay; (2) aggregates, determined by size exclusion chromatography; (3) residual free drug, determined by reversed-phase HPLC; (4) drug-antibody ratio (DAR), determined by hydrophobic interaction chromatography.

[0173] Example 2 - Fabrication of an ADC named 3H9-vc-MMAE

[0174] An ADC was prepared comprising MMAE as a cytotoxic drug, mAb 3H9 as the Doppel targeting moiety, and a cathepsin B-cleavable peptide linker composed of Val-Cit, thus yielding an ADC named 3H9-vc-MMAE.

[0175]

[0176] The synthesis method of ADC 3H9-vc-MMAE is described below, and its overall structure is shown in the figure above.

[0177] To prepare this ADC, mAb 3H9 was partially reduced with tris(2-carboxyethyl)phosphine (TCEP), and then the reduced cysteine ​​residues were reacted with a maleimide-terminated linker-loador (maleimide hexanoic acid-valine-citrulline-p-aminobenzyloxycarbonyl-MMAE, also known as mc-Val-Cit-PABA-MMAE, available from MedChemExpress).

[0178] Specifically, 4.2 molar excess of tris(2-carboxyethyl)phosphine (TCEP) was added to a solution of 100 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid buffer) (pH 7.0) and 1 mM diethylenetriaminepentaacetic acid (DTPA), and the mixture was reacted at 37 °C for 1 hour to partially reduce 3H9 mAb. The concentration of the reduced antibody was determined by the BCA method of the solution, and the thiol concentration was determined by reacting with DTNB and measuring the absorbance at 412 nm, thereby determining the thiol / antibody ratio.

[0179] The reduced mAb was cooled on ice. Maleimide-terminated linker-load (mc-Val-Cit-PABA-MMAE, MedChemExpress) was added to the reaction mixture at a linker-load / mAb-thiol molar ratio of 2:1, and the reaction was continued at 4ºC for 1 hour. After 1 hour of incubation, the reaction mixture was concentrated by centrifugation and ultrafiltration, and purified by elution with PBS via desalting G25 at 4 °C. Then, 3H9-vc-MMAE was filtered aseptically through a 0.2 μm filter and immediately frozen at -80 °C. The ADC (3H9-vc-MMAE) was analyzed as follows: (1) concentration, determined by BCA protein assay; (2) aggregates, determined by size exclusion chromatography; (3) residual free drug, determined by reversed-phase HPLC; (4) drug-antibody ratio (DAR), determined by hydrophobic interaction chromatography.

[0180] Example 3 - Preparation of an ADC named 3H9-KGDEVD-Exatecan (Name = SEQ ID NO: 75)

[0181] The cytotoxic drug ethanotecan was linked to the anti-Doppel human monoclonal antibody 3H9 via a caspase-cleavable peptide linker composed of Lys-Gly-Asp-Glu-Val-Asp (SEQ ID NO: 8), resulting in an ADC named 3H9-KGDEVD-ethanotecan (name = SEQ ID NO: 75).

[0182]

[0183] (SEQ ID NO: 76)

[0184] The synthesis method of 3H9-KGDEVD-ethanotecan (name = SEQ ID NO: 75) is described below, and its overall structure is shown in the figure above (SEQ ID NO: 76).

[0185] A. Synthesis of maleimide-KGDEVD-ethanotecan (SEQ ID NO: 2) drug-connector conjugate

[0186] The preparation method of the maleimide-KGDEVD-ethanotecan (SEQ ID NO: 2) conjugate is as described in U.S. Patent No. 10,357,572 (the entire contents of which are incorporated herein by reference). Specifically, ε-maleimide hexanoate and ethanotecan are linked to the amino and C-terminus of the lysine side chain of the AcKGDEVD peptide (SEQ ID NO: 1), respectively.

