Drug conjugates containing α-enolase antibodies and their use

Abstract

The immunoconjugate comprises an anti-ENO1 antibody or binding fragment thereof and a therapeutic agent or label and has the formula: Ab-(LD) m where Ab is an anti-ENO1 antibody or binding fragment thereof, L is a linker or direct bond, D is a therapeutic agent or label, and m is an integer from 1 to 12. The antibody can be a monoclonal antibody, which can be humanized or fully human. A method for treating inflammatory diseases, immune disorders, or cancer comprises administering to a subject in need of such treatment a pharmaceutically effective amount of an immunoconjugate comprising an antibody against ENO1, or a binding fragment thereof, and a therapeutic agent covalently attached to the antibody.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 022,702, filed on 11 May 2020, which is incorporated herein by reference for all purposes.

[0002] This invention relates to antibody-drug conjugates containing anti-human alpha-enolase protein (ENO1) antibodies, and their use in therapy. The invention also relates to methods for treating inflammatory diseases or immune disorders, or for suppressing tumor growth and metastasis, by administering anti-ENO1 ADCs to subjects. [Background technology]

[0003] Antibody-drug conjugates (ADCs) enable targeted therapy for various diseases and conditions, including cancer. An ADC is a complex molecule in which a bioactive agent, such as a cytotoxic agent or drug, is bound to an antibody. By combining the antibody's inherent targeting ability with the drug's therapeutic effect, antibody-drug conjugates can distinguish between normal cells and cancer cells, thereby minimizing side effects. ADCs typically contain a cytotoxic agent (e.g., a tubulin inhibitor or DNA alkylating agent) bound to an antibody that specifically targets a marker, such as a tumor marker. The antibody identifies these proteins in the body and attaches to the surface of cancer cells. When the antibody binds to the target protein (antigen), a signal is activated in the tumor cell, causing the ADC to enter the cell. Once inside the cell, the cytotoxic agent is released, potentially killing cancer cells. Due to its specific targeting, drug side effects are reduced. α-enolase (enolase 1, ENO1) is a multifunctional protein that was first discovered as a major enzyme in glycolysis. Under normal conditions, ENO1 is expressed in the cytosol. However, ENO1 is also known to be expressed as a plasminogen receptor on the surface of many cancer cells, as well as on activated hematopoietic cells such as neutrophils, lymphocytes, and monocytes. Upregulation of the plasminogen receptor protein can trigger a cascade reaction of the urokinase plasminogen activation system, which is known to lead to extracellular matrix degradation. As a result, cancer cell metastasis and immune cell invasion increase. Inflammatory stimuli, such as LPS, upregulate ENO1 cell surface expression on human blood monocytes and U937 mononuclear cells through post-translational modification and translocation to the cell surface. ENO1 migration is thought to be regulated by the MAP kinase signaling pathway. This suggests that increased ENO1 expression on the cell surface may play an important role in inflammatory diseases. Autoantibodies against ENO1 have been found in various autoimmune and inflammatory diseases, including lupus erythematosus, systemic sclerosis, Behçet's disease, ulcerative disease, and Crohn's disease. ENO1 is known to play a crucial role in the progression of rheumatoid arthritis by enhancing the infiltration activity of monocytes and macrophages through its plasminogen receptor activity. In short, monocytes with upregulated ENO1 expression as a plasminogen receptor on their cell surface, resulting in increased invasiveness, are crucial for the progression of multiple sclerosis, rheumatoid arthritis, and related immune disorders. Therefore, targeting ENO1 on the cell surface of monocytes may be effective in treating inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, and systemic lupus erythematosus, or related immune disorders such as chronic obstructive pulmonary disease (COPD), asthma, allergies, psoriasis, type 1 diabetes, atherosclerosis, and osteoporosis. Furthermore, the expression of ENO1 as a plasminogen receptor on the surface of cancer cells may enhance the invasive activity of cancer cells. Therefore, ENO1 is also a potential target for cancer treatment. While antibodies against ENO1 are beneficial, there is still a need to improve therapeutic strategies using anti-ENO1 ADCs. [Overview of the project]

[0004] This invention relates to antibody-drug conjugates containing ENO1 antibody, and their use in therapy. One aspect of the present invention relates to an immune complex. An immune complex according to one embodiment of the present invention comprises an anti-ENO1 antibody or a binding fragment thereof, and a therapeutic agent or label, with the formula: Ab-(LD) m The formula comprises, where Ab is an anti-ENO1 antibody or a conjugated fragment thereof, L is a linker or direct conjugate, D is a therapeutic agent or label, and m is an integer from 1 to 12. According to any embodiment of the present invention, Ab may comprise a heavy chain variable domain having three complementary regions including HCDR1(GYTFTSCVMN; SEQ ID NO: 1), HCDR2(YINPYNDGTKYNEKFKG; SEQ ID NO: 2), and HCDR3(EGFYYGNFDN; SEQ ID NO: 3), and a light chain variable domain having three complementary regions including LCDR1(RASENIYSYLT; SEQ ID NO: 4), LCDR2(NAKTLPE; SEQ ID NO: 5), and LCDR3(QHHYGTPYT; SEQ ID NO: 6). According to any embodiment of the present invention, Ab may comprise a heavy chain variable domain having three complementary regions including HCDR1(GYTFTSXVMN, where X is any amino acid other than cysteine; SEQ ID NO: 7), HCDR2(YINPYNDGTKYNEKFKG; SEQ ID NO: 2), and HCDR3(EGFYYGNFDN; SEQ ID NO: 3), and a light chain variable domain having three complementary regions including LCDR1(RASENIYSYLT; SEQ ID NO: 4), LCDR2(NAKTLPE; SEQ ID NO: 5), and LCDR3(QHHYGTPYT; SEQ ID NO: 6). The linker L may be a direct link that directly connects (conjugates) the payload D to the antibody or its binding fragment. The linker may be any linker commonly used in protein modification or binding, such as a short peptide (e.g., val-cit) or a short organic molecule linker (e.g., SMCC, succinimidyl-4(N-maleimidomethyl)cyclohexane-1-carboxylate). The payload D may be a therapeutic agent such as a cytotoxic agent. Examples of cytotoxic agents that can be used in embodiments of the present invention include maytansinoids (e.g., DM1 or DM4), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and paclitaxel. The payload D may be a diagnostic or imaging label or drug. Examples of contrast agents include DTPA (diethylenetriaminepentaacetic acid) or DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid).

