Use of subcutaneous injection formulation comprising hyaluronidase used in combination with antibody-drug conjugate, and method for treating disease by using same

Abstract

The present invention relates to: a use of a subcutaneous injection formulation comprising hyaluronidase and used in combination with an antibody-drug conjugate; a subcutaneous injection kit comprising same; and a method for treating diseases including cancer using same. Preferably, the present invention relates to a method for treating diseases by subcutaneously injecting an antibody-drug conjugate, in which an antibody against a cancer-specific antigen and a drug having the characteristics of killing or inhibiting the growth of cancer cells are conjugated, together with an enzyme having hyaluronidase activity, and a subcutaneous administration kit, and presents an injection route that minimizes side effects, particularly systemic toxicity (e.g., neutrophil reduction) and local toxicity of drugs, through subcutaneous injection using hyaluronidase.

Description

Use of a subcutaneous administration formulation containing hyaluronidase used in combination with an antibody-drug conjugate, and a method for treating a disease using the same

[0001] The present invention relates to a subcutaneous administration formulation comprising hyaluronidase used in combination with an antibody-drug conjugate (hereinafter referred to interchangeably as “ADC” in the specification and drawings) comprising one or more drug portions conjugated to an antibody or an antigen-binding antibody fragment, a subcutaneous administration kit comprising the same, and a method for treating a disease using the same.

[0002] The present invention relates to a method for treating diseases including cancer by subcutaneously administering an antibody-drug conjugate, preferably in the form of a drug (payload) having the property of killing cancer cells or inhibiting their growth, together with a protein having hyaluronidase activity, preferably hyaluronidase, and a kit for subcutaneous administration.

[0003]

[0004] Targeted anticancer therapy is a treatment method that eliminates cancer cells by delivering drugs that target specific genes, proteins, or signaling pathways that play a key role in the development of cancer cells. Since the targeted substances are present only in cancer cells rather than normal cells and exert effects on important molecular-level pathways, they can selectively eliminate cancer cells without damaging normal cells. Targeted anticancer therapies include monoclonal antibodies, small molecule agents, and antibody-drug conjugates (ADCs); ADCs are a treatment method that enhances the therapeutic effect of drugs on cancer cells by leveraging the cancer-selective properties of monoclonal antibodies.

[0005]

[0006] The advantages of using antibody-drug conjugates (ADCs) are as follows: (a) The chemotherapy drug itself is structurally well-defined. (b) The chemotherapy drug is linked to the monoclonal antibody protein using very well-defined conjugation chemistry, often at specific sites far removed from the antigen-binding domain of the monoclonal antibody. (c) Antibody-drug conjugates are more reproducible and generally have lower immunogenicity than monoclonal antibodies or chemical drugs containing bacterial or plant toxins, making commercial development and regulatory approval easier. (d) Antibody-drug conjugates have significantly less systemic toxicity than drugs or radionuclide conjugates.

[0007] Most currently used anticancer drugs are administered intravenously (IV); however, intravenous administration is known to require improvement in terms of patient convenience, such as the long time required for administration. Accordingly, research is actively underway to administer anticancer drugs in a more convenient subcutaneous (SC) form, and there have already been reports of several antibody products having been converted from intravenous to subcutaneous formulations.

[0008] Currently, very few cytotoxic drugs are administered via subcutaneous injection for cancer treatment (e.g., Leveque et al., 2014, Anticancer Res 34:1579-86). This is because most known anticancer cytotoxic agents are stimulants and / or blistering agents, which are known to cause local damage to subcutaneous or subdermal tissues following extravasation (Leveque et al., 2014). While conjugation with antibodies can reduce local toxicity, the low absorption rates of monoclonal antibodies such as trastuzumab and alemtuzumab can make subcutaneous administration of antibody-drug conjugates (ADCs) difficult (Leveque et al., 2014). Another challenge is that maintaining a low injection volume for subcutaneous administration requires high concentrations / high doses of ADCs to demonstrate a therapeutic effect (Leveque et al., 2014).

[0009] When such high concentrations / high doses of antibody-drug conjugates are used, particularly for subcutaneous administration, adverse effects caused by the toxic drug, such as serious local toxicity including tissue necrosis at the injection site, are observed.

[0010] Antibody-drug conjugates (ADCs) are being applied to the treatment of various types of cancer as therapeutic agents that combine the target specificity of antibodies with the potent efficacy of cytotoxic drugs. However, due to the nature of ADCs, toxicity originating from the antibody and cytotoxic drug (payload) portions, as well as rare but potentially severe toxicity, acts as a significant limitation to their clinical application.

[0011] Regarding ADC administration, rare but potentially life-threatening adverse effects have been reported. Representative examples include hepatotoxicity (a spectrum ranging from elevated liver enzymes to hepatic veno-occlusive syndrome and liver failure), interstitial lung disease, hemorrhage, progressive multiple leukoencephalopathy, cutaneous toxicity (e.g., SJS / TEN), cardiovascular toxicity, tumor lysis syndrome, pancreatitis, hyperglycemia and diabetic ketoacidosis, and other neurological complications. Although these side effects occur infrequently, they can lead to serious complications or death if they do occur, so they must be given special consideration during ADC therapy.

[0012] Side effects attributed to the immunological characteristics of the antibodies themselves or their Fc-effector functions are also reported. These include fever, infection, ophthalmic toxicity, dermal toxicity, fluid exudation, and peripheral edema, which may occur as antibodies interact with the immune system or induce local inflammatory responses.

[0013] There are also toxicities typically observed depending on the mechanism of action of cytotoxic drugs (payloads). These include cytopenia, fatigue, diarrhea, nausea and vomiting, decreased appetite, hair loss, constipation, and spider angiectasia. These side effects are primarily directly associated with mechanisms of action such as microtubule inhibitors or DNA inhibitors. In particular, peripheral neuropathy has been reported to occur only with drugs belonging to the microtubule inhibitor class.

[0014] In addition to these side effects, it is known that high concentration / high dose administration is very difficult due to physical properties that have solubility limitations.

[0015]

[0016] Meanwhile, hyaluronidase is known to be a method that can be usefully employed to change the administration route of drugs, such as anticancer agents, from intravenous to subcutaneous administration, and can also increase the administration volume.

[0017] Accordingly, the inventors have completed the present invention by identifying that when an antibody-drug conjugate, which is known to be difficult to administer subcutaneously due to the occurrence of side effects such as local toxicity, is administered simultaneously or sequentially with a high concentration of hyaluronidase, side effects such as local toxicity are surprisingly reduced even when a high concentration / high dose of the antibody-drug conjugate is administered subcutaneously.

[0018]

[0019] Summary of the Invention

[0020] The object of the present invention is to provide a subcutaneous administration formulation comprising a protein having hyaluronan degradation activity used in combination with an antibody-drug conjugate.

[0021] Another objective of the present invention is to provide a subcutaneous administration formulation comprising an antibody-drug conjugate and hyaluronidase.

[0022] In addition, another objective of the present invention is to provide a subcutaneous injection kit comprising an antibody-drug conjugate and hyaluronidase, and a method for treating a disease by subcutaneous administration of the antibody-drug conjugate.

[0023] In addition, another objective of the present invention is to provide a method of administration that minimizes side effects, particularly systemic toxicity (e.g., neutropenia) and local toxicity of the drug, through subcutaneous administration of an antibody-drug conjugate using hyaluronidase.

[0024] The present invention relates to a subcutaneous administration formulation comprising a protein having hyaluronan degradation activity (used interchangeably with "hyaluronidase" in the title, description, summary, and drawings of the present invention) used in combination with an antibody-drug conjugate (ADC) comprising one or more drug portions conjugated to an antibody or an antigen-binding antibody fragment, and preferably, said formulation is,

[0025] (a) comprising a protein having hyaluronan degradation activity of 4,000–16,000 unit / mL; and / or

[0026] (b) a protein having hyaluronan degradation activity administered in an activity range of 10 to 470,000 units per 1 mg of drug; the invention relates to a subcutaneous administration formulation characterized by this.

[0027]

[0028] The subcutaneous administration formulation containing the protein having hyaluronan degradation activity is characterized by being administered simultaneously or sequentially with an antibody-drug conjugate (ADC), wherein the sequential administration is characterized by the subcutaneous administration formulation containing the protein having hyaluronan degradation activity being administered first, followed by the subcutaneous administration of a formulation containing an antibody-drug conjugate (ADC) to substantially the same site after a certain period of time has elapsed, but is not limited thereto.

[0029] In the present invention, “substantially identical site” refers to a site where the activity of a protein having hyaluronan degradation activity is maintained at the site of administration at the time of pre-administration, and, for example, may refer to a site within 0.01 to 10 cm from the site of administration at the time of pre-administration, but is not limited thereto.

[0030] The above sequential administration may be characterized by the fact that, after a subcutaneous administration formulation containing a protein having hyaluronan degradation activity is administered first, a formulation containing an antibody-drug conjugate (ADC) is administered later after 30 seconds to 10 hours, preferably 40 seconds to 5 hours, and more preferably 50 seconds to 1 hour.

[0031] In the above sequential administration, the antibody-drug conjugate (ADC) at the subsequent administration may be administered subcutaneously alone or together with a protein having hyaluronan degradation activity, and

[0032] When administering a subcutaneous formulation containing a protein having hyaluronan degradation activity and an antibody-drug conjugate (ADC) simultaneously or sequentially, the antibody-drug conjugate (ADC) and the protein having hyaluronan degradation activity may be characterized by being administered subcutaneously as individual formulations or as a single mixed formulation.

[0033]

[0034] When the above subcutaneous administration formulation is co-administered with an antibody-drug conjugate (ADC), the activity range of the protein having hyaluronan degradation activity per 1 mg of drug is 10 to 470,000 units, preferably 150 to 470,000 units, more preferably 1,000 to 200,000 units, even more preferably 10,000 to 150,000 units, and most preferably 15,000 to 70,000 units.

[0035] When administered sequentially, the active range of the protein having hyaluronan degradation activity per 1 mg of drug may be characterized as 10 to 470,000 units, preferably 10 to 50,000 units, more preferably 15 to 20,000 units, and most preferably 20 to 4,000 units, but is not limited thereto.

[0036]

[0037] In addition, the present invention relates to a mixed subcutaneous administration formulation comprising an antibody-drug conjugate (ADC) and a protein having hyaluronan degradation activity, wherein such a mixed subcutaneous administration formulation may be used for simultaneous administration with the antibody-drug conjugate (ADC) or for subsequent administration in sequential administration.

[0038] The above mixed administration formulation is.