[0187] B. Coupling with mAb 3H9

[0188] To prepare this ADC, mAb 3H9 was partially reduced with tris(2-carboxyethyl)phosphine (TCEP), and then the reduced cysteine ​​residues were reacted with a maleimide-KGDEVD-ethatecan (SEQ ID NO: 2) conjugate (e.g., a maleimide-terminated linker-load).

[0189] Specifically, 4.2 molar excess of tris(2-carboxyethyl)phosphine (TCEP) was added to a solution of 100 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid buffer) (pH 7.0) and 1 mM diethylenetriaminepentaacetic acid (DTPA), and the mixture was reacted at 37 °C for 1 hour to partially reduce 3H9 mAb. The concentration of the reduced antibody was determined by the BCA method of the solution, and the thiol concentration was determined by reacting with DTNB and measuring the absorbance at 412 nm, thereby determining the thiol / antibody ratio.

[0190] The reduced mAb was cooled on ice. Maleimide-terminated linker-load was added to the reaction mixture at a linker-load / mAb-thiol molar ratio of 2:1, and the reaction was continued at 4°C for 1 hour. After 1 hour of incubation, the reaction mixture was concentrated by centrifugation and ultrafiltration, and purified by elution with PBS via desalting G25 at 4°C. Then, ADC 3H9-KGDEVD-ethanotecan (name = SEQ ID NO: 75; structure = SEQ ID NO: 76) was aseptically filtered through a 0.2 μm filter and immediately frozen at -80°C. The ADC was analyzed as follows: (1) concentration, determined by BCA protein assay; (2) aggregates, determined by size exclusion chromatography; (3) residual free drug, determined by reversed-phase HPLC; (4) drug-antibody ratio (DAR), determined by hydrophobic interaction chromatography.

[0191] Example 4 - Cytotoxicity Assay

[0192] The ability of the anti-Doppel ADC described in this paper to deliver potent cytotoxic drugs and eliminate cells expressing Doppel was evaluated.

[0193] The HCT116 cell line expressing Doppel was selected and cultured with different concentrations of ADC. Cell viability was assessed for each culture after 72 hours. IC50 50Values ​​were calculated using logistic nonlinear regression and reported in nM. The ADCs 3H9-KGDEVD-MMAE (name = SEQ ID NO: 65; structure = SEQ ID NO: 66) and 3H9-vc-MMAE prepared as described above inhibited the growth of the Doppel-expressing cell line (HCT116), while 3H9 mAb alone did not have this effect. Figure 1 )

[0194] Table 6

[0195]

[0196] Example 5 - Endocytosis of anti-Doppel antibody in Doppel-expressing cell lines

[0197] The endocytosis of anti-Doppel mAb in human colorectal tumor endothelial cells (HCTEC) was investigated using immunofluorescence microscopy. 1×10⁻⁶ cells were used. 4 HCTEC cells cultured on coverslips were treated with 0.1 mg / ml FITC-labeled 3H9 mAb. After incubation for 3 hours, the cells were washed with PBS and fixed with 10% neutral-buffered formalin. The cells were then stained overnight at 4°C with 5 μg / ml WGA-Texas Red. Subsequently, the cells were washed with PBS, mounted in situ with DAPI-containing mounting medium, and observed under a microscope. The results showed that anti-Doppel mAb 3H9 was internalized into Doppel-expressing HCTEC cells. Figure 2 )

[0198] Example 6 - Lysosomal colocalization of anti-Doppel antibody in Doppel-expressing cell lines

[0199] Immunofluorescence microscopy was used to investigate the lysosomal colocalization of anti-Doppel mAb in human colorectal tumor endothelial cells (HCTEC). 1×10 4 HCTEC cells cultured on coverslips were treated with 0.1 mg / ml Cy5.5-labeled 3H9 mAb. After incubation for 3 hours, the cells were washed with PBS and treated with lysosomal probes at 37ºC for 2 hours. The cells were then washed again with PBS and fixed with 10% neutral-buffered formalin. After washing with PBS, in situ mounting medium containing DAPI was added, and the cells were observed under a microscope. The results showed that anti-Doppel mAb 3H9 co-localized with lysosomal markers in Doppel-expressing HCTEC cells. Figure 3 )