[0005] According to some embodiments of the present invention, the antibody may be a monoclonal antibody, which may be a humanized antibody or a fully human antibody. One aspect of the present invention relates to a method for diagnosing or imaging cells or tissues expressing ENO1. A method according to one embodiment of the present invention may include administering the above-mentioned immune complex to a subject. According to certain embodiments of the present invention, human ENO1 protein-related diseases or disorders may be any conditions resulting from abnormal activation or expression of the human ENO1 protein. Examples of such diseases include abnormal interactions between the human ENO1 protein and its ligand, thereby altering cell adhesion or cell signaling. Such alterations in cell adhesion or cell signaling may lead to neoplastic and / or inflammatory or immune disorders. One aspect of the present invention relates to a method for treating inflammatory diseases or immune disorders such as multiple sclerosis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, and systemic lupus erythematosus, or related immune disorders such as chronic obstructive pulmonary disease (COPD), atopic dermatitis, idiopathic pulmonary fibrosis, non-alcoholic fatty liver disease, asthma, allergies, psoriasis, psoriatic arthritis, type 1 diabetes, atherosclerosis, osteoporosis, systemic sclerosis, viral pneumonia, or macrophage activation syndrome. One aspect of the present invention relates to a method for treating cancer. A method according to one embodiment of the present invention may include administering a pharmaceutically effective amount of the above-mentioned immune complex to a subject in need of cancer treatment. The cancers are those that highly express ENO1, such as lung cancer, breast cancer, pancreatic cancer, liver cancer, colorectal cancer, and prostate cancer. Those skilled in the art will understand that the effective pharmaceutically acceptable dose varies depending on various factors such as the patient's condition, age, disease state, and route of administration, and that such an effective dose can be determined in routine practice based on these factors without excessive experimentation. Other aspects of the present invention will become apparent in the following description. [Brief explanation of the drawing]

[0006] [Figure 1] Graph showing the PLRP-HPLC results for HuL001-SMCC-DM1. Example 7 describes that the binding reaction was substantially complete, with only residual amounts of anti-ENO1 antibody and ADC remaining. [Figure 2] Graph showing PLRP-HPLC results for HuL001-SPP-DM4. Example 7 describes that the binding reaction was substantially complete, with only residual amounts of anti-ENO1 antibody and ADC remaining. [Figure 3] A graph showing the HIC results for HuL001-mal-vc-MMAE. Example 7 describes that the binding reaction was substantially complete, with only residual amounts of anti-ENO1 antibody and ADC remaining. [Figure 4] A graph showing the PLRP-HPLC results for HuL001-Ph-MMAF. Example 7 describes that the binding reaction was substantially complete, with only residual amounts of anti-ENO1 antibody and ADC remaining. [Figure 5] Graph showing the HIC results of HuL001-mal-vc-steroid. In Example 7, it is described that the conjugation reaction was substantially completed and only the remaining amounts of the anti-ENO1 antibody and the ADC remained. [Figure 6] Graph showing the LC / MS results of HuL001-SMCC-DM1. Details are described in Example 8. [Figure 7] Graph showing the LC / MS results of HuL001-SPP-DM4. Details are described in Example 8. [Figure 8] Graph showing the LC / MS results of HuL001-mal-vc-MMAE. Details are described in Example 8. [Figure 9] Graph showing the LC / MS results of HuL001-Ph-MMAF. Details are described in Example 8. [Figure 10] Graph showing the LC / MS results of HuL001-mal-vc-steroid. Details are described in Example 8. [Figure 11] Graph showing the in vitro cytotoxicity results of HuL001-SPP-DM4 in the LPS-stimulated B cell cancer cell line DHL-4. Details are described in Example 9. [Figure 12] Graph showing that there is no detectable in vitro cytotoxicity of HuL001-SMCC-DM1 in isolated normal human B cells regardless of LPS stimulation. Details are described in Example 9. [Figure 13] Graph showing that there is no detectable in vitro cytotoxicity of HuL001-mal-vc-MMAE in isolated normal human B cells regardless of LPS stimulation. Details are described in Example 9. [Figure 14] Graph showing the superior anti-inflammatory effect of HuL001-mal-vc-steroid compared to HuL001 on TNF-α and CCL2 secretion in LPS-treated human monocyte cell line THP-1. Details are described in Example 10. [Figure 15]It shows the in vivo efficacy of HuL001-SMCC-DM1 in a PC-3 xenograft prostate cancer model. Treatment with HuL001-SMCC-DM1 was able to inhibit tumor growth compared to the vehicle control group. The detailed procedure was carried out as described in Example 11. [Figure 16] Graph showing the in vivo efficacy of HuL001-SMCC-DM1 in a C57BL / 6 EAE disease model. Treatment with HuL001-SMCC-DM1 was able to delay the progression of disease symptoms in EAE mice compared to the PBS control group and further the COPAXONE® group. The detailed procedure was carried out as described in Example 12. [Figure 17] Graph showing the in vivo efficacy of HuL001-SMCC-DM1 in a C57BL / 6 bleomycin-induced pulmonary fibrosis disease model. Treatment with HuL001-SMCC-DM1 was able to attenuate the weight loss and increase in lung mass of pulmonary fibrosis mice compared to the bleomycin treatment group. The detailed procedure was carried out as described in Example 13.

Modes for Carrying Out the Invention

[0007] General Definitions[[ID=!15]] In carrying out the present invention, unless otherwise indicated, prior art in molecular biology, microbiology, recombinant DNA, and immunology within the scope of the art will be used. Such art is well described in the literature. For example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning, Volumes I and II (DNGlover ed., 1985); Culture Of Animal Cells (RIFreshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press,1986);B.Perbal,A Practical Guide To Molecular Cloning(1984);the treatise,Methods In Enzymology(Academic Press,Inc.,NY);Gene Transfer Vectors For Mammalian Cells(JHMiller and MP Calos eds.,1987,Cold Spring Harbor Laboratory);Methods In Enzymology,Vols.154 and 155(Wu et al.eds.), Immunochemical Methods In Cell And See Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Antibodies: A Laboratory Manual, by Harlow and Lane (Cold Spring Harbor Laboratory Press, 1988); and Handbook of Experimental Immunology, Volumes I-IV (DMWeir and CCBlackwell, eds., 1986).

[0008] The terms “antibody” and “immunoglobulin” are used interchangeably in a broad sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, monovalent antibodies, multivalent antibodies, and polyspecific antibodies (e.g., bispecific antibodies insofar as they exhibit the desired biological activity), and may also include specific antibody fragments (described in more detail herein). Antibodies may be chimeric, human, humanized, and / or affinity-mature antibodies. The term "variable" refers to the fact that certain portions of the variable domain exhibit wide-ranging sequence differences between antibodies, which are used for the binding and specificity of each particular antibody to a specific antigen. However, variability is not uniformly distributed throughout the entire variable domain of an antibody. In both the light chain and heavy chain variable domains, it is concentrated in three regions called complementarity-determining regions (CDRs) or hypervariable regions. The more highly conserved portions of the variable domain are called frameworks (FRs). The natural heavy and light chain variable domains each consist of four FR regions, mostly β-sheet structures, connected by three CDRs. The CDRs form loops that connect, and sometimes form part of, the β-sheet structure. The CDRs of each chain are held in close proximity to each other by the FR regions and, together with the CDR of the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domain does not directly participate in antibody binding to antigens, but it exhibits various effector functions, such as the involvement of antibodies in antibody-dependent cytotoxicity.