[0039] (a) comprising a protein having hyaluronan degradation activity of 4,000–16,000 unit / mL; and / or

[0040] (b) may be characterized by including an activity range of a protein having hyaluronan degradation activity per 1 mg of drug of 10 to 470,000 units, preferably 150 to 470,000 units, more preferably 1,000 to 200,000 units, even more preferably 10,000 to 150,000 units, most preferably 15,000 to 70,000 units, but is not limited thereto.

[0041]

[0042] In addition, the present invention comprises an antibody-drug conjugate comprising one or more drug portions conjugated to an antibody or an antigen-binding antibody fragment, a hyaluronidase having hyaluronan degradation activity, and an instruction sheet.

[0043] In the above instruction manual, subcutaneous administration of the antibody-drug conjugate is performed in a pre-administration - post-administration manner, and

[0044] The above pre-administration is the subcutaneous administration of hyaluronidase alone at the site of subcutaneous administration of the antibody-drug conjugate-hyaluronidase mixed formulation prior to subcutaneous administration of the antibody-drug conjugate-hyaluronidase mixed formulation, and

[0045] The present invention relates to a kit for subcutaneous administration of an antibody-drug conjugate, wherein the above subsequent administration involves subsequently administering an antibody-drug conjugate and a hyaluronidase mixed formulation or an antibody-drug conjugate and a hyaluronidase to the hyaluronidase administration site, respectively, within 30 seconds to 10 hours of the above prior administration.

[0046] The term "hyaluronidase administration site" above refers to an area where the viscosity of the interstitial tissue is reduced due to the administration of hyaluronidase, and in addition to the precise injection site, it refers to a range within a radius of about 5 cm, preferably 4 cm, more preferably 3 cm, more preferably 2 cm, and more preferably 1 cm.

[0047] In addition, the present invention, according to the instructions, administers the above-mentioned pre-administration at a dose of 1,000 to 16,000 unit / mL based on hyaluronidase activity, preferably 4,000 to 16,000 unit / mL, and

[0048] The present invention relates to a kit for subcutaneous administration of an antibody-drug conjugate, wherein the above-mentioned subsequent administration is described as administering 1,000 to 16,000 unit / mL, preferably 4,000 to 16,000 unit / mL based on hyaluronidase activity, when administering a mixed formulation of the antibody-drug conjugate and hyaluronidase, or when administering the antibody-drug conjugate and hyaluronidase separately.

[0049] In addition, the present invention relates to a kit for subcutaneous administration of an antibody-drug conjugate, wherein the instruction manual describes that the subcutaneous administration of the antibody-drug conjugate according to the above pre-administration-post-administration method includes an initiation administration performed daily for one week or 2 to 5 times a week for two weeks, and a maintenance administration performed at least once every 2 weeks to 6 months after the initiation administration.

[0050] In addition, the present invention relates to a kit for subcutaneous administration of an antibody-drug conjugate, wherein the kit comprises a mixed formulation of the antibody-drug conjugate and hyaluronidase and a hyaluronidase-alone formulation.

[0051] In addition, the present invention relates to a kit for subcutaneous administration of an antibody-drug conjugate, wherein the kit comprises an antibody-drug conjugate alone formulation and a hyaluronidase alone formulation.

[0052] In addition, the present invention relates to a kit for subcutaneous administration of an antibody-drug conjugate in which the aforementioned pre-dose and / or post-dose doses are described in the instructions as effective doses for producing a therapeutic effect.

[0053]

[0054] The drug (payload) included in the antibody-drug conjugate according to the present invention is a microtubule (tubulin) inhibitor, metansinoid or its derivative, anthracycline, topoisomerase inhibitor I or II inhibitor, camptothecin or its derivative, calicemycin, auristatin, nitrogen mustard, ethyleneimine derivative, alkyl sulfonate, nitrosourea, triagen, folic acid analog, taxane, COX-2 inhibitor, pyrimidine analog, purine analog, antibiotic, enzyme inhibitor, epipodophyllotoxin, platinum coordination complex, vinca alkaloid, substituted urea, methylhydrazine derivative, adrenocorticosteroid inhibitor, hormone antagonist, anmetatar, alkylating agent, antimitotic agent, anti-angiogenic agent, tyrosine kinase inhibitor, mTOR inhibitor, heat shock protein (HSP90) inhibitor, proteosome inhibitor, HDAC inhibitor, and It may be one or more selected from the group consisting of proapotosis agents, preferably tubulin inhibitors, but is not limited thereto.

[0055] Preferably, the above drug is maytansine analogs such as MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F), DM1 and DM4, 5-fluorouracil, apatinib, aplidin, azaribin, anastrozole, anthracycline, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan, calicemycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatinum, COX-2 inhibitor, irinotecan (CPT-11), SN-38, Dxd, exatecan, carboplatin, cladribin, camptothecan, crizotinib, cyclophosphamide, It may be one or more selected from the group consisting of cytarabine, dacarbazine, dasatinib, dinaclicb, docetaxel, dactinomycin, and daunorubicin, and more preferably may be MMAE, MMAF, DM1 or DM4, but is not limited thereto.

[0056]

[0057] In addition, the present invention relates to the antibody-drug conjugate comprising ADAM9, AG7, ALCAM, ALPG, AXL, CCR2, CCR7, CD7, CD19, CD22, CD25, CD33, CD37, CD38, CD46, CD48, CD70, CD74, CD79B, CD248, CD274, CD276, CDH3, CDH6, CEACAM5, CLDN6, CLDN18.2, cMET, DLL3, DPEP3, DUX4, EGFR, EPCAM, FAP, FGFR2, FN1, FOLH1, FOLR1, GCC, Globo H, GPNMB, GPR20, GPRC5D, HAVCR1, HER2, HER3, ICAM1, IGF1R, IL3RA, ITGB6, KAAG1, KIT, KLK3, LGALS3BP, LRRC15, It may include, but is not limited to, antibodies or antigen-binding antibody fragments against LY6E, LY75, LYPD3, MELTF, MS4A1, MSLN, MUC1, NCAM1, NECTIN4, NT5E, PRLR, PROM1, PTK7, RNF43, ROR1, ROR2, S. aureus, SDC1, SEZ6, SLC34A2, SLC39A6, SLITRK6, ST8SIA1, TACSTD2, TDGF1, TFRC, TM4SF1, TNF, TNFRSF1A, TNFRSF8, TNFRSF17, TPBG, TRV6, VEGFA, VTCN1, or tissue factor.

[0058]

[0059] In addition, the present invention relates to the antibody-drug conjugate comprising IMGC-936, AbGn-107, Pralusatamab Lavtansine, SGN-ALPV, Enapotamab Vedotin, Mekbotamab Vedotin, Mifacetamab Uzoftyrin, LCB17-0877, TAK-500, JBH-492, Grisnilimab Cetaritox, Roncastuximab Tesirin, Coltuximab Lavtansine, ABBV-319, IKS-03, Inotuzumab Ozogamicin, Moxetumomab Pasodotox, Efratuzumab-SN38, ADCT-602, TAC-001, BAY-1862864, TRPH-222, RM-1995, Kamidanlumab Tesirin, Gemtuzumab Ozogamicin, Lintuzumab-Ac-225, BL-M11D1, Naratuximab Emtansine, AGS-67E, STI-6129, CD38 ADC, FOR-46, SGN-CD48A, PRO-1160, ARX-305, STRO-001, Folatuzumab Vedotin, Iladatuzumab Vedotin, NBT-508, SHR-A1912, MP-ENDOS-ADC, SGN-PDL1V, Vobramitamab Duocamazine, Ifinatamab Deruxtecan, Mirzotamab Clezutoclax, HS-20093, IBI129, MHB088C, BAT-8009, YL201, BC3195, DS-6000, Tusamitamab Lavtansine, Lavetuzumab Goritecan, EBC-129, M-9140, DS-9606, TORL-1-23, LM-302, RC-118, SHR-A1904, SOT-102, XNW-27011, ATG-022, BA1301, CMG-901, CPO-102, IBI343, JS107, SKB-315, SYSA-1801, TORL-2-307-ADC, TQB2103, Telisotuzumab Vedotin, RC-108, REGN5093-M114, BYON-3521, MYTX-011, SHR-A1403, TR1801-ADC, Rovalfituzumab Tesyrin, SC-002, Tamlintamab Famozirin, AOC-1020, Cetuximab Sarotalocan, Departuxizumab Mapodotin, Losatuxizumab Vedotin, Cerclutamab Talirin, MRG-003, AVID-100, BB-1705, EGFR-EDV-RRM1,Oportuzumab Monatox, OMTX-705, Aprutumab Ixadotin, PYX-201, MEDI-3726, Mirvetuximab Sorabtansine, Paletuzumab Echteribulin, Rubeltamab Tazebibulin, AZD-5335, PRO-1184, AMT-151, BAT-8006, IMGN151, TAK-164, OBI-999, Glembatumumab Vedotin, DS-6157a, LM-305, CDX-014, Dicitamab Vedotin, Trastuzumab Deruxtecan, Trastuzumab Emtansine, Trastuzumab Duocamazine, Pertuzumab Zubotulimod, A-166, ARX-788, DP-303c, LCB14-0110, MRG-002, SHR-A1811, B-003, BAT-8001, BB-1701, BDC-1001, DB-1303, DX126-262, FDA-022, GQ-1001, IBI-354, ALT-P7, BAT-8010, BI-CON-02, BL-M07D1, FDA022-BB05, GB-251, GQ-1005, GQ-1007, HS630, MT-5111, NJH-395, PF-06804103, SHR-A1201, TQB2102, ZV0203, Patritumab Deruxtecan, BL-B01D1, SHR-A2009, Enrimomab Pegol, Ronigutamab Ugodotine, IGF-MTX, Pibekimab Sunilin, SGN-B6A, ADCT-901, MGTA-117, ARX-517, MP-LGS-ADC, Samrotamab Vedotin, RG-7841, 16A5-MCC-DM1, MEN-1309, Rupartumab Amadintin, SGN-CD228A, CON-4619, MRG-001, TRS-005, Anetumab Lavtansine, BMS-986148, RC-88, DAC-005, DXC-005, Lorbotuzumab Mertansine, Enfortumab Vedotin, 9MW-2821, BAT-8007, SHR-A2102, SKB-410, SYS6002, BB-1709, Rolinsatamab Talirin, OXS-1650, Cofetuzumab Felidotin, SC-006, Zilovertamab Vedotin, NBE-002, ABL-202, LCB-71, Ozuriftamab Vedotin, RG-7861,Indatuximab lavatansine, SC-011, Upipitamab rilsodotin, XMT-1592, Radiratuzumab vedotin, Sirtratumab vedotin, PF-06688992, Sacituzumab govitecan, Datopotamab deruxtecan, SKB-264, BIO-106, DB-1305, ESG-401, MHB036C, SHR-A1921, BAT-8008, BL-M02D1, DAC-002, FDA-018, YL202, BIIB-015, CX-2029, AGX101, ABBV-154, ABBV-3373, Brentuzumab vedotin, F0002-ADC, Belantamab mafodotin, Espectamab devotansine, HDP-101, AMG-224, MEDI-2228, ASN-004, SYD-1875, AB-160, AZD-8205, HS-20089, SGN-B7H4V, XMT-1660, Tisotumab Vedotin, MRG-004A, XB-002, CBP1008 for FOLR1 and TRV6, AZD-9592 for EGFR and cMET, M-1231 for EGFR and MUC1, Zanitamab Zobodotin, JSKN-003, KM-501, CBX-12, CPO-204, DAN-222, DB-1202, DS001, DXC-009, DXC004A, MG1901, MG2001, MG2002A, It may include, but is not limited to, SGN-STNV, SHR-4602, SYS6010, TORL-3-600, TORL-4-500, or TUB-030.