[0200] Example 7 - In vivo efficacy of anti-Doppel ADC in xenograft models

[0201] To evaluate the antitumor efficacy of anti-Doppel ADC, an HCT116 xenograft tumor model was used. HCT116 cells (1×10⁻⁶) were used. 7 (1 cell / mouse) was injected into the left ventricular region of Balb-c / nu mice. When the average tumor volume reached 70-100 mm... 3 During the experiment, ADC was administered intravenously every 4 days for a total of 4 times. Tumor volume was measured every 4 days until the end of the experiment.

[0202] The ADCs evaluated were 3D1-vc-MMAE, 3D5-vc-MMAE, 3H9-vc-MMAE, and 4D1-vc-MMAE. The preparation methods for ADCs 3D1-vc-MMAE, 3D5-vc-MMAE, and 4D1-vc-MMAE were the same as those for ADC 3H9-vc-MMAE, but different anti-Doppel antibodies (mAb 3D1, mAb 3D5, and mAb 4D1, whose sequences are described above) were used; these monoclonal antibodies are also described in U.S. Patent Application No. 17 / 350,763 (the entire contents of which are incorporated herein by reference).

[0203] The results showed that ADC treatment in each test effectively inhibited tumor growth. Figure 4 )

[0204] Dose-dependent studies were conducted on the ADCs 3D1-vc-MMAE, 3D5-vc-MMAE, and 3H9-vc-MMAE using the same tumor models and procedures. The results showed that each tested ADC exhibited a dose-dependent effect. Figure 5 )

[0205] In vivo efficacy tests were also conducted comparing ADCs prepared using different connectors. The ADCs used were 3H9-KGDEVD-MMAE (name = SEQ ID NO: 65; structure = SEQ ID NO: 66), 3H9-DEVD-MMAE (name = SEQ ID NO: 73; structure = SEQ ID NO: 74), and 3H9-vc-MMAE. The preparation method of ADC 3H9-DEVD-MMAE (name = SEQ ID NO: 73; structure = SEQ ID NO: 74) was the same as that of 3H9-KGDEVD-MMAE (name = SEQ ID NO: 65; structure = SEQ ID NO: 66), except that maleimide-DEVD-MMAE (SEQ ID NO: 3) was used instead of maleimide-KGDEVD-MMAE (SEQ ID NO: 77).

[0206] The results showed that ADCs 3H9-KGDEVD-MMAE (name = SEQ ID NO: 65; structure = SEQ ID NO: 66), 3H9-DEVD-MMAE (name = SEQ ID NO: 73; structure = SEQ ID NO: 74), and 3H9-vc-MMAE all effectively inhibited tumor growth, while the non-Doppel ADC (hIgG-vc-MMAE, which contains the human IgG portion instead of the antibody portion targeting Doppel) did not show effective anti-tumor activity. Figure 6 )

[0207] Using the same tumor model described above, the efficacy of other anti-Doppel ADCs was evaluated using ADCs 3D1-KGDEVD-MMAE (name = SEQ ID NO: 67; structure = SEQ ID NO: 68), 3D5-KGDEVD-MMAE (name = SEQ ID NO: 69; structure = SEQ ID NO: 70), 3H9-KGDEVD-MMAE (name = SEQ ID NO: 65; structure = SEQ ID NO: 66), and 4D1-KGDEVD-MMAE (name = SEQ ID NO: 71; structure = SEQ ID NO: 72) (prepared as described above, but with anti-Doppel antibodies mAb 3D1, mAb 3D5, mAb 3H9, and mAb 4D1 used instead of the antibodies mentioned above). The results showed that each tested ADC effectively inhibited tumor growth. Figure 7 ).

[0208] The structure of the anti-Doppel ADC described in these embodiments is shown in the figure below.