[0009] The antibody may be full-length, or it may include, but is not limited to, Fab, F(ab')2, Fab', F(ab)', Fv, short-chain Fv (scFv), bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fd, dAb fragments (e.g., Ward et al, Nature, 341:544-546 (1989)), CDR, diabody, triabody, tetrabody, linear antibody, short-chain antibody molecule, and polyspecific antibody formed from antibody fragments, as well as antibody fragments (or multiple fragments) having an antigen-binding moiety. Short-chain antibodies produced by linking antibody fragments using a recombinant method or synthetic linker are also included in the present invention (Bird et al. Science, 1988, 242:423-426. Huston et al. Proc. Natl. Acad. Sci. USA, 1988, 85:5879-5883). An antibody having variable heavy chain regions and variable light chain regions that are at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or at least 100% homologous to the variable heavy chain region and variable light chain region of an antibody produced by a reference antibody, and which can also bind to ENO1. The homology can exist at either the amino acid or nucleotide sequence level. The terms "Kabat variable domain residue numbering" or "Kabat amino acid position numbering" and their variations refer to the numbering system used for heavy-chain or light-chain variable domains in antibody collection, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortenings or insertions of FR or HVR in the variable domain. For example, a heavy-chain variable domain may contain a single amino acid insertion after H2 residue 52 (residue 52a in Kabat) and an inserted residue after heavy-chain FR residue 82 (e.g., residues 82a, 82b, and 82c in Kabat). Kabat numbering of residues can be determined for a given antibody by aligning the antibody sequence with a "standard" Kabat numbered sequence in homologous regions.

[0010] "Cancer" and "malignant" generally refer to or describe a physiological condition in mammals characterized by uncontrolled cell growth / proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas (e.g., Hodgkin lymphoma and non-Hodgkin lymphoma), blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, lymphoproliferative disorders such as leukemia, and various head and neck cancers. As used herein, "treatment" refers to a clinical intervention for altering the natural course of an individual or cell being treated, and can be performed for prevention or during the course of a clinical pathology. Desirable effects of treatment include preventing the onset or recurrence of a disease, alleviating symptoms, reducing the direct or indirect pathological consequences of a disease, preventing or reducing inflammation and / or tissue / organ damage, reducing the rate of disease progression, restoring or alleviating a disease state, and improving remission or prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of a disease or disorder. An "individual" or "subject" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, domestic animals (such as cows), sport animals, pets (such as cats, dogs, horses, etc.), primates, mice, and rats. In certain embodiments, the vertebrate is a human.

[0011] "Effective amount" refers to an amount effective at the dosage and for the period required to achieve the desired therapeutic or prophylactic result. The "therapeutically effective amount" of a substance / molecule of the present invention can vary depending on factors such as the condition, age, sex, and weight of the individual, as well as the ability of the substance / molecule to elicit the desired response in the individual. A therapeutically effective amount is also an amount where the therapeutically beneficial effects of the substance / molecule outweigh the toxic or harmful effects. A "preventively effective amount" refers to an amount effective at the dosage and for the period required to achieve the desired preventive result. Usually, but not always, since preventive dosages are typically used in subjects before the onset or at an early stage of a disease, a preventively effective amount is less than a therapeutically effective amount. As used herein, the term "therapeutic agent" refers to a substance that inhibits or blocks the function of a cell and / or causes cell destruction. This term includes radioisotopes (e.g., 211 At, 131 I, 125 I, <000,005>Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 60 C, as well as lutetium 177, strontium 89, and samarium ( 153It is intended to include radioactive isotopes of Sm), immunomodulators, cytotoxic agents, and toxins such as low-molecular-weight toxins or enzyme-active toxins of bacterial, fungal, plant, or animal origin (including synthetic analogs and derivatives thereof).

[0012] "Cytotoxic agents" are compounds useful in the treatment of cancer. Examples of chemotherapy drugs include maytansinoid 1 (DM1), maytansinoid 4 (DM4), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), anthracyclines, pyrrolobenzodiazepines, α-amanitin, tubulisin, benzodiazepines, erlotinib (TARCEVA®, Genentech / OSI Pharm.), and bortezomib (VELCADE®, Millennium). Pharm.), fulvestrant (FASLODEX®, Astrazeneca), sunitinib (SUTENT®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK222584 (Novartis), oxaliplatin (ELOXATIN®, Sanofi), leucovorin, rapamycin (Sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), ronafarnib (SARASAR®, SCH66336), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs.), and gefitinib (IRESSA®, Astrazeneca), AG1478, AG1571 (SU5271; Sugen), alkylating agents, e.g., thiotepa and CYTOXAN®, cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan, and pigosulfan; aziridines, e.g., benzodopa, carbocone, metsuredopa, and uredopa; ethyleneimines and methylamelamines, e.g., altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomellamine; acetogenins (especially bratacin and bratacinone); camptothecin (including its synthetic analog topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogs adzeresin, karzeresin, and biceresin);Cryptophycin (especially cryptophycin 1 and cryptophycin 8); dorastatin; duocalmycin (including synthetic analogs KW-2189 and CB1-TM1); eryuterobin; pancratistatin; sarcodicin; spongistatin; nitrogen mustards, e.g., chlorambucil, chlornafadin, colophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, fenestrine, prednimustine, trophosphamide, uracil mustard; nitrothreas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, e.g., engine antibiotics (e.g., calitiamycin, especially calitiamycin gamma 1I and calitiamycin omega I1 (Angew) Chem.Intl.Ed.Engl.(1994)33:183-186); Dinemycin, e.g., Dinemycin A; Bisphosphonates, e.g., Chlorophenate; Esperamicin; and Neocardinostatin chromophores and related pigment proteins (Endiine antibiotic luminescent phosphatides), Aclacinomycin, Actinomycin, Anthramycin, Azaserin, Bleomycin, Kakutinomycin, Carabicin, Kaminomycin, Cardinophilin, Chromomycinis, Dactinomycin, Daunorubicin, Detrubicin, 6-Diazo-5-Oxo-L-Norleucine, ADRIAMYCIN (Registered Trademark) Standard doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zorubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folic acid analogs, e.g., denopterin, pteropterin, trimethrexate;Purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, phloxuridine; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal drugs, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folinic acid acid); acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisanthren; edatraxate; defofamine; demecoltin; diazicone; elformitin; eriptinium acetate; epotilon; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidynin; meitansinoids, e.g., meitansin and anthamitosin; mitogwazone; mitoxantrone; mopidammole; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Oregon); Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic Acid; Triadicone; 2,2',2”-Trichlorotriethylamine; Trichothecin (especially T-2 Toxin, Beraclin A, Loridine A, and Anguidin); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., TAXOL® Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, New Jersey), ABRAXANE® Albumin-Conjugated Paclitaxel in Cremofoll-Free Nanoparticle Formulations (American Pharmaceuticals) Partners (Schaumburg, Illinois), and TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, e.g., cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, e.g., retinoic acid; capecitabine (Xeloda®, Roche); and any pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0013] Exemplary ADC linker Suitable exemplary linkers for ADCs are described, for example, in U.S. Patent No. 7,595,292 (International Publication No. 2005 / 007197). The entire description of linkers is incorporated herein by reference. The linker (L) links the antibody to the drug moiety via one or more covalent bonds that do not contain disulfide groups. The linker is a difunctional or polyfunctional group that can be used to conjugate one or more therapeutic agents or labels (D) to an antibody unit (Ab) to form an antibody-drug conjugate (ADC) of formula (I). Antibody-drug conjugates (ADCs) can be easily prepared using linkers that have reactive functionality for binding to the drug and antibody. The N-terminus or amino acid side chain of the antibody (Ab), such as cysteine ​​thiol or amine, e.g., lysine, can form a bond with the functional group of the linker reagent, the drug moiety, or the drug-linker reagent. The linker is preferably stable extracellularly. Until transported or delivered intracellularly, the antibody-drug conjugate (ADC) is preferably stable in its complete form, i.e., the antibody remains bound to the drug moiety. The linker is stable extracellularly and can be cleaved intracellularly at a reasonably effective rate. An effective linker: (i) maintains the specific binding properties of the antibody; (ii) enables intracellular delivery of the conjugate or drug moiety; (iii) maintains stability and integrity, i.e., is not cleaved until the conjugate is delivered or transported to the target site; and (iv) maintains the cytotoxicity, cytotoxicity, or cytostatic effect of the therapeutic or labeled moiety. The stability of the ADC can be measured by standard analytical techniques such as mass spectrometry, HPLC, and separation / analysis techniques such as LC / MS.