[0060]

[0061] In addition, the present invention relates to a kit for subcutaneous administration of an antibody-drug conjugate, wherein the instruction manual states that subcutaneous administration of the antibody-drug conjugate can be performed at various sites.

[0062] In addition, the present invention relates to a method for treating a disease by subcutaneously administering an antibody-drug conjugate comprising one or more drug portions conjugated to an antibody or an antigen-binding antibody fragment, wherein

[0063] Subcutaneous administration of antibody-drug conjugates is carried out in a pre-administration - post-administration manner, and

[0064] The above pre-administration is the subcutaneous administration of hyaluronidase alone at the site of subcutaneous administration of the antibody-drug conjugate-hyaluronidase mixed formulation prior to subcutaneous administration of the antibody-drug conjugate-hyaluronidase mixed formulation, and

[0065] The present invention relates to a method for treating a disease by subcutaneous administration of an antibody-drug conjugate, wherein the above-mentioned subsequent administration involves administering a mixed formulation of the antibody-drug conjugate and hyaluronidase, or the antibody-drug conjugate and hyaluronidase, respectively, to the site of administration of the hyaluronidase after the above-mentioned prior administration.

[0066] In addition, the present invention relates to a method for treating a disease by subcutaneous administration of an antibody-drug conjugate, wherein the subsequent administration is performed within 30 seconds to 10 hours after the prior administration.

[0067] In addition, the present invention relates to a method for treating a disease by subcutaneous administration of an antibody-drug conjugate, wherein the above-mentioned pre-administration is administered at a dose of 1,000 to 16,000, preferably 4,000 to 16,000 unit / mL based on hyaluronidase activity, and the above-mentioned subsequent administration is administered by administering a mixed formulation of the antibody-drug conjugate and hyaluronidase, or by administering the antibody-drug conjugate and hyaluronidase separately at a dose of 1,000 to 16,000 unit / mL, preferably 4,000 to 16,000 unit / mL based on hyaluronidase activity.

[0068] In addition, the present invention relates to a method for treating a disease by subcutaneous administration of an antibody-drug conjugate, wherein the subcutaneous administration of the antibody-drug conjugate according to the above pre-administration-post-administration method comprises an initiation administration performed daily for 1 week or 2 to 5 times a week for 2 to 3 weeks, and a maintenance administration performed at least once every 2 weeks to 6 months after the initiation administration.

[0069] In addition, the present invention relates to a method for treating a disease by subcutaneous administration of an antibody-drug conjugate, wherein the dose administered at the time of pre-administration and / or the dose administered at the time of administration is an effective dose for exhibiting a therapeutic effect.

[0070] In addition, the present invention relates to a method for treating a disease by subcutaneous administration of an antibody-drug conjugate, wherein the subcutaneous administration of the antibody-drug conjugate is performed at multiple sites.

[0071]

[0072] The configuration of the present invention will be described in more detail below.

[0073] Meanwhile, each description and embodiment disclosed herein may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the invention. Furthermore, the scope of the invention is not to be limited by the specific descriptions provided below.

[0074] Furthermore, a person skilled in the art can recognize or identify a number of equivalents to the specific embodiments of the invention described in this application using only ordinary experiments. Furthermore, such equivalents are intended to be included in the invention.

[0075]

[0076] As an embodiment of the present invention for solving the above problem, the present invention provides a subcutaneous injection kit capable of improving the pharmacokinetic / pharmacokinetic activity of an antibody-drug conjugate and alleviating side effects.

[0077]

[0078] Antibody-drug conjugates (ADCs) consist of a cytotoxic drug conjugated to an antibody portion by a linker.

[0079] The “antibody portion” may be a molecule acting as an “antibody,” including a monoclonal antibody, an antigen-binding antibody fragment, a bispecific antibody or other polyvalent antibody, or other antibody-based molecules and peptides. The antibody may be various isoforms, preferably human IgG1, IgG2, IgG3, or IgG4, and more preferably may contain human IgG1 hinge and constant region sequences. The antibody or its fragment may be a chimeric, humanized, or human antibody, or a variant thereof such as an anti-IgG4 antibody (hereinafter referred to as “unibody”) as described by van der Neut Kolfschoten et al. (Science 2007; 317:1554-1557). More preferably, the antibody portion may be designed or selected to contain a human constant region sequence belonging to a specific allotype, thereby reducing the immunogenicity of the ADC when administered to human subjects. Preferred allotypes for administration include non-G1m1 allotypes (nG1m1) such as G1m3, G1m3,1, G1m3,2 or G1m3,1,2. More preferably, the allotype may be selected from the group consisting of nG1m1, G1m3, nG1m1,2 and Km3 allotypes (Jefferies and Lefranc, 2009, mAbs 1(4):1-7).

[0080] The antibody portion of an ADC enables specific binding to, for example, each of the following antigens; examples of ADCs that have been released or are currently in clinical development for each antigen are as follows.

[0081] IMGC-936 is the ADC for ADAM9,

[0082] AbGn-107 as an ADC for AG7,

[0083] Pralusatamab ravtansine is an ADC for ALCAM.

[0084] SGN-ALPV is the ADC for ALPG,

[0085] ADCs for AXL include enafotamab vedotin, mekbotamab vedotin, mifasetamab uzoftyrin, and LCB17-0877.

[0086] TAK-500 as an ADC for CCR2,

[0087] The JBH-492 is the ADC for the CCR7,

[0088] As an ADC for CD7, there is grisnilimab cetaritox,

[0089] ADCs for CD19 include roncastuximab tesirin, coltuximab ravtansine, ABBV-319, and IKS-03.

[0090] ADCs targeting CD22 include inotuzumab ozogamicin, moxetumomab pasodotox, efratuzumab-SN38, ADCT-602, TAC-001, BAY-1862864, TRPH-222,

[0091] ADCs targeting CD25 include Kamidanlumab Tesirin and RM-1995.

[0092] ADCs targeting CD33 include gemtuzumab ozogamicin, lintuzumab-Ac-225, and BL-M11D1.

[0093] ADCs for CD37 include naratuximab emtansine and AGS-67E.

[0094] ADCs for the CD38 include the STI-6129 and CD38 ADC.

[0095] FOR-46 is the ADC for CD46,

[0096] The ADC for the CD48 is the SGN-CD48A,

[0097] ADCs for the CD70 include the PRO-1160 and ARX-305.

[0098] STRO-001 as the ADC for CD74,

[0099] ADCs for CD79B include polatuzumab vedotin, iladatuzumab vedotin, NBT-508, SHR-A1912,

[0100] MP-ENDOS-ADC as the ADC for CD248,

[0101] SGN-PDL1V as the ADC for CD274,

[0102] ADCs for CD276 include vobramitamab duocamazine, ifinatab deruxtecan, mirzotamab clezutoclax, HS-20093, MHB088C, IBI129, BAT-8009, YL201,

[0103] BC3195 is the ADC for CDH3,

[0104] The DS-6000 is the ADC for the CDH6,

[0105] ADCs for CEACAM5 include tusamitamab ravtansine, labetuzumab govitecan, EBC-129, and M-9140.

[0106] ADCs for CLDN6 include the DS-9606 and TORL-1-23.

[0107] ADCs for CLDN18.2 include LM-302, RC-118, SHR-A1904, SOT-102, XNW-27011, ATG-022, BA1301, CMG-901, CPO-102, IBI343, JS107, SKB-315, SYSA-1801, TORL-2-307-ADC, TQB2103,

[0108] ADCs for cMET include telisotuzumab vedotin, RC-108, REGN5093-M114, BYON-3521, MYTX-011, SHR-A1403, TR1801-ADC,

[0109] ADCs for DLL3 include rovalfituzumab tesirin, SC-002,

[0110] ADCs for DPEP3 include tamlintamab famozirin,

[0111] The AOC-1020 is the ADC for the DUX4,

[0112] ADCs for EGFR include cetuximab sarotalocan, departuxizumab mafodotin, losatuxizumab vedotin, cerclutamab talirin, MRG-003, AVID-100, BB-1705, EGFR-EDV-RRM1,

[0113] Ofortuzumab Monatox is an ADC for EPCAM,

[0114] For the ADC for FAP, the OMTX-705,

[0115] ADCs for FGFR2 include aftumab ixadotin,

[0116] PYX-201 as the ADC for FN1,

[0117] MEDI-3726 is the ADC for FOLH1,

[0118] ADCs for FOLR1 include mirvetuximab sorabtansine, paletuzumab ecteribulin, rubeltamab tazebibulin, AZD-5335, PRO-1184, AMT-151, BAT-8006, IMGN151,

[0119] TAK-164 as an ADC for GCC,

[0120] OBI-999 is an ADC for Globo H,

[0121] Glembatumumab vedotin is an ADC for GPNMB,

[0122] DS-6157a is an ADC for GPR20,

[0123] The LM-305 is the ADC for the GPRC5D,

[0124] CDX-014 as the ADC for HAVCR1,

[0125] HER2-targeted ADCs include dicitamab vedotin, trastuzumab deruxtecan, trastuzumab emtansine, trastuzumab duocamazine, pertuzumab jubotulimod, A-166, ARX-788, DP-303c, LCB14-0110, MRG-002, SHR-A1811, B-003, BAT-8001, BB-1701, BDC-1001, DB-1303, DX126-262, FDA-022, GQ-1001, IBI-354, ALT-P7, BAT-8010, BI-CON-02, BL-M07D1, FDA022-BB05, GB-251, GQ-1005, GQ-1007, HS630, MT-5111, NJH-395, PF-06804103, SHR-A1201, TQB2102, ZV0203,

[0126] ADCs against HER3 include patritumab deruxtecan, BL-B01D1, and SHR-A2009.