[0209] 1. 3H9-KGDEVD-MMAE: SEQ ID NO: 65 (name) / SEQ ID NO: 66 (structure)

[0210] 2. 3D1-KGDEVD-MMAE: SEQ ID NO: 67 (name) / SEQ ID NO: 68 (structure)

[0211] 3. 3D5-KGDEVD-MMAE: SEQ ID NO: 69 (name) / SEQ ID NO: 70 (structure)

[0212] 4. 4D1-KGDEVD-MMAE: SEQ ID NO: 71 (name) / SEQ ID NO: 72 (structure)

[0213] 5. 3H9-vc-MMAE

[0214] 6. 3D1-vc-MMAE

[0215] 7. 3D5-vc-MMAE

[0216] 8. 4D1-vc-MMAE

[0217] 9. 3H9-DEVD-MMAE: SEQ ID NO: 73 (name) / SEQ ID NO: 74 (structure)

[0218] 10. 3H9-KGDEVD-Exanotecan: SEQ ID NO: 75 (name) / SEQ ID NO: 76 (structure)

Claims

1. An anti-Doppel antibody-drug conjugate (ADC), comprising: (i) A Doppel targeting portion coupled with a Doppel, the Doppel targeting portion being connected directly or via a connector to... (ii) a pyrolytic connector, said pyrolytic connector being directly or via a connector connected to (iii) Therapeutic agents.

2. The ADC according to claim 1, wherein, The Doppel targeting portion is selected from monoclonal antibodies, polyclonal antibodies, single-chain antibodies, chimeric antibodies, humanized antibodies, modified antibodies, and any Doppel-binding fragment thereof.

3. The ADC according to claim 1, wherein, The Doppel targeting portion is a Doppel targeting antibody selected from human monoclonal antibody A12, human monoclonal antibody B2, human monoclonal antibody E9, human monoclonal antibody 3D5, human monoclonal antibody 3D1, human monoclonal antibody 4D1, human monoclonal antibody 3H9, and any Doppel-binding fragment thereof.

4. The ADC according to claim 1, wherein, The cleavable linker comprises a caspase-cleavable peptide linker.

5. The ADC according to claim 4, wherein, The caspase-cleavable peptide linker contains four C-terminal amino acid residues selected from Asp-Xaa-Xaa-Asp, Leu-Xaa-Xaa-Asp, and Val-Xaa-Xaa-Asp, where Xaa represents any amino acid residue.

6. The ADC according to claim 5, wherein, The four C-terminal amino acid residues of the caspase cleavable peptide linker are selected from Asp-Glu-Val-Asp (SEQ ID NO: 4), Asp-Leu-Val-Asp (SEQ ID NO: 5), Asp-Glu-Ile-Asp (SEQ ID NO: 6) and Leu-Glu-His-Asp (SEQ ID NO: 7).

7. The ADC according to claim 1, wherein, The amino acid sequence of the caspase-cleavable peptide is Lys-Gly-Asp-Glu-Val-Asp (SEQ ID NO: 8).

8. The ADC according to claim 1, wherein, The cleavable linker can be cleaved by intracellular proteases.

9. The ADC according to claim 1, wherein, The cleavable linker is selected from cleavable dipeptide linkers, hydrazone linkers that hydrolyze at pH less than 5.5, and disulfide linkers; the dipeptide is selected from valine-citrulline, valine-alanine, and phenylalanine-lysine.

10. The ADC according to claim 1, wherein, The therapeutic agent contains a chemotherapeutic agent that induces tumor cell apoptosis.