[0014] For covalent bonding between an antibody and a therapeutic agent or labeled moiety, the linker must have two reactive functional groups, i.e., be divalent in terms of reactivity. Divalent linker reagents useful for conjugating two or more functional groups or bioactive moieties such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups are known, and methods for obtaining complexes have been described (Hermanson, GT (1996) Bioconjugate Techniques; Academic Press: New York, pp. 234-242). Typically, antibody-drug conjugate compounds include a linker unit between the therapeutic or labeled unit and the antibody unit. In some embodiments, the linker is cleavable under intracellular conditions, and cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In yet other embodiments, the linker unit is incleavable, and the drug is released, for example, by the degradation of the antibody. In some embodiments, the linker can be cleaved by cleavage agents present in the intracellular environment (e.g., within lysosomes, endosomes, or caveoleas). The linker may be a peptidyl linker cleaved by intracellular peptidase or protease enzymes, including but not limited to lysosomal or endosomal proteases. In some embodiments, the peptidyl linker is at least two amino acid long or at least three amino acid long. The cleavage agents may include cathepsins B and D, and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release active drugs in target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Most commonly, the peptidyl linker is cleavable by enzymes present in 158P1D7-expressing cells. For example, peptidyl linkers that can be cleaved by the thiol-dependent protease cathepsin B, which is highly expressed in cancer tissue, can be used (e.g., Phe-Leu or Gly-Phe-Leu-Gly linkers). Other examples of such linkers are described, for example, in U.S. Patent No. 6,214,345, which is incorporated herein by reference in its entirety for all purposes. In certain embodiments, the peptidyl linker cleaved by an intracellular protease is the Val-Cit linker or the Phe-Lys linker (see, for example, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using the val-cit linker). One advantage of using intracellular proteolytic release of therapeutic agents is that the drug is usually attenuated upon binding, and the serum stability of the complex is usually high.

[0015] In other embodiments, the cleavable linker is pH-sensitive, i.e., readily hydrolyzable at a specific pH value. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-unstable linkers that are hydrolyzable in lysosomes (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitamide, orthoester, acetal, ketal, etc.) can be used (see, for example, U.S. Patent No. 5,122,368; U.S. Patent No. 5,824,805; U.S. Patent No. 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as in blood, but unstable at pH 5.5 or below 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether bound to the therapeutic agent via an acylhydrazone bond (see, for example, U.S. Patent No. 5,622,929)). In yet other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). Various disulfide linkers are known in the art, for example, SATA (N-succinimidyl-S-acetylthioacetic acid), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionic acid), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyric acid), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene), SPDB, and SMPT can be used to form these (see, for example, Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer, CWVogel ed., Oxford U. Press, 1987; further see U.S. Patent No. 4,880,935). In yet another specific embodiment, the linker is a malonic acid linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimide benzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12). In yet another embodiment, the linker unit is indestructible, and the drug is released by antibody degradation (see U.S. Patent Application Publication No. 2005 / 0238649, which is incorporated herein by reference in its entirety for all purposes).

[0016] Typically, linkers are substantially insensitive to the extracellular environment. As used herein in the context of linkers, the phrase "substantially insensitive to the extracellular environment" means that when an antibody-drug conjugate is present in an extracellular environment (e.g., plasma), approximately 20% or less, typically approximately 15% or less, more typically approximately 10% or less, and even more typically approximately 5% or less, approximately 3% or less, or approximately 1% or less of the linker in an antibody-drug conjugate sample is cleaved. Whether a linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating the antibody-drug conjugate with plasma for a predetermined time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free drug present in the plasma. In other non-mutually exclusive embodiments, the linker facilitates intracellular translocation. In certain embodiments, the linker facilitates intracellular translocation when bound to the therapeutic agent (i.e., in the environment of the linker-therapeutic portion of the antibody-drug conjugate compound described herein). Various exemplary linkers that can be used in conjunction with the configuration and method of the present invention are described in International Publication No. 2004-010957, U.S. Patent Application Publication No. 2006 / 0074008, U.S. Patent Application Publication No. 20050238649, and U.S. Patent Application Publication No. 2006 / 0024317 (all of which are incorporated herein by reference in their entirety for all purposes).