[0127] Enrimomab Pegol is an ADC for ICAM1,

[0128] ADCs targeting IGF1R include ronigutamab ugodotin and IGF-MTX,

[0129] Pibekimab sunilin is an ADC for IL3RA,

[0130] SGN-B6A is the ADC for ITGB6,

[0131] ADCT-901 as the ADC for KAAG1,

[0132] The MGTA-117 is the ADC for the KIT,

[0133] ARX-517 as an ADC for KLK3,

[0134] MP-LGS-ADC as an ADC for LGALS3BP,

[0135] Samrotamab vedotin is an ADC for LRRC15,

[0136] The RG-7841 is the ADC for the LY6E,

[0137] ADCs for the LY75 include the 16A5-MCC-DM1 and MEN-1309.

[0138] Lupartumab amadatin is an ADC for LYPD3,

[0139] SGN-CD228A is an ADC for MELTF,

[0140] ADCs for MS4A1 include CON-4619, MRG-001, and TRS-005.

[0141] ADCs for MSLN include anetumab ravtansine, BMS-986148, and RC-88.

[0142] For the ADCs for MUC1, DAC-005, DXC-005,

[0143] ADCs targeting NCAM1 include lorbotuzumab mertansine,

[0144] ADCs for NECTIN4 include enfortumab vedotin, 9MW-2821, BAT-8007, SHR-A2102, SKB-410, SYS6002,

[0145] BB-1709 as the ADC for NT5E,

[0146] ADCs for PRLR include rolinsatamab talirin,

[0147] The OXS-1650 is used as the ADC for PROM1,

[0148] Cofetuzumab felidotin is an ADC for PTK7,

[0149] SC-006 is the ADC for RNF43,

[0150] ADCs for ROR1 include zilovertamab vedotin, NBE-002, ABL-202, LCB-71,

[0151] Ozuriftamab Vedotin is an ADC for ROR2,

[0152] RG-7861 is an ADC for S. aureus,

[0153] Indatuximab ravtansine is an ADC for SDC1.

[0154] SC-011 as the ADC for SEZ6,

[0155] ADCs for SLC34A2 include Upipitamab rilsodotin and XMT-1592,

[0156] Radiratuzumab vedotin is an ADC for SLC39A6,

[0157] Sirtratumab vedotin is an ADC for SLITRK6,

[0158] PF-06688992 is the ADC for ST8SIA1,

[0159] ADCs for TACSTD2 include sacituzumab govitecan, datopotamab deruxtecan, SKB-264, BIO-106, DB-1305, ESG-401, MHB036C, SHR-A1921, BAT-8008, BL-M02D1, DAC-002, FDA-018, YL202,

[0160] BIIB-015 is an ADC for TDGF1,

[0161] The CX-2029 is the ADC for TFRC,

[0162] AGX101 as the ADC for TM4SF1,

[0163] ABBV-154 is an ADC for TNF,

[0164] ABBV-3373 is the ADC for TNFRSF1A,

[0165] ADCs for TNFRSF8 include brentuzimab vedotin and F0002-ADC,

[0166] ADCs for TNFRSF17 include belantamab mafodotin, espectamab devotansin, HDP-101, AMG-224, and MEDI-2228.

[0167] ADCs for TPBG include ASN-004, SYD-1875,

[0168] AB-160 is an ADC for VEGFA,

[0169] ADCs for VTCN1 include AZD-8205, HS-20089, SGN-B7H4V, XMT-1660,

[0170] ADCs for tissue factor include tisotumab vedotin, MRG-004A, XB-002,

[0171] ADCs using dual antibodies include CBP1008 against FOLR1 and TRV6, AZD-9592 against EGFR and cMET, M-1231 against EGFR and MUC1, and ADCs using dual antibodies against HER2 include zanidatamab zobodotin, JSKN-003, and KM-501.

[0172] ADCs for which the developer has not precisely disclosed the target of the antibody include CBX-12, CPO-204, DAN-222, DB-1202, DS001, DXC-009, DXC004A, MG1901, MG2001, MG2002A, SGN-STNV, SHR-4602, SYS6010, TORL-3-600, TORL-4-500, and TUB-030.

[0173] The ADC mentioned above is merely an example, and it is obvious to those skilled in the art that the ADC of the present invention is not limited to the ADC described above.

[0174] A peptide-drug conjugate (PDC) is a therapeutic agent formed by combining a tumor-homing peptide, which targets receptors overexpressed in cancer cells, with a cytotoxic drug via a linker. Conceptually, PDCs are similar to antibody-drug conjugates (ADCs) in that they deliver drugs to targeted cancer cells. However, PDCs have the following advantages over ADCs: (1) While the substances that can be used as drugs in ADCs are limited to a small number of highly toxic substances (MMAE, DM-1, etc.), PDCs can use a variety of drugs (adriamycin, paclitaxel, camptothecin, cisplatin, etc.). (2) Tumor homing peptides have cell penetrating capability because they bind to target receptors, making it easy to deliver drugs into cancer cells and enhancing the drug's effect. (3) Due to their low molecular weight, they have low renal, bone marrow, and hepatotoxicity. (4) They can be expressed in situ or chemically synthesized, making production costs relatively low. However, due to the low molecular weight described above, they have a short half-life, and there is a clear limitation in that the peptide is degraded before the drug's effect appears. Therefore, there are not many peptide-drug conjugates currently on the market. Lutetium 177-dothetate (Lutathera®) for SSTR-positive cells, which are directly related to cancer, received FDA approval for gastric cancer, pancreatic cancer, and neuroendocrine tumors. Melphalen flufenamide (Melflufen®) for aminopeptidase received FDA accelerated approval, but was withdrawn from the US market after it was confirmed that the effect was minimal in Phase 3 trials. In addition, ANG1005, which prevents brain metastasis of cancer, and NGR015, which prevents angioplasty of cancer, are currently undergoing Phase 3 clinical trials.Furthermore, peptide-drug conjugates (PDCs) cause inconvenience to medical staff and patients because oral administration is difficult and they must be administered via intravenous injection. Therefore, if administered subcutaneously using the pharmaceutical composition containing hyaluronidase described in this invention, the versatility of peptide-drug conjugates (PDCs) can be increased.

[0175]

[0176] Linkers used to design ADCs include cleavage-type and non-cleavage-type linkers. Cleavage-type linkers are a type of linker that releases a drug by cleaving the binding in vivo under certain conditions or by biological enzymes; examples include, but are not limited to, pH-cleavage linkers, thiol-reactive cleavage linkers, Caaz motif cleavage linkers, cathepsin cleavage linkers, beta-glucourinidase cleavage linkers, enzyme cleavage linkers, polymer cleavage linkers, and peptide cleavage linkers.

[0177] Non-cleavage linkers are types of linkers that are not cleaved in vivo but are cleaved within cell organelles to release drugs within the cell. Examples include thiol-reactive linkers, amino-reactive linkers, amino / thiol dual-reactive linkers, acetylphenylalanine linkers, BG dual-action linkers, chelating linkers, sorbase linkers, and pulling linkers, but are not limited to these.

[0178]

[0179] Examples of drugs to be conjugated to antibodies or antibody fragments include microtubule (tubulin) inhibitors, metansinoids or their derivatives, anthracyclines, topoisomerase inhibitors I or II, camptothecin, calicemycin, auristatin, nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, triagenes, folate analogs, taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs, antibiotics, enzyme inhibitors, epipodophyllotoxin, platinum coordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenocorticotropic inhibitors, hormone antagonists, anmetatars, alkylating agents, antimitotic agents, anti-angiogenic agents, tyrosine kinase inhibitors, mTOR inhibitors, heat shock protein (HSP90) inhibitors, proteosome inhibitors, HDAC inhibitors, and proapotogenic agents, in which the drug is present in the nanomolar range of cells In most cases, they are toxic.

[0180] Examples of specific drug groups available include maytansine analogs such as MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F), DM1, and DM4, 5-fluorouracil, apatinib, aplidin, azaribin, anastrozole, anthracycline, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan, calicemycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatinum, COX-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecan, crizotinib, cyclophosphamide, cytarabine, and dacarbazine. You can choose from a group consisting of dasatinib, dinacileb, docetaxel, dactinomycin, and daunorubicin.

[0181] The types of drugs conjugated to ADCs that have been released or are in clinical development are as follows, but are not limited thereto. Toxic drugs include microtubule inhibitors, DNA inhibitors, and other drugs, each of which can be subdivided into the following categories, but are not limited thereto. (Cancer Cell 40(11) 1255-1263 (2022), Biomedicine & Pharmacotherapy 161: 114408 (2023), Acta Pharmaceutica Sinica B13(10), 4025-4059 (2023), Chem. Pharm. Bull. 67, 173-185 (2019), European Journal of Pharmaceutical Sciences 198: 106793 (2024))

[0182] Microtubule inhibitors that inhibit cell division by inhibiting the development of microtubules include drugs of the methansinoid class, tubulosin class, auristatin class, eribulin class, and hemiasterlin class. Methansinoid class drugs include drugs of the methansin (DM1) class and drugs of the ravtansin (DM4) class, with an IC50 of 0.05 to 0.1 nM. Drugs of the methansinoid class or antibody-drug conjugates containing them include trastuzumab emtansin and naratuximab emtansin. Drugs of the ravtansin (DM4) class include OBT076, TRPH-222, mirvetuximab soravtansin, pralusatamab ravtansin, and tusamitamab ravtansin. Tublicin-class drugs have an IC50 of 0.1 to 1 nM and include tublicin and tublicin B derivatives, while auristatin-class drugs have an IC50 of 0.05 to 0.1 nM and include MMAE, MMAF, Auristatin 0101, Auristatin W derivatives, AF-HPA, Dolastatin 100, TZT-1027, Brentuzumab Vedotin, Polatuzumab Vedotin, Enfortumab Vedotin, Tisotumab Vedotin, Dicitamab Vedotin, Telisotuzumab Vedotin, Radiratuzumab Vedotin, Zilovertamab Vedotin, MRG001, MRG002, MRG003, Mekbotamab Vedotin, Ozuriftamab Vedotin, FOR46, OBI999, CX-2029, ALT-P7, There are A-166, belantamab mafodotin, ARX-788, cofetuzumab felidotin, upipitamab lylsodotin, etc., and eribulin-class drugs include eribulin mesylate (E7389), eribulin-LF (E7389-LF), and paletuzumab ecteribulin, and hemiasterin-class drugs include HTI-286, E7974, and rubeltamab tazebibulin.