11. The ADC of claim 10, wherein, The chemotherapy agents are selected from 5-FU, afatinib, aprilidine, azaribin, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, briostatin-1, busulfan, carlicicillin, ethacryne, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatin, and COX-2 inhibitors. Irinotecan, SN-38, Cladribine, Crizotinib, Cyclophosphamide, Cytarabine, Dacarbazine, Dasatinib, Denaciline, Docetaxel, Actinomycin D, Daunorubicin, Doxorubicin, Podophyllotoxin, Erlotinib, Entenolol, Estrogen Receptor Binding Agents, Etoposide, Exemestane, Fingolimod, Fluuridine, Fludarabine, Flutamide, Flurapirib, Fositatinib, Ganetinib, GDC-0834 GS-1101, Gefitinib, Gemcitabine, Hydroxyurea, Ibrutinib, Idarubicin, Edraliximab, Ifosfamide, Imatinib, L-Asparaginase, Lapatinib, Lenalidomide, Leucovorin, LFM-A13, Lomustine, Nitrogen Mustard, Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone, Scirpus, Mitomycin, Mitotan, Naveline, Neratinib, Nilotinib, Nitrosourea, Olaparib Priligy, Procarbazine, Paclitaxel, PCI-32765, Pentostatin, PSI-341, Raloxifene, Semustine, Sorafenib, Levozocin, SU11248, Sunitinib, Tamoxifen, Temozolomide, Transplatinum, Thalidomide, Thioguanine, Thiotepa, Teniposide, Topotecan, Uracil Mustard, Vatalani, Vinorelbine, Vincristine, Vincristine Alkaloids, and ZD183.

12. The ADC according to claim 10, wherein, The chemotherapeutic agents are selected from anthracyclines, antibiotics, alkylating agents, platinum-based drugs, antimetabolites, topoisomerase inhibitors, and mitotic inhibitors.

13. The ADC according to claim 10, wherein, The chemotherapeutic agents are selected from doxorubicin, daunorubicin, epirubicin, idarubicin, vararubicin, and their derivatives.

14. The ADC of claim 10, wherein, The chemotherapeutic agents are selected from actinomycin D, bleomycin, mitomycin C, calichiomycin, and their derivatives.

15. The ADC of claim 10, wherein, The chemotherapeutic agents are selected from cyclophosphamide, nitrogen mustard, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozotocin, busulfan, dacarbazine, temozolomide, thiotepa, atratamine, ducamycin, cisplatin, carboplatin, nedaplatin, oxaliplatin, satroplatin, triplatinum tetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cytarabine, fluorouracil, fludarabine, gemcitabine. Tabin, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, esanotecan, camptothecin, topotecan, irinotecan, etoposide, teniposide, mitoxantrone, paclitaxel, docetaxel, ixaprone, vincristine, vinblastine, vinorelbine, estradiol, maytansine, DM1 (mettansine), DM4, dolalastatin, oliquistatin E, oliquistatin F, monomethyl oliquistatin E (MMAE), and their derivatives.

16. The ADC of claim 10, wherein, The chemotherapy agent is monomethylolpropionate E (MMAE).

17. The ADC of claim 10, wherein, The chemotherapy agent is ethathecan.

18. The ADC according to claim 1, wherein, The therapeutic agent contains an immunomodulator.

19. The ADC of claim 18, wherein, The immunomodulators are selected from cytokines, lymphokines, monokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony-stimulating factor (CSF), interferon (IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, pro-relaxanthin, follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), liver growth factor, prostaglandins, fibroblast growth factor, prolactin, placental lactogen, OB protein, transforming growth factor (TGF), TGF-α, TGF-β, and insulin-like growth factor. Factors (IGF), erythropoietin, thrombopoietin, tumor necrosis factor (TNF), TNF-α, TNF-β, Müllerian duct inhibitor, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrin, interleukin (IL), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-α, interferon-β, interferon-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL- 8. IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, IL-25, LIF, kit ligand, FLT-3, angiostatin, thromboretin, endostatin, Toll-like receptor (TLR) agonists (selected from CU-T12-9, Pam3CSK4, FSL-1, Pam2CSK4, and CL429), Poly(A:U), Poly(I:C), lipids Polysaccharides (LPS), MPLA-SM, CRX-527, flagellin, thiazoquinoline derivatives, imidazoquinoline derivatives (selected from CL097, gardiquimod, imiquimod and resiquimod), adenine analogs, guanosine analogs, thymidine analogs, benzozazepine analogs and CpG oligodeoxynucleotides (ODNs) (selected from ODN1585, ODN2216, ODN2336, ODN1668, ODN1826, ODN2006, ODN2007, ODN BW006, ODND-SL01, ODN2395, ODNM362 and ODND-SL03).