[0017] Embodiments of the present invention relate to antibody-drug conjugates containing an ENO1 antibody and their use in therapy. ENO1 is a multifunctional protein known to be expressed as a plasminogen receptor on the surface of many cancer cells, as well as on activated hematopoietic cells such as neutrophils, lymphocytes, and monocytes. Therefore, antibody-based ADCs against ENO1 may be useful diagnostic and / or therapeutic agents. However, rapid intracellular translocation of therapeutic antibodies or a lack of ADCC activity can lead to antibody inactivation and resistance. Therefore, it is necessary to enhance the therapeutic effect of anti-ENO1-based therapies. One method is to conjugate a payload with an anti-ENO1 antibody (i.e., an antibody-drug conjugate). By conjugating an anti-ENO1 antibody with a payload (i.e., an ADC), embodiments of the present invention become more potent than a naked anti-ENO1 antibody, thereby reducing the amount of antibody used. According to embodiments of the present invention, anti-ENO1 antibodies or their conjugated fragments can be conjugated to drugs, diagnostic agents, or therapeutic agents. Accordingly, the term “antibody-drug conjugate” (ADC) as used herein may refer to an antibody portion (which may be the entire antibody or its conjugated fragment) conjugated to a payload (which may be a drug, diagnostic agent, or therapeutic agent). The ADCs of the present invention include a payload designed for therapeutic or diagnostic applications. These ADCs will have better biological activity and will be able to achieve the desired effect at lower doses compared to naked anti-ENO1 antibodies. Embodiments of the present invention will be described below according to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and that other modifications and variations are possible without departing from the scope of the present invention. [Examples]

[0018] Unless otherwise stated, 1¹H NMR data were obtained at 500 MHz. Unless otherwise specified, the following abbreviations are used herein: Bu: butyl; Bn: benzyl; BOC: t-butyloxycarbonyl; BOP: benzotriazole-1-yloxytri / dimethylamino-phosphonium hexafluorophosphate; DCC: dicyclohexylcarbodiimide; DMF: N,N-dimethylformamide; DMAP: 4-dimethylaminopyridine; EDC: 1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride; Â: ethyl acetate; Eq.: equivalent; HOBt: hydroxybenztriazole; LAH: lithium aluminum hydride; MeOH: methanol; MHz: megahertz; MS(ES): mass spectrometer (electrospray); NMP: N-methylpyrrolidinone; Ph: phenyl; Pr: propyl; TEA: triethylamine; THF: tetrahydrofuran; TLC: thin-layer chromatography; tetrakis(triphenylphosphine)palladium.

[0019] Example 1 Preparation of anti-ENO1 antibody According to embodiments of the present invention, a general method for producing an anti-ENO1 antibody involves obtaining a hybridoma that produces a monoclonal antibody against ENO1. Methods for producing monoclonal antibodies are known in the art and are not described in detail herein. Briefly, mice are exposed to an antigen (ENO1) with a suitable adjuvant. Splenocytes from the immunized mice are then collected and fused with the hybridoma. Positive clones can be confirmed to be able to bind to the ENO1 antigen using known methods such as ELISA. In one embodiment, the anti-ENO1 antibody is HuL001. An exemplary antibody HuL001 is described in U.S. Patent Application Publication No. 2019 / 0322762, the contents of which are incorporated herein by reference in their entirety. The antibody-drug conjugates (ADCs) of the claimed invention can specifically target ENO1. These ADCs can use any antibody that specifically binds to ENO1. For example, the ADCs of the claimed invention may use mouse or humanized anti-ENO1 antibodies, or their scFv or Fab fragments. An exemplary anti-ENO1 antibody, for example, HuL001, may include a heavy chain variable domain having three complementary regions including HCDR1(GYTFTSCVMN; SEQ ID NO: 1), HCDR2(YINPYNDGTKYNEKFKG; SEQ ID NO: 2), and HCDR3(EGFYYGNFDN; SEQ ID NO: 3), and a light chain variable domain having three complementary regions including LCDR1(RASENIYSYLT; SEQ ID NO: 4), LCDR2(NAKTLPE; SEQ ID NO: 5), and LCDR3(QHHYGTPYT; SEQ ID NO: 6). Another exemplary anti-ENO1 antibody may include a heavy chain variable domain having three complementary regions including HCDR1(GYTFTSXVMN, where X is any amino acid other than cysteine; SEQ ID NO: 7), HCDR2(YINPYNDGTKYNEKFKG; SEQ ID NO: 2), and HCDR3(EGFYYGNFDN; SEQ ID NO: 3), as well as a light chain variable domain having three complementary regions including LCDR1(RASENIYSYLT; SEQ ID NO: 4), LCDR2(NAKTLPE; SEQ ID NO: 5), and LCDR3(QHHYGTPYT; SEQ ID NO: 6). In embodiments of the present invention, the antibody may be a mouse antibody. Alternatively, the antibody may be a chimeric antibody (e.g., a constant region of a human antibody ligated to a variable region of a mouse antibody), a humanized antibody (e.g., a mouse CDR transplanted into the framework region of a human immunoglobulin), or a fully human antibody. Monoclonal antibodies can be humanized by obtaining the CDR sequence from a hybridoma and cloning the CDR sequence into a human framework sequence to produce a humanized antibody. Any common method known in the art may be used to identify the CDR sequence. The CDR region in the present invention is identified using the Kabat number scheme. First, an anti-ENO1 hybridoma (e.g., a mouse hybridoma) was prepared. Such a hybridoma can be prepared using a standard procedure for producing monoclonal antibodies. Next, the total RNA of the hybridoma was isolated using, for example, a TRIzol® reagent. Then, for example, a first-strand cDNA synthesis kit (Superscript III) and oligonucleotides (dT 20 cDNA was synthesized from total RNA using primers or Ig-3' constant region primers. Next, the heavy-chain and light-chain variable regions of immunoglobulin genes were cloned from cDNA. For example, the VH and VL variable regions of anti-ENO1 mAb were amplified from mouse hybridoma cDNA by PCR using a mouse Ig-5' primer set (Novagen). The PCR product could be directly cloned into a suitable vector (e.g., pJET1.2 vector) using the CloneJet® PCR cloning kit (Ferments). The pJET1.2 vector contains a lethal insert, and only if the desired gene is cloned into this lethal region can it survive under selective conditions. This facilitates the selection of recombinant colonies. Finally, the recombinant colonies were screened to obtain the desired clones, and the DNA of these clones was isolated and sequenced. The nucleotide sequences of immunoglobulins (IG) can be analyzed on the international ImMunoGeneTics information system (IGMT) website.

[0020] Antibody expression and purification For antibody production, isolated clones can be expressed in any suitable cells. As an example, F293 cells (Life Technologies) were transfected with an anti-ENO1 mAb expressing a plasmid and cultured for 7 days. The anti-ENO1 antibody was purified from the culture medium using a protein A affinity column (GE). Protein concentrations could be determined using a Bio-Rad protein assay kit and analyzed by 12% SDS-PAGE using procedures known in the art or according to the manufacturer's instructions. According to embodiments of the present invention, antibody-drug conjugates (ADCs) can be prepared using any of these anti-ENO1 antibodies, as described in the following examples.