[0183] DNA inhibitors include the duocamycin class, which modifies gene structure through DNA alkylation; the caliceamycin class, which breaks the double helix; the PBD class, which binds to the double helix; and the camptothecin class, which are DNA topoisomerase inhibitors. Duokamycin-class drugs have an IC50 of 10 to 100 pM and include SYD1875, seco-DUBA, KW-2189, U-80244, trastuzumab duocamazine, and vobramitamab duocamazine; caliceamycin-class drugs have an IC50 of 0.1 to 1 uM and include N-acetyl-gamma caliceamycin and caliceamycin valeric acid derivatives; and PBD-class drugs have an IC50 of 0.1 to 1 pM and include dPBD, SC-DR002, SG3199, SG2000, and loncastuximab There are Tesirin, Kamidanlumab Tesirin, Pibekimab Sunirin, etc., and Camptothecin class drugs include Irinotecan (CPT-11), Sacituzumab Gobitecan, Topotecan, Belotecan, Exatecan, and Exatecan derivative class drugs, and Exatecan derivative class drugs with an IC50 of 1 to 10 uM include Trastuzumab Deruxtecan, Patritumab Deruxtecan, Datopotamab Deruxtecan, Ipinatamab Deruxtecan, Laludotatuk Deruxtecan, SKB-264, etc.

[0184] Other substances include immunoagents and RNA inhibitors. Immunagents include STING agonists and TLR agonists; STING agonist drugs have an IC50 of 100 nM or less, and TLR agonists have an IC50 of 1 uM or less; in addition, there are T785, T-CpG, and TLR7 agonists. RNA inhibitors include RNA splicing inhibitors and RNA polymerase II inhibitors. RNA splicing inhibitors include tyrantatin and its derivatives, with an IC50 of 0.1 to 1 nM; derivatives include tyrantatin A, B, and C. RNA polymerase II inhibitors include amatoxins such as α-amanitine and β-amanitine. In addition, proximity-based proteolysis targeting chimera (PROTAC) substances, Bcl-xL inhibitors, niacinamide phosphate ribose transferase (NAMPT) inhibitors, and proteasome activity inhibitors are being studied and developed as novel ADC drugs.

[0185]

[0186] Cytotoxic drugs exhibit systemic and local toxicity, resulting in a therapeutic window (maximum tolerable toxicity minus the therapeutic dose). The therapeutic window is mostly determined by the Drug-to-Antibody Ratio (DAR) of the antibody-drug conjugate and the characteristics of the drug itself. However, assuming that the maximum tolerable toxicity is determined by the administered drug concentration and the therapeutic dose by the drug dosage, the width of the therapeutic window can be expanded if the maximum tolerable toxicity can be increased by changing the drug concentration of the finished product or the route of administration.

[0187]

[0188] Antibody-drug conjugates are generally administered via intravenous infusion (IV infusion). This route is chosen as a safe route because it allows for lower drug concentrations while increasing therapeutic doses. However, this method can cause inconvenience during treatment due to the prolonged administration time and the need for appropriate medical support during the administration period. Subcutaneous administration can be considered as an alternative, but it is avoided due to concerns about toxicity, as using standard subcutaneous administration requires increasing the concentration of the antibody-drug conjugate.

[0189]

[0190] Subcutaneous injection is the most attractive alternative to intravenous injection. Compared to intravenous injection, it can reduce the burden on patients by allowing them to administer the injection themselves, thereby reducing the time and effort required for treatment. Currently, 30% of approved monoclonal antibodies are in subcutaneous formulations. On the other hand, clinical experience shows that antibody-drug conjugates (ADCs) are limited in terms of subcutaneous administration. According to data published to date, case studies have been conducted only on the following antibody-drug conjugates.

[0191] According to U.S. Patent No. 10,799,597, an antibody-drug conjugate containing SN-38 as a drug can be injected subcutaneously. The patent demonstrated that antibody-drug conjugates containing SN-38 as a drug are effective when administered subcutaneously to various patients against antibodies against various antigens expressed by cancer cells, such as Trop-2, CEACAM5, and HLA-DR. First, in the case of sacituzumab govitecan (IMMU-132), when administered subcutaneously at a dose of 2 to 4 mg / kg for 1 week daily or 3 times a week for 2 weeks as maintenance therapy after induction to triple-negative breast cancer patients who had failed standard therapy two or more times, a 35% reduction in tumor volume was observed after two cycles, no antibodies against hRS7 (anti-Trop-2 antibody) were detected in the blood, and no local adverse effects were observed at the injection site. Next, when IMMU-130 (anti-CEACAM5 antibody + SN-38) was administered subcutaneously at a dose of 4 mg / kg three times a week for 2 weeks to a 52-year-old male patient with colorectal cancer metastasized to the left and right lobes of the liver and the right lung, the tumor size of the three target lesions decreased by 25%. Finally, when IMMU-140 (anti-HLA-DR antibody+SN-38) was administered subcutaneously at a dose of 3 mg / kg to a 75-year-old patient with metastatic stage 4 colorectal cancer for more than 20 times (5 cycles), with one cycle consisting of 4 administrations per cycle followed by a 1-week rest, it was confirmed that the index tumor lesion decreased by 21% at week 8 and by 27% at week 13.

[0192] In addition, in a clinical study (NCT04460456) conducted with SBT6050, which used a toll-like receptor 8 agonist as the payload and was conjugated with the HER2-targeting monoclonal antibody pertuzumab, a manageable safety profile was reported when administered subcutaneously at a dose of 0.3 to 1.2 mg / kg once every two weeks as a monotherapy or in combination with pembrolizumab, and the most frequent adverse event was injection site reaction (Grade 1 50%, Grade 2 33%, Grade 3 3%).

[0193] These cases are limited to specific drugs or ADCs, and it is difficult to expect similar results with all ADCs; this implies that an evaluation of local toxicity is essential when administering antibody-drug conjugates subcutaneously.

[0194]

[0195] Generally, when antibody therapies are switched from intravenous to subcutaneous injection, hyaluronidase is used; in this case, pharmacokinetic advantages enabling efficient absorption and improvements in side effects are expected.

[0196] The hyaluronidase described above may include, but is not limited to, the following. In some embodiments, the hyaluronidase enzyme is a mammalian-type hyaluronidase, such as endo-beta-N-acetylhexosaminidase, having tetrasaccharides and hexasaccharides as major end products. In some cases, the mammalian hyaluronidase possesses both hydrolytic and transglycosidase activity and can degrade hyaluronan and chondroitin sulfate. In other embodiments, the hyaluronidase enzyme is a bacterial hyaluronidase, such as endo-beta-N-acetylhexosaminidase, which produces a disaccharide end product by beta removal. In yet another embodiment, the hyaluronidase enzyme is an endo-beta-glucuronidase that produces tetrasaccharide and hexasaccharide end products through the hydrolysis of β-1-3 linkages. In some embodiments, the hyaluronidase enzyme in the target antibody-drug polymer composition comprises a mammalian hyaluronidase having a neutral active site or an acidic active site. In certain embodiments, the composition of interest comprises a recombinant human hyaluronidase enzyme. In certain cases, the recombinant human hyaluronidase enzyme is a PH20 recombinant human hyaluronidase enzyme (rHuPH20). In some embodiments, the hyaluronidase enzyme (e.g., a soluble hyaluronidase glycoprotein) facilitates the subcutaneous administration of the target composition. In some cases, the hyaluronidase enzyme is present in an amount that rapidly depolymerizes hyaluronan in the extracellular space, reduces the viscosity of the interstitial tissue to increase hydrodynamic conductivity, and allows a large amount of the drug to be administered into the subcutaneous tissue. In certain embodiments, the increased hydrodynamic conductivity induced by the hyaluronidase enzyme through reduced interstitial viscosity allows for greater dispersion, thereby increasing the systemic bioavailability of the subcutaneously administered antibody-drug polymer described herein.

[0197] In certain embodiments, the composition comprises one or more hyaluronidase enzymes (e.g., soluble hyaluronidase glycoproteins), such as those described in International Patent Publications No. WO 2004 / 078140 and WO 2006 / 091871 and U.S. Patent No. 7,767,429, the disclosures thereof of which are incorporated herein by reference.

[0198] In some embodiments, the hyaluronidase enzyme is a variant or fragment of recombinant human hyaluronidase that is active and capable of degrading hyaluronan. The sequence of the wild-type human PH20 hyaluronidase enzyme (Sequence No. 1) is shown in Table 1 below.

[0199]

[0200] In certain embodiments, the hyaluronidase is a soluble hyaluronidase. Soluble hyaluronidase includes any that exist in a soluble form upon expression and secretion from a cell. Such soluble hyaluronidase includes, but is not limited to, non-human soluble hyaluronidase, bacterial soluble hyaluronidase, bovine PH20, sheep PH20, and variants thereof. Among soluble hyaluronidases, the soluble modified human PH20 polypeptide is included. For example, hyaluronidase such as human PH20 containing a glycophosphatidylinositol (GPI) anchor may be made soluble by cleavage and removal of all or part of the GPI anchor. In one example, human hyaluronidase PH20, which is typically membrane-bound via a GPI anchor, is made soluble by cleavage and removal of all or part of the GPI anchor at the C-terminus.

[0201] Soluble hyaluronidases also include neutrally active hyaluronidases such as soluble human PH20 polypeptides. In certain examples, the hyaluronidase for use in the compositions, combinations, and methods of the present invention is a soluble neutrally active hyaluronidase. Examples of hyaluronidases include soluble forms of PH20 from any species, such as the soluble form of PH20. Soluble forms of PH20 are known in the art. These include sheep and bovine PH20 polypeptides, and the soluble form of human PH20 of SEQ ID NO. 1. The soluble form of human PH20 of SEQ ID NO. 1. Such soluble forms include variants of its truncated form lacking all or part of the C-terminal GPI anchor, provided that the hyaluronidase is soluble (secreted upon expression) and retains hyaluronidase activity. Such forms are also typically mature forms lacking the signal peptide when expressed in cells. Full-length mature human PH20 (residues 36-509 of Sequence No. 1) occurs as a GPI-fixed polypeptide. As is known in the art, it becomes soluble by cleavage at the C-terminus. Such cleavage may remove all GPI anchor attachment sequences or only a portion of the GPI anchor attachments. However, the resulting polypeptide is soluble. If the soluble hyaluronidase retains a portion of the GPI anchor attachment signal sequence, 1, 2, 3, 4, 5, 6, 7 or more amino acid residues of the GPI anchor attachment signal sequence may be retained as long as the polypeptide is soluble. A polypeptide containing one or more amino acids of the GPI anchor is designated as an extended soluble hyaluronidase. A person skilled in the art can determine whether the polypeptide is GPI-fixed using methods well known in the art.These methods include, but are not limited to, the steps of predicting the presence and location of the GPI anchor attachment signal sequence and the ω-site using a known algorithm, and performing solubility analysis before and after digestion with phosphatidylinositol-specific phospholipase C (PI-PLC) or D (PI-PLD).