20. The ADC according to claim 1, wherein, The treatment agent contains a toxin.

21. The ADC according to claim 20, wherein, The toxins are selected from ricin, abrinogen, ribonuclease (RNase), DNase 1, Staphylococcus aureus enterotoxin A, pokeweed antiviral protein, gel toxin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

22. The ADC according to claim 1, wherein, The therapeutic agent contains a radionuclide.

23. The ADC of claim 22, wherein, The radionuclides are selected from 11 C 13 N、 15 O、 32 P, 33 P, 47 Sc、 51 Cr 57 Co、 58 Co、 59 Fe、 62 Cu、 67 Cu、 67 Ga、 75 Br、 75 Se、 76 Br、 77 As、 77 Br、 80m Br、 89 Sr、 90 Y、 95 Ru、 97 Ru、 99 Mo、 99m Tc, 103m Rh、 103 Ru、 105 Rh、 105 Ru、 107 Hg, 109 Pd, 109 Pt, 111 Ag、 111 In、 113m In、 119 Sb, 121m Te、 122m Te、 125 I, 125m Te、 126 I, 131 I, 133 I, 142 Pr, 143 Pr, 149 Pm, 152 Dy、 153 Sm、 161 Ho、 161 Tb, 165 Tm、 166 Dy、 166 Ho、 167 Tm、 168 TM、 169 Er、 169 Yb、 177 Lu、 186 Re、 188 Re、 189m Os、 189 Re、 198 Ir 194 Ir 197 Pt 198 Au 199 Au 203 Hg 211 At 211 Bi 211 Pb 212 Bi 212 Pb 213 Bi 215 Po 217 At 219 Rn 221 Fr 223 Ra 225 Ac 227 Th and 255 Fm 24. The ADC according to claim 1, wherein, The therapeutic agent is a DNA cross-linking agent selected from indobenzobenzodiazepine dimer (IGN), pyrrolobenzodiazepine (PBD), and their derivatives.

25. The ADC according to claim 1, wherein, The ADC is selected from 3D1-KGDEVD-MMAE (name = SEQ ID NO: 67; structure = SEQ ID NO: 68), 3D5-KGDEVD-MMAE (name = SEQ ID NO: 69; structure = SEQ ID NO: 70), 3H9-KGDEVD-MMAE (name = SEQ ID NO: 65; structure = SEQ ID NO: 66), 4D1-KGDEVD-MMAE (name = SEQ ID NO: 71; structure = SEQ ID NO: 72); 3H9-vc-MMAE; 3D1-vc-MMAE; 3D5-vc-MMAE; 4D1-vc-MMAE; 3H9-DEVD-MMAE (name = SEQ ID NO: 73; structure = SEQ ID NO: 74) and 3H9-KGDEVD-ethanotecan (name = SEQ ID NO: 75; structure = SEQ ID NO: 76).

26. A pharmaceutical composition comprising the ADC of claim 1 and a pharmaceutically acceptable carrier.

27. The pharmaceutical composition according to claim 26, wherein, The pharmaceutical composition is formulated for intravenous administration.

28. A method of treating a subject with a Doppel-related disease or condition, comprising administering the ADC of claim 1 to the subject in need.

29. The method according to claim 28, wherein, The Doppel-related diseases or conditions are selected from asthma, tuberculosis, atherosclerosis, and pulmonary hypertension (PAH).

30. The method according to claim 28, wherein, The doppel-related disease or condition is cancer, in which the cancer cells express doppel.

31. A kit comprising the ADC of claim 1 contained in a container, optionally further comprising instructions for use.

Images