[0021] Example 2 Preparation of the HuL001-SMCC-DM1 complex [ka] In this example, the ADC contains DM1, a maytansinoid developed for cancer treatment. Maytansine, a benzoansamcrolide, is a very potent microtubule-targeting compound that induces mitotic arrest and kills tumor cells at sub-nanomole concentrations. DM1 binds to the tips of microtubules and suppresses their dynamicity, i.e., inhibits microtubule assembly. DM1 is a maytansinoid with lower systemic toxicity than maytansine. In this example, an antibody-drug conjugate is created by reacting SMCC-DM1, which is DM1 conjugated with a reactive linker SMCC, with an antibody. SMCC-DM1 is commercially available from MedKoo Biosciences, Inc. or ALB Technology, among others. For example, the HuL001 buffer (70 mg) was replaced with sodium citrate buffer at pH 6.5 to adjust the concentration to 5 mg / mL. A solution of SMCC-DM1 (5 mM in DMA, 16 eq) was slowly added to the HuL001 solution. The reaction mixture was incubated in a 37°C shaking incubator (150 rpm) for 18 hours. The HuL001-SMCC-DM1 mixture was concentrated and SMCC-DM1 was removed using an Amicon Ultra-15 centrifuge filter with an NMWL of 30 kDa and 25 mM sodium citrate at pH 6.5. ADC concentration: 5.478 mg / mL, ADC yield: 16.5 mg (23.5%), mean DAR: 3.87.

[0022] Example 3 Preparation of HuL001-SPP-DM4 complex [ka] In this embodiment, ADC contains DM4, another meitansine analog. DM4 is also a potent microtubule-targeting compound that inhibits cell proliferation during mitosis. DM4 may be used in some embodiments of the present invention. In this example, the HuL001 buffer (70 mg) was replaced with sodium citrate buffer at pH 6.5 to adjust the concentration to 5 mg / mL. A solution of SPP-DM4 (10 mM in DMA, 15 eq) was slowly added to the HuL001 solution. The reaction mixture was incubated in a 37°C shaking incubator (150 rpm) for 18 hours. The HuL001-SPP-DM4 mixture was concentrated and SPP-DM4 was removed using an Amicon Ultra-15 centrifuge filter with an NMWL of 30 kDa and 25 mM sodium citrate at pH 6.5. ADC concentration: 3.65 mg / mL, ADC yield: 34.3 mg (49%), mean DAR: 3.18.

[0023] Example 4 Preparation of the HuL001-mal-vc-MMAE complex [ka] Monomethyl auristatin E (MMAE) is an anti-cancer drug that inhibits cell division by blocking tubulin polymerization. It is derived from a peptide (drastatin) that occurs in marine shellless mollusks. MMAE has been shown to be a beneficial payload for ADCs. The linker of ADCs can have a significant impact on their biological activity. For example, in in vivo studies, peptide-binding conjugates have been shown to induce regression and cure in engrafted tumors with a therapeutic index of up to 60 times. These conjugates demonstrate the importance of linker technology, efficacy, and binding methods in the development of safe and effective mAb-drug conjugates for cancer treatment. Some embodiments of the present invention relate to MMAE conjugated to an antibody via valine-citrulline (vc), a lysosome-cleavable dipeptide, which has been shown to improve the efficacy of ADCs. In this example, 1 mg of HuL001 buffer was replaced with PBS / EDTA buffer at pH 7.4 to adjust the concentration to 5 mg / mL. An aqueous solution of TCEP (10 mM, 3 eq) was slowly added to the HuL001 solution. The mixture was incubated at 37°C for 2 hours to reduce the disulfide bonds of the antibody. A solution of mal-PEG2-vc-PAB-MMAE (10 mM in DMA, 10 eq) was then slowly added to the protein solution. The reaction mixture was incubated in a shaking incubator (150 rpm) at 25°C for 2 hours. The excess mal-vc-steroid was then quenched using 100 mM NAC (20 eq). Concentration of HuL001-mal-vc-MMAE and removal of mal-PEG2-vc-PAB-MMAE were performed using an Amicon Ultra-15 centrifugation filter with an NMWL of 10 kDa and buffer (pH 6.0 PBS, 137 mM NaCl). ADC concentration: 3.7 mg / mL, ADC yield: 0.481 mg (48.1%), mean DAR: 3.64.

[0024] Example 5 Preparation of the HuL001-Ph-MMAF complex [ka] Some embodiments of the present invention relate to an ADC containing monomethyl auristatin F (MMAF), an analog of MMAE. A buffer of HuL001 (37 mg) was replaced with PBS buffer at pH 7.4 to adjust the concentration to 5 mg / mL. A solution of OSu-ph-MMAF (5 mM in DMA, 5 eq) was slowly added to the protein solution. The reaction mixture was incubated in a shaking incubator (150 rpm) at 37°C for 1 hour. Concentration of HuL001-ph-MMAF and removal of OSu-ph-MMAF were performed using an Amicon Ultra-15 centrifuge filter with an NMWL of 30 kDa and 25 mM sodium citrate at pH 6.5. ADC concentration: 7.8 mg / mL, ADC yield: 34.1 mg (92.1%), mean DAR: 3.09.

[0025] Example 6 Preparation of HuL001-mal-vc-steroid complex [ka] In this example, the buffer for HuL001 (1 mg) was replaced with PBS / EDTA buffer at pH 7.4 to adjust the concentration to 10 mg / mL. An aqueous solution of TCEP (10 mM, 4 eq) was slowly added to the protein solution. The solution was incubated at 37°C for 1.5 hours to reduce the disulfide bonds of the antibody. Next, a solution of mal-vc-steroid (10 mM in DMSO, 10 eq) was slowly added to the protein solution. The reaction mixture was incubated at 0°C in a shaking incubator (150 rpm) for 18 hours. Then, the excess mal-vc-steroid was quenched using 100 mM NAC (20 eq). The HuL001-mal-vc-steroid was concentrated and the mal-vc-steroid was removed using an Amicon Ultra-15 centrifugation filter with an NMWL of 30 kDa and a buffer (pH 6.0 PBS, 137 mM NaCl). ADC concentration: 2.2 mg / mL, ADC yield: 0.33 mg (33%), average DAR: 5.2.

[0026] Example 7 PLRP-HPLC or HIC analysis Various ADCs according to the embodiments of the claimed invention were analyzed by PLRP-HPLC or HIC. Figures 1-5 show that the binding reaction was substantially complete, with only residual amounts of anti-ENO1 antibody and ADC remaining.