[0202] An example of a soluble hyaluronidase is soluble human PH20. A soluble form of recombinant human PH20 has been produced and may be used in the compositions, combinations, and methods described herein. Descriptions and production of such soluble forms of PH20 are described in U.S. Patent Nos. 7,767,429, 8,202,517, 8,431,380, 8,431,124, 8,450,470, 8,765,685, 8,772,246, 7,871,607, 7,846,431, 7,829,081, 8,105,586, 8,187,855, 8,257,699, 8,580,252, 9,677,061, and 9,677,062, which are incorporated herein by reference, for example.

[0203] A recombinant soluble form of human PH20 has been produced and can be used in the compositions, combinations, and methods provided herein. For example, referring to SEQ ID NO. 1, which presents a sequence of a full-length precursor PH20 comprising a signal sequence (residues 1-35), the soluble form is a C-terminal cleavage of human PH20 presented in SEQ ID NO. 1 having C-terminal amino acid residues 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 of the amino acid sequence presented in SEQ ID NO. 1. Polypeptide, or polypeptides exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity thereof, comprising but not limited to, having activity at neutral pH and being soluble (secreted into medium when expressed in mammalian cells). Soluble forms of human pH20 generally include those containing amino acids 36–464 as presented in SEQ ID NO. 1. For example, when expressed in mammalian cells, the 35-amino acid N-terminal signal sequence is cleaved during processing, and the mature form of the protein is secreted. Accordingly, mature soluble polypeptides include those containing amino acids 36 to 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482 and 483 of SEQ ID NO. 1.Examples of soluble hyaluronidase include a soluble PH20 polypeptide having an amino acid sequence presented as amino acid residues 36-482, 36-477, 36-478, 36-479, 36-480, 36-481, and 36-483 of SEQ ID NO. 1, a soluble human PH20 polypeptide having a length of 442, 443, 444, 445, 446, 447, or 448 amino acids, and a soluble human PH20 polypeptide having an amino acid length of 442, 443, 444, 445, 446, 447, or 448 amino acids that retain hyaluronidase activity and, for example, an amino acid sequence presented as amino acid residues 36-482, 36-477, 36-478, 36-479, 36-480, 36-481, and 36-483 of SEQ ID NO. 1 and at least 85%, 86%, 87%, 88%, It is a variant of this having 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. These available forms of recombinant human PH20 are described, for example, in U.S. Patent Nos. 7,767,429, 8,202,517, 8,431,380, 8,431,124, 8,450,470, 8,765,685, 8,772,246, 7,871,607, 7,846,431, 7,829,081, 8,105,586, 8,187,855, 8,257,699, 8,580,252, 9,677,061, and 9,677,062, which are incorporated herein by reference. Because glycosylation is important for the catalytic activity and stability of hyaluronidase, the soluble form of PH20 is typically produced using a protein expression system that facilitates precise N-glycosylation to ensure the polypeptide maintains its activity. These cells include, for example, Chinese hamster ovary cells (CHO, e.g., DG44 CHO cells).

[0204] Soluble recombinant human PH20, including a recombinant form of human PH20, can be produced recombinantly. One such product is designated as rHuPH20; rHuPH20 generally refers to a composition produced upon expression in cells, e.g., CHO cells, of a nucleic acid encoding residues 36–482 of SEQ ID NO. 1, linked to a natural or heterogeneous signal sequence (residues 1–35 of SEQ ID NO. 1). rHuPH20 is produced by the expression of a nucleic acid molecule such as one encoding amino acids 1–482 (presented in SEQ ID NO. 1). Post-translational processing removes the 35-amino acid signal sequence, leaving a polypeptide or a polypeptide mixture. As produced in culture medium, the product designated as rHuPH20 has C-terminal heterogeneity to include a mixture of species terminating at residues 477, 478, 479, 480, 481, and 482 in varying abundances, with reference to SEQ ID NO. 1. Hyaluronidase rHuPH20 is selected from the group consisting of polypeptides referring to SEQ No. 1 corresponding to amino acid residues 36-477; amino acid residues 36-478, c amino acid residues 36-479, amino acid residues 36-480, amino acid residues 36-481 and amino acid residues 36-482. Generally, the most abundant species is the 446-amino acid polypeptide corresponding to residues 36-481 of SEQ No. 1.

[0205] Soluble human PH20 polypeptides include those referred to as extended soluble hyaluronidases. Extended soluble hyaluronidases can be produced by preparing a C-terminal cleavage of any natural GPI-fixed hyaluronidase such that the resulting polypeptide is soluble and contains one or more amino acid residues from the GPI anchor attachment signal sequence (see, e.g., U.S. Patent No. 8,927,249). Extended soluble human PH20 polypeptides include those terminating at any of residues approximately 495–500 of SEQ ID NO. 1. The mature form begins at residue 36. Extended soluble human PH20 polypeptides are neutrally active and soluble. These may contain amino acid substitutions and exhibit at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence homology with an extended soluble PH20 polypeptide.

[0206] Hyaluronidase can be produced recombinantly or purified or partially purified from natural sources, such as testicular extracts, for example. Methods for producing recombinant proteins containing recombinant hyaluronidase are well known in the field. Soluble pH20 is produced in cells that facilitate precise N-glycosylation to maintain activity, such as CHO cells (e.g., DG44 CHO cells).

[0207] Glycosylation, including N- and O-linking glycosylation, can be important for the catalytic activity and stability of some hyaluronidases, including soluble pH 20 hyaluronidases. For some hyaluronidases, the removal of N-linking glycosylation can result in the near-complete inactivation of hyaluronidase activity. N-linked oligosaccharides are classified into several basic types (oligomannose, complex, hybrid, sulfated), all of which possess a (Man) 3-GlcNAc-GlcNAc-core attached via the amide nitrogen of an Asn residue belonging to the -Asn-Xaa-Thr / Ser- sequence (where Xaa is not Pro). Glycosylation at the -Asn-Xaa-Cys- site has been reported for coagulation protein C. In some cases, hyaluronidases such as PH20 hyaluronidase may contain N-glycosidic and O-glycosidic linkages. For example, PH20 has N-linked oligosaccharides as well as O-linked oligosaccharides. N82, N166, N235, N254, N368, and N393 of the human PH20 exemplified in SEQ ID NO. 1 have six potential N-linked glycosylation sites.

[0208] In certain embodiments, the hyaluronidase enzyme is a variant or fragment of hyaluronidase PH20 having one or more amino acid residue additions, deletions, or substitutions in the amino acid sequence of mature wild-type PH20, such as hyaluronidase PH20 having one or more amino acid residue additions, deletions, or substitutions in the amino acid sequence of wild-type PH20. In some embodiments, the variant or fragment hyaluronidase PH20 comprises one or more amino acid substitutions, additions, or deletions located in the alpha-helix 8 region of PH20. In some embodiments, the variant or fragment hyaluronidase PH20 comprises one or more amino acid substitutions, additions, or deletions located in the linker region between alpha-helix 7 and alpha-helix 8 of PH20. In certain embodiments, the variant or fragment hyaluronidase PH20 comprises the amino acid sequence from L36 to S490 of wild-type PH20 (i.e., L36 to S490 of SEQ ID NO. 1).

[0209] In some embodiments, a PH20 variant or a fragment thereof exhibits amino acid sequence homology with the amino acid sequence of the wild-type PH20 enzyme of SEQ NO. 1, comprising at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

[0210] In some embodiments, the variant or fragment hyaluronidase PH20 comprises, but is not limited to, one or more amino acid residue substitutions at one or more positions selected from T341 to N363, such as T341, L342, S343, I344, M345, S347, M348, K349, L352, L353, L354, D355, N356, E359, 1361 and N363. For example, amino acid residue substitutions at one or more positions selected from T341, L342, S343, I344, M345, S347, M348, K349, L352, L353, L354, D355, N356, E359, 1361 and N363 may be, but are not limited to, one or more amino acid residue substitutions selected from T341A, T341C, T341D, T341G, T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, D355K, E359D, I361T and N363G. In an embodiment, an amino acid residue substitution is described by a code number and letter such as "T455S", which means that at number position 455 of a given sequence number, the amino acid residue threonine ("T") is substituted with the amino acid residue serine ("S"). In a specific embodiment, the variant or fragment hyaluronidase PH20 comprises one or more amino acid residue substitutions selected from T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T.In certain embodiments, the variant or fragment hyaluronidase PH20 comprises one or more amino acid residue substitutions selected from M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and may further comprise one or more amino acid residue substitutions selected from the group consisting of T341A, T341C, T341D, T341G, T341S, L342W, S343E, I344N, and N363G, but is not limited thereto.

[0211] In some embodiments, variant or fragment hyaluronidase PH20 is (a) T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T; (b) L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T; (c) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, and N363G; (d) T341G, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (e) T341A, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (f) T341C, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (g) T341D, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (h) I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T; and (i) one or more amino acid residue substitutions selected from S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T.

[0212] In some embodiments, the variant or fragment hyaluronidase PH20 comprises a cleavage of the amino acid sequence of SEQ ID NO. 1 prior to F38 at the N-terminus. In some cases, the variant or fragment of hyaluronidase PH20 comprises a cleavage of the amino acid sequence of SEQ ID NO. 1 prior to amino acid residues selected from M1 to P42. For example, the variant or fragment of hyaluronidase PH20 comprises a cleavage at the N-terminus prior to amino acid residues L36, N37, F38, R39, A40, P41, or P42 such that one or more amino acid residues are deleted at the N-terminus. In the embodiments, the phrase "cleavage prior to amino acid residues selected from M1 to P42 at the N-terminus" means that amino acid residues immediately prior to amino acid residues G2 to P42 at the N-terminus are cleaved and deleted.

[0213] In some embodiments, a variant or fragment of hyaluronidase PH20 comprises a cleavage of the amino acid sequence of SEQ ID NO. 1 following an amino acid selected from V455 to S490. For example, a variant or fragment of hyaluronidase PH20 comprises a cleavage of the amino acid sequence of SEQ ID NO. 1 following amino acid residues V455, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, E477, P478, Q479, I480, F481, Y482, N483, A484, P486, T488, or S490 such that one or more amino acid residues are deleted at the C-terminus. In an embodiment, the phrase "cleavage after an amino acid residue selected from V455 to S490 at the C-terminus" means that an amino acid residue immediately following amino acid residues V455 to S490 at the C-terminus is cleaved and deleted.