[0027] Example 8 intact or reduced LC / MS analysis The evaluation of the drug-antibody ratio (DAR) is important for monitoring the payload binding efficiency to the target antibody. The drug-antibody ratio can affect the therapeutic effect of anti-ENO1 ADC products. Intact liquid chromatography-mass spectrometry (LC-MS) is the preferred method for determining the drug-antibody ratio (DAR) and drug load distribution of lysine-binding antibody-drug conjugates (ADCs). Reduced LC-MS is the preferred method for determining the DAR and drug load distribution of cysteine-binding ADCs. Peak area percentages represent the relative distribution of a particular drug-loading ADC species. Next, the weighted average DAR is calculated using peak area percentage information and the number of drug loads. Figures 5-10 show examples of LC-MS analysis of ADCs (anti-ENO1 ADCs) according to embodiments of the claimed invention. This shows the distribution of the number of drugs bound to the antibody, with the most common being 1-12 drugs bound to the antibody. The mean drug-antibody ratio (DAR) ranges from 3.18 to 5.2 in these examples. If multiple drugs are bound to a single antibody, the drugs can be delivered more efficiently into the cell.

[0028] Example 9 Cytotoxicity assay The cytokine-induced cytotoxic effects of anti-ENO1 ADC products have been demonstrated in ENO1-dependent cell lines derived from various human cancers, including lymphoma, lung cancer, breast cancer, pancreatic cancer, and diffuse large B-cell lymphoma (DLBCL). Various cell lines, such as U937, A549, MDA-MB-231, MCF-7, MBA-MD-453, MBA-MD-175, PANC-1, Raji, SU-DHL-4, Toledo, GA-10, or HT, were activated for 4 hours with the cytokines TGF-β, MCP-1, or IL-6, respectively, to mimic the inflammatory tumor microenvironment, and then incubated for a further 72 hours using serially diluted antibody solutions. Cell viability was measured using a cell counting (CCK-8) kit, and IC50 was measured. 50 The value was calculated. IC of HuL001 in the tested cells. 50 The results exceeded the highest concentration tested (1000 nM). The anti-ENO1 ADC product specifically suppressed cytokine-inducing (20 ng / mL TGF-β, 100 ng / mL MCP-1, 50 ng / mL IL-6) pro-inflammatory surface ENO1 in various cell lines. Tables 1 and 2 show the IC50 of HuL001-SMCC-DM1 and HuL001-mal-vc-MMAE in most cell lines. 50 However, after activation, it dramatically decreased to a single digital level, reflecting the cytotoxic effect. Figure 11 shows IC in DLBCL cell line DHL-4. 50 This shows HuL001-SPP-DM4 at a single digital level. Figures 12 and 13 show that neither HuL001 nor HuL001-SMCC-DM1 nor HuL001-mal-vc-MMAE exhibit detectable in vitro cytotoxicity in normal human B cells isolated in the presence or absence of LPS stimulation. In summary, the ADCs according to this disclosure effectively kill cancer cells, particularly in response to cytokine stimulation, without affecting the viability of normal cells. This makes them promising candidates for targeted cancer therapy. [Table 1] [Table 2]

[0029] Example 10 Cytokine assay The anti-inflammatory effects of anti-ENO1 steroid ADCs were demonstrated in the human monocyte cell line THP-1. THP-1 cells were stimulated with LPS to induce surface expression of ENO1 and secretion of pro-inflammatory cytokines TNF-α and CCL2. Figure 14 shows the superior anti-inflammatory effect of anti-ENO1 steroid ADCs compared to anti-ENO1 antibodies.

[0030] Example 11 Prostate cancer model with anti-ENO1 ADC HuL001-SMCC-DM1 was evaluated in an experimental castration-resistant prostate cancer model. 4-6 week old male nude (nu / nu) mice were used (Lasco Co., Ltd., Taiwan). Before inoculation, human castration-resistant prostate cancer cell line PC-3 cells were washed with PBS, resuspended in 1:1 PBS and Matrigel, and the final concentration was 10 7 The concentration was set to cells / mL. Cells (10 6 The tumor (100 μL) was subcutaneously transplanted into the right flank of mice. The average tumor volume was 100 mm². 3 After reaching a certain stage (6 days after transplantation), mice were randomly assigned to either a control group or a treatment group and administered either PBS (5 mL / kg) or HuL001-SMCC-DM1 (1 or 9 mg / kg), respectively. HuL001-SMCC-DM1 was administered intraperitoneally twice, once every 6 days until the end of the study. Body weight and tumor volume were measured daily. Subcutaneous tumor volume was determined according to the following formula: Tumor volume = Short diameter 2 ×Longest diameter / 2. Administered by intraperitoneal injection. Figure 15 shows that HuL001-SMCC-DM1 inhibits tumor growth without causing weight loss.

[0031] Example 12 Anti-ENO1 ADC EAE disease model HuL001-SMCC-DM1 was evaluated using experimental autoimmune encephalomyelitis (EAE) in the C57BL / 6 mouse, the most commonly used experimental model of multiple sclerosis, a human inflammatory demyelinating disease. Female C57BL / 6 mice aged 10-12 weeks were subcutaneously administered 100 μg of MOG p35-55 in complete Freud's adjuvant, followed by intraperitoneal injection of 100 ng of pertussis toxin. On day 2, another 100 ng of pertussis toxin was administered. Mice were observed daily, and clinical symptoms were evaluated as follows: 0: No signs; decreased tail tone; 2: Mild incomplete monoplegia or parephritis; 3: Severe incomplete paraplegia; 4: Paraplegia and / or quadriplegia (quadparaparesis); 5: Near death or death. Mice were randomly assigned to three groups: a control group of 10 mice, a COPAXONE® (Teva Pharmaceuticals) group of 10 mice, and a HuL001-SMCC-DM1 group of 5 mice. Day 1 was defined as the day of disease onset (disease score ≥ 1). EAE mice in the HuL001-SMCC-DM1 group received subcutaneous administration on days 1, 8, and 15. EAE mice in the COPAXONE group received daily subcutaneous administration from day 1 to day 18. Figure 16 shows that treatment with HuL001-SMCC-DM1 slowed the progression of disease symptoms in EAE mice compared to the PBS control group and the COPAXONE® group.

[0032] Example 13 IPF disease model with anti-ENO1 ADC HuL001-SMCC-DM1 was evaluated in bleomycin-induced pulmonary fibrosis using the C57BL / 6 mouse, the most commonly used experimental model for idiopathic pulmonary fibrosis, a human fibrotic disease. Male C57BL / 6 mice aged 7-9 weeks were administered a single intratracheal dose of bleomycin (3 mg / kg). The mice were randomly assigned to three groups (3 sham mice, 6 bleomycin mice, and 6 HuL001-SMCC-DM1 mice). The day of exposure to bleomycin was defined as day 0. Mice in the HuL001-SMCC-DM1 group received subcutaneous administration on days 1, 8, and 15. Figure 17 shows that treatment with HuL001-SMCC-DM1 attenuated weight loss and lung mass increase compared to the bleomycin group. The hydroxyproline content in the lungs and the TGF-β concentration in bronchoalveolar lavage fluid (BALF) were reduced by HuL001-SMCC-DM1 treatment.

[0033] Unless otherwise defined, all technical and scientific terms and any abbreviations used herein have the same meaning as those generally understood by those skilled in the art of the present invention. Any composition, method, kit, and means for conveying information similar or equivalent to those described herein may be used in carrying out the present invention, but preferred compositions, methods, kits, and means for conveying information are described herein. All references herein are incorporated herein by reference to the maximum extent permitted by law. The consideration of these references is intended solely to summarize the claims made by their authors. No reference is made to acknowledge that any reference (or any part thereof) constitutes relevant prior art. The applicant reserves the right to challenge the accuracy and validity of any referenced references.

Claims

1. A pharmaceutical composition for use in the treatment of ENO1-expressing cancer, comprising an immune complex that specifically binds to ENO1, The aforementioned immune complex General formula: Ab-(LD) m (I) included, In the formula, Ab is an anti-ENO1 antibody or a conjugated fragment thereof, L is a linker or direct conjugate, D is a therapeutic agent, and m is an integer from 1 to 12. The aforementioned antibody HCDR1 (GYTFTSCVMN; Sequence ID 1), HCDR2 (YINPYNDGTKYNEKFKG; SEQ ID NO: 2), and HCDR3 (EGFYYGNFDN; SEQ ID NO: 3) A heavy chain variable domain having three complementary regions including; and LCDR1 (RASENIYSYLT; Sequence ID 4), LCDR2 (NAKTLPE; Sequence ID No. 5), and LCDR3 (QHHYGTPYT; Sequence ID 6) A light chain variable domain having three complementary regions including; or HCDR1 (GYTFTSXVMN, where X is any amino acid other than cysteine; SEQ ID NO: 7), HCDR2 (YINPYNDGTKYNEKFKG; SEQ ID NO: 2), and HCDR3 (EGFYYGNFDN; SEQ ID NO: 3) A heavy chain variable domain having three complementary regions including; and LCDR1 (RASENIYSYLT; Sequence ID 4), LCDR2 (NAKTLPE; Sequence ID No. 5), and LCDR3 (QHHYGTPYT; Sequence ID 6) Light chain variable domain having three complementary regions including including, The aforementioned pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the antibody is a monoclonal antibody.

3. The pharmaceutical composition according to claim 1, wherein the antibody is a mouse antibody, a chimeric antibody, a humanized antibody, or an antibody fragment thereof.

4. The pharmaceutical composition according to claim 1, wherein the therapeutic agent comprises a cytotoxic agent, an immunomodulator, a radioisotope, and a toxin.

5. The cytotoxic agents mentioned above include maytansinoid 1 (DM1), maytansinoid 4 (DM4), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), anthracyclines, pyrrolobenzodiazepines, α-amanitin, tubulisin, benzodiazepines, erlotinib, bortezomib, fulvestrant, sunitinib, letrozole, imatinib mesylate, PTK787 / ZK222584, oxaliplatin, leucovorin, rapamycin, lapatinib, ronafarnib, sorafenib, gefitinib, AG1478, and AG1571. , alkylating agents; alkyl sulfonates; aziridines; ethyleneimine and methylamelamine; acetogenins; camptothecin; bryostatin; calistatin; CC-1065; cryptophycin; dorastatin; duocalmycin; eryuterobin; pancratistatin; sarcodicin; spongistatin; nitrogen mustard; nitrothrea; antibiotics; dinemisin; bisphosphonates; esperamicin; and neocardinostatin chromophores and related pigment proteins enediin antibiotic chromophores, acrasinomycin, actinomycin, and Tramycin, azacerin, bleomycin, kactinomycin, carabicin, caminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detrubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin; antimetabolites; folic acid analogs selected from the group consisting of denopterin, pteropterin, and trimethrexate; purine analogs selected from the group consisting of fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine. Forms; pyrimidine analogs selected from the group consisting of ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine and phloxuridine; androgens; anti-adrenal drugs; folic acid supplements; acegraton; aldofrosphamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisantren; edatraxate; defofamine; demecoltin; diaziquan; elformitin; eriptinium acetate; epotilon; etogluside; gallium nitrate; hydroxyurea;Lentinan; Ronidynin; Maytansinoid; Mitoguazone; Mitoxantrone; Mopidammol; Nitraerine; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; Polysaccharide complex; Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadicone; 2,2',2"-Trichlorotriethylamine; Trichothecin; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside; Cyclophosphamide; Thiotepa; Taxoids, Paclitaxel, Albumin-bound Paclitaxel nanoparticles The pharmaceutical composition according to claim 4, comprising the following formulations, and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs selected from the group consisting of cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids; capecitabine; and any pharmaceutically acceptable salt, acid, or derivative thereof.

6. A pharmaceutical composition according to claim 1, comprising:

7. The pharmaceutical composition according to claim 1, wherein the ENO1-expressing cancer includes leukemia, multiple myeloma, gastric carcinoma, skin cancer, lung cancer, melanoma, kidney cancer, liver cancer, myeloma, prostate cancer, breast cancer, colorectal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, hematological cancer, lymphoma, leukemia, ovarian cancer, bladder cancer, urothelial carcinoma, head and neck cancer, or metastatic lesions of the said cancer.

8. The use of an immune complex for manufacturing a drug to treat ENO1-expressing cancer, wherein the immune complex is General formula: Ab-(L-D)m (I) In the formula, Ab is an anti-ENO1 antibody or a conjugated fragment thereof, L is a linker or direct conjugate, D is a therapeutic agent, and m is an integer from 1 to 12. The aforementioned antibody HCDR1 (GYTFTSCVMN; Sequence ID 1), HCDR2 (YINPYNDGTKYNEKFKG; SEQ ID NO: 2), and HCDR3 (EGFYYGNFDN; SEQ ID NO: 3) A heavy chain variable domain having three complementary regions including; and LCDR1 (RASENIYSYLT; Sequence ID 4), LCDR2 (NAKTLPE; Sequence ID No. 5), and LCDR3 (QHHYGTPYT; Sequence ID 6) A light chain variable domain having three complementary regions including; or HCDR1 (GYTFTSXVMN, where X is any amino acid other than cysteine; SEQ ID NO: 7), HCDR2 (YINPYNDGTKYNEKFKG; SEQ ID NO: 2), and HCDR3 (EGFYYGNFDN; SEQ ID NO: 3) A heavy chain variable domain having three complementary regions including; and LCDR1 (RASENIYSYLT; Sequence ID 4), LCDR2 (NAKTLPE; Sequence ID No. 5), and LCDR3 (QHHYGTPYT; Sequence ID 6) Light chain variable domain having three complementary regions including The aforementioned use, including.

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