[0214] In some embodiments, a variant or fragment of hyaluronidase PH20 may have the amino acid sequence of SEQ ID NO. 1 and may include one or more amino acid residue substitutions selected from T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and may include a cleavage up to F38 at the N-terminus and a cleavage down to F468 at the C-terminus (also known as HP46, ALT-B4, SEQ ID NO. 6). In some cases, a variant or fragment of hyaluronidase PH20 may have the amino acid sequence of SEQ ID NO. 1 and may include one or more amino acid residue substitutions selected from T341A, T341C, T341G, S343E, M345T, K349E, L353A, L354I, N356E, and I361T. In certain cases, one or more amino acid residue substitutions are located in the alpha-helix 8 region (S347 to C381) and / or the linker region (A333 to R346) between alpha-helix 7 and alpha-helix 8. For example, amino acid substitution in the linker region between alpha-helix 7 and alpha-helix 8 may include substitution of one or more amino acid residues in the region composed of amino acid residues T341 to N363, T341 to 1361, L342 to I361, L342 to I361, S343 to I361, I344 to I361, M345 to I361, or M345 to N363.

[0215] In some embodiments, a variant or fragment of hyaluronidase PH20 may be substituted with some amino acid residues of the amino acid sequence of the human HYAL1 region as presented in Tables 2 and 3 having SEQ ID NO. 2, having one or more amino acid residue substitutions located in the alpha-helix 8 region (S347 to C381) and / or the linker region (A333 to R346) between alpha-helix 7 and alpha-helix 8 of wild-type PH20 (e.g., mature wild-type PH20).

[0216] Table 2 below is the amino acid sequence of wild-type human Hyal1 (Sequence No. 2).

[0217]

[0218] Table 3 below compares the alpha helix and amino acid sequences of PH20 and Hyal1.

[0219]

[0220] In certain embodiments, variants or fragments of hyaluronidase PH20 are described in U.S. Application No. 16 / 628,258, which is incorporated herein by reference. An exemplary variant of hyaluronidase PH20 is an ALT-B4 variant having the sequence of SEQ ID NO. 6.

[0221]

[0222] In some embodiments, the hyaluronidase variant or fragment is a hyaluronidase enzyme comprising, instead of a signal peptide of wild-type PH20 consisting of M1 to T35 but not limited thereto, a human growth hormone signal peptide having the amino acid sequence MATGSRTSLLLAFGLLCLPWLQEGSA of SEQ No. 3 at the N-terminus as shown in Table 5, a human serum albumin signal peptide having the amino acid sequence MKWVTFISLLFLFSSAYS of SEQ No. 4, or a human Hyal1 signal peptide having the amino acid sequence MAAHLLPICALFLTLLDMAQG of SEQ No. 5. In the embodiments, the signal peptide of wild-type PH20 (amino acid residues M1 to T35) is partially or completely deleted. In some embodiments, when a portion of the N-terminus is additionally deleted, for example, when cleavage occurs before residues N37, F38, R39, A40, P41, or P42, additional deletion of the N-terminus occurs along with deletion of the wild-type PH20 signal peptide. Table 4 below shows the amino acid sequences of human growth hormone, human serum albumin, or human Hyal1 signal peptide.

[0223]

[0224] In some embodiments, the variants or fragments of hyaluronidase include a variant having a C-terminus with a 6×His-tag attached (“HM”), a variant without a 6×His-tag (“HP”), a mature wild-type PH20 (L36-S490) having a C-terminus with a 6×His-tag attached (“WT”), and a mature wild-type PH20 (L36 to Y482) having a C-terminus without a 6×His-tag and cleavage occurring after Y482 (“HW2”).

[0225] In certain embodiments, the composition of the present invention comprises one or more variants or fragments of a hyaluronidase enzyme, such as those described in European Patent Publication No. EP3636752A1 and International Patent Publication No. WO 2020 / 197230, the disclosures of which are incorporated herein by reference.

[0226] In certain embodiments, the amount of hyaluronidase enzyme present in the composition may vary and may be a hyaluronidase enzyme comprising 100 units or more, e.g., 250 units or more, e.g., 500 units or more, e.g., 750 units or more, e.g., 1000 units or more, e.g., 1500 units or more, e.g., 2000 units or more, e.g., 2500 units or more, e.g., 3000 units or more, e.g., 3500 units or more, e.g., 4000 units or more, e.g., 4500 units or more, e.g., 5000 units or more, e.g., 10,000 units or more, e.g., 20,000 units or more, e.g., 30,000 units or more, e.g., 40,000 units or more, and e.g., 50,000 units or more.In some embodiments, the composition comprises about 500 units of hyaluronidase enzyme, about 600 units, about 700 units, about 800 units, about 900 units, about 1,000 units, about 1,100 units, about 1,200 units, about 1,300 units, about 1,400 units, about 1,500 units, about 1,600 units, about 1,700 units, about 1,800 units, about 1,900 units, about 2,000 units, about 2,100 units, about 2,200 units, about 2,300 units, about 2,400 units, about 2,500 units, about 2,600 units, about 2,700 units, about 2,800 units, about 2,900 units, about 3,000 units, about 3,100 units, It contains about 3,200 units, about 3,300 units, about 3,400 units, about 3,500 units, about 3,600 units, about 3,700 units, about 3,800 units, about 3,900 units, about 4,000 units, about 4,100 units, about 4,200 units, about 4,300 units, about 4,400 units, about 4,500 units, about 4,600 units, about 4,700 units, about 4,800 units, about 4,900 units, or about 5,000 units of hyaluronidase enzyme.For example, the amount of hyaluronidase enzyme in the composition may be a range of hyaluronidase enzymes including 50 units to 50,000 units, e.g., 100 units to 45,000 units, e.g., 250 units to 40,000 units, e.g., 500 units to 35,000 units, e.g., 750 units to 30,000 units, e.g., 1000 units to 25,000 units, e.g., 1500 units to 20,000 units, e.g., 2000 units to 15,000 units, e.g., 2500 units to 10,000 units, and 3000 units to 5000 units. For example, the amount of hyaluronidase enzyme in the composition may be a range of hyaluronidase enzymes including 50 units to 50,000 units, e.g., 100 units to 40,000 units, e.g., 300 units to 30,000 units, e.g., 500 units to 20,000 units, e.g., 700 units to 10,000 units, e.g., 800 units to 5,000 units, e.g., 900 units to 4,000 units, e.g., 1,000 units to 3,000 units, e.g., 1,500 units to 2,500 units, and 1,700 units to 2,200 units.

[0227] In certain embodiments, the concentration of hyaluronidase enzyme in the composition is 50 unit / mL or more, e.g., 100 unit / mL or more, e.g., 250 unit / mL or more, e.g., 500 unit / mL or more, e.g., 750 unit / mL or more, e.g., 1000 unit / mL or more, e.g., 2000 unit / mL or more, e.g., 2500 unit / mL or more, e.g., 3000 unit / mL or more, e.g., 3500 unit / mL or more, e.g., 4000 unit / mL or more, e.g., 4500 unit / mL or more, e.g., 5000 unit / mL or more.In some cases, the concentration of hyaluronidase enzyme is approximately 100 unit / mL, 200 unit / mL, 300 unit / mL, 400 unit / mL, 500 unit / mL, approximately 600 unit / mL, approximately 700 unit / mL, approximately 800 unit / mL, approximately 900 unit / mL, approximately 1,000 unit / mL, approximately 1,100 unit / mL, approximately 1,200 unit / mL, approximately 1,300 unit / mL, approximately 1,400 unit / mL, approximately 1,500 unit / mL, approximately 1,600 unit / mL, approximately 1,700 unit / mL, approximately 1,800 unit / mL, approximately 1,900 unit / mL, approximately 2,000 unit / mL, approximately 2,100 unit / mL, approximately 2,200 unit / mL, approximately 2,300 unit / mL, approximately 2,400 unit / mL, approx. 2,500 unit / mL, approx. 2,600 unit / mL, approx. 2,700 unit / mL, approx. 2,800 unit / mL, approx. 2,900 unit / mL, approx. 3,000 unit / mL, approx. 3,100 unit / mL, approx. 3,200 unit / mL, approx. 3,300 unit / mL, approx. 3,400 unit / mL, approx. 3,500 unit / mL, approx. 3,600 unit / mL, approx. 3,700 unit / mL, approx. 3,800 unit / mL, approx. 3,900 unit / mL, approx. 4,000 unit / mL, approx. 4,100 unit / mL, approx. 4,200 unit / mL, approx. 4,300 unit / mL, approx. 4,400 unit / mL, approx. 4,500 unit / mL, approx. It is 4,600 unit / mL, about 4,700 unit / mL, about 4,800 unit / mL, about 4,900 unit / mL, or about 5,000 unit / mL.For example, the concentration of hyaluronidase enzyme in the composition may be in a range of 50 unit / mL to 5000 unit / mL, e.g. 100 unit / mL to 4500 unit / mL, e.g. 250 unit / mL to 4000 unit / mL, e.g. 500 unit / mL to 3500 unit / mL, e.g. 750 unit / mL to 3000 unit / mL, 1000 unit / mL to 2000 unit / mL, and 1500 unit / mL to 2500 unit / mL.

[0228]

[0229] The objective of the present invention is to provide a method of administration that minimizes side effects, particularly systemic toxicity (e.g., neutropenia) and local toxicity of the drug, through subcutaneous administration of an antibody-drug conjugate using hyaluronidase, as presented in the embodiments of the present invention. Since intravenous administration can lead to a rapid increase in the blood concentration of the antibody-drug conjugate and consequently systemic toxicity, subcutaneous administration can serve as an alternative to prevent such a rapid rise in blood concentration. However, in this case, if the diffusion of the antibody-drug conjugate at the injection site is delayed, the possibility of local toxicity increases; therefore, to prevent this and to accelerate the diffusion of the antibody-drug conjugate, a subcutaneous administration method containing hyaluronidase can be presented as a method to minimize systemic and local toxicity. The inventors have revealed that, preferably, hyaluronidase is administered first to facilitate the diffusion of the drug within the skin tissue, and subsequently, subcutaneous administration of a formulation containing both the antibody-drug conjugate and hyaluronidase can accelerate the diffusion of the antibody-drug conjugate at the injection site, thereby enabling safer drug use.

[0230] It is evident that in subcutaneous administration formulations containing hyaluronidase, if the active unit of hyaluronidase is the same, the pharmacokinetic results are similar. Hyaluronidase identical to and / or similar to that described above may be used. In the case of subcutaneous injection of antibodies, an active unit of hyaluronidase of 1,000 to 16,000 unit / mL, preferably 1,500 to 12,000 unit / mL, and more preferably 2,000 to 12,000 unit / mL is used. However, in conclusion, the inventors have invented using 3,000 to 16,000 unit / mL, preferably 4,000 to 12,000 unit / mL, and more preferably 6,000 to 12,000 unit / mL in the case of a subcutaneous administration method of antibody-drug conjugates such as the following examples, in order to minimize local toxicity. In addition, the inventors use an active unit of 1,000 to 16,000 unit / mL when hyaluronidase is administered first, preferably 1,500 to 12,000 unit / mL, more preferably 2,000 to 12,000 unit / mL, and most preferably 4,000 to 16,000 unit / mL, and subsequently, an active unit of hyaluronidase in a mixed formulation with an antibody-drug conjugate is 1,000 to 16,000 unit / mL, preferably 1,500 to 12,000 unit / mL, more preferably 2,000 to 12,000 unit / mL, and most preferably 4,000 to 16,000 unit / mL.

[0231]

[0232] The ADCs used in the embodiments of the present invention are ALT-P7 developed by Alteogen and Kadcyla developed by Roche. ALT-P7 is an ADC in which MMAE is conjugated to the antibody trastuzumab as a drug using a cathepsin-cleaving linker, and Kadcyla is an ADC in which mertansine DM1 is conjugated to trastuzumab as a drug using an amino / thiol dual-reactive linker (non-cleaving type). However, the effects according to the present invention can be applied not only to ADCs associated with trastuzumab but also to all types of ADCs described above. Here, MMAE and mertansine DM1 were selected for experiments because they are substances that inhibit the development of intracellular microtubules and may exhibit significant local toxicity.

[0233]

[0234] Effects of the invention

[0235] A subcutaneous injection formulation containing an antibody-drug conjugate and hyaluronidase according to the present invention can enhance the safety of an antibody-drug conjugate that exhibits an efficient anticancer therapeutic effect.

[0236]

[0237] Figure 1 presents the results of a pharmacokinetic study of an anti-Her2 monoclonal antibody in rats.

[0238] Figure 1(A) presents the PK profile, and Figure 1(B) presents each factor of the pharmacokinetic study results. Here, A-1 is the group administered Herceptin subcutaneously mixed with hyaluronidase (Herceptin Hylecta, Roche), and A-2 is the group administered Trastuzumab mixed with the hyaluronidase variant ALT-B4 (Alteogen). It was confirmed that the pharmacokinetics of the Herceptin subcutaneously administered group and the Trastuzumab mixed with ALT-B4 were similar in rats.

[0239] Figure 2 presents the results of a pharmacokinetic study of an anti-Her2 monoclonal antibody in minipigs. Figure 2(A) presents the PK profile, and Figure 2(B) presents each factor of the pharmacokinetic study results. Here, B-1 is the group administered Herceptin subcutaneously mixed with hyaluronidase (Herceptin Hylecta, Roche), and B-2 is the group administered trastuzumab mixed with the hyaluronidase variant ALT-B4 (Alteogen). Similar to rats, it was confirmed that the pharmacokinetics of the Herceptin subcutaneously injected group and the trastuzumab mixed with ALT-B4 were similar in minipigs.

[0240] Figure 3 presents the results of a pharmacokinetic study of the anti-Her2 monoclonal antibody-drug (MMAE) conjugate ALT-P7 in monkeys. Figure 3(A) presents the PK profile, and Figure 3(B) presents each factor of the pharmacokinetic study results. Here, C-ADC represents the result of measuring the ADC of ALT-P7, C-Total Ab represents the result of measuring the Total Ab of ALT-P7, and C-MMAE represents the result of measuring MMAE, the drug of ALT-P7. As with other ADCs, a decrease in blood Cmax, AUC, and half-life of the ADC relative to the Total Ab was confirmed.

[0241] Figure 4 presents the results of the first pharmacokinetic study of the anti-Her2 monoclonal antibody-drug (MMAE) conjugate in minipigs.

[0242] Figure 4(A) presents the PK profile for the ADC, Figure 4(B) presents the factors of the pharmacokinetic test results for the ADC, Figure 4(C) presents the PK profile for Total Ab, Figure 4(D) presents the factors of the pharmacokinetic test results for Total Ab, Figure 4(E) presents the PK profile for the drug MMAE, and Figure 4(F) presents the factors of the pharmacokinetic test results for MMAE. Here, D-1 is the intravenous administration group of ALT-P7, D-2 is the subcutaneous administration group of ALT-P7 in the inguinal region, and D-3 is the mixed subcutaneous administration group of ALT-P7 and ALT-B4 in the inguinal region. The AUC of the subcutaneous administration group compared to the intravenous administration group was at the 100% level, and the pharmacokinetics of the administration group mixed with ALT-B4 and the group not mixed were similar. This suggests that local toxicity and hematotoxicity are evaluated under similar conditions of in vivo drug exposure.

[0243] Figure 5 presents the results of the second pharmacokinetic study of the anti-Her2 monoclonal antibody-drug (MMAE) conjugate in minipigs. Figure 5(A) presents the PK profile for the ADC, Figure 5(B) presents the factors of the pharmacokinetic study results for the ADC, Figure 5(C) presents the PK profile for Total Ab, Figure 5(D) presents the factors of the pharmacokinetic study results for Total Ab, Figure 5(E) presents the PK profile for the drug MMAE, and Figure 5(F) presents the factors of the pharmacokinetic study results for MMAE. Here, E-1 is the intravenous administration group of ALT-P7, E-2 is the mixed inguinal subcutaneous administration group of ALT-P7 and ALT-B4 (2,000 unit / mL), E-3 is the mixed inguinal subcutaneous administration group of ALT-P7 and ALT-B4 (6,000 unit / mL), and E-4 is the sequential inguinal subcutaneous administration group of ALT-B4 (2,000 unit / mL) and ALT-P7. Compared to the intravenous administration group using the high-concentration (52 mg / mL) sample, the ADC AUC of the subcutaneous administration groups was analyzed to be at the 90% level for all groups, without any difference depending on the administration method. No differences in pharmacokinetic parameters were observed among the subcutaneous administration groups.

[0244] Figure 6 presents the results of the third pharmacokinetic study in minipigs of the anti-Her2 monoclonal antibody-drug (DM1) conjugate and the anti-Her2 monoclonal antibody-drug (MMAE). Figure 6(A) presents the PK profile for the ADC, Figure 6(B) presents the factors of the pharmacokinetic study results for the ADC, Figure 6(C) presents the PK profile for Total Ab, Figure 6(D) presents the factors of the pharmacokinetic study results for Total Ab, Figure 6(E) presents the PK profile for the drugs DM1 and MMAE, and Figure 6(F) presents the factors of the pharmacokinetic study results for DM1 and MMAE. Here, F-1 is the intravenous administration group of Kadcyla 4 mg / kg, F-2 is the mixed inguinal subcutaneous administration group of Kadcyla 4 mg / kg and ALT-B4 (2,000 unit / mL), F-3 is the mixed inguinal subcutaneous administration group of Kadcyla 4 mg / kg and ALT-B4 (6,000 unit / mL), and F-4 is the mixed inguinal subcutaneous administration group of ALT-P7 4 mg / kg and ALT-B4 (6,000 unit / mL). Pharmacokinetics were analyzed by administering at a drug concentration of 20 mg / mL. For Kadcyla, the AUC of the ADC was approximately 40% when administered subcutaneously compared to intravenous administration. ALT-P7 was analyzed to be approximately 20% higher compared to the AUCs in Figures 4d and 5b. A tendency for the AUC to increase slightly as the ALT-B4 units per mg of ALT-P7 increased was confirmed.

[0245] Figure 7 presents the results of the fourth pharmacokinetic study of the anti-Her2 monoclonal antibody-drug (MMAE) in minipigs. Figure 7(A) presents the PK profile for the ADC, Figure 7(B) presents the factors of the pharmacokinetic study results for the ADC, Figure 7(C) presents the PK profile for Total Ab, Figure 7(D) presents the factors of the pharmacokinetic study results for Total Ab, Figure 7(E) presents the PK profile for the drug MMAE, and Figure 6(F) presents the factors of the pharmacokinetic study results for MMAE. Here, G-1 is an inguinal subcutaneous administration group in which ALT-B4 was administered first at 3,000 unit / mL followed by sequential administration of ALT-P7 4 mg / kg, and G-2 is an inguinal subcutaneous administration group in which ALT-B4 was administered first at 3,000 unit / mL followed by sequential administration of a mixture of ALT-P7 4 mg / kg and ALT-B4 (2,000 unit / mL). G-3 is an inguinal subcutaneous administration group in which ALT-B4 was administered first at 3,000 unit / mL followed by sequential administration of a mixture of ALT-P7 4 mg / kg and ALT-B4 (4,000 unit / mL). ADC analysis results confirmed that both Cmax and AUC increased proportionally as the units of ALT-B4 per mg of ALT-P7 increased. In particular, a clear reduction in Tmax was observed in G-2 and G-3.

[0246] Figure 8 presents the results of the 5th pharmacokinetic study of anti-Her2 monoclonal antibody-drug (MMAE) dorsal administration in minipigs. Figure 8(A) presents the PK profile for the ADC, Figure 8(B) presents the factors of the pharmacokinetic study results for the ADC, Figure 8(C) presents the PK profile for Total Ab, Figure 8(D) presents the factors of the pharmacokinetic study results for Total Ab, Figure 8(E) presents the PK profile for the drug MMAE, and Figure 8(F) presents the factors of the pharmacokinetic study results for MMAE. Here, H-1 is the dorsal (right) subcutaneous administration group in which ALT-B4 was administered first at 3,000 unit / mL, followed by sequential administration of a mixture of ALT-P7 4 mg / kg and ALT-B4 (6,000 unit / mL). H-2 is the dorsal (right) subcutaneous administration group of a mixture of ALT-P7 4 mg / kg and ALT-B4 (6,000 unit / mL). H-3-1 is the dorsal (left / right) subcutaneous administration group of a mixture of ALT-P7 8 mg / kg and ALT-B4 (6,000 unit / mL). H-3-2 is the dorsal (left / right) subcutaneous administration group of a mixture of ALT-P7 4 mg / kg and ALT-B4 (6,000 unit / mL). When the administration site was changed, the PK parameter exhibited first-order kinetics, and it was observed that the concentration at the Cmax level persisted for up to 120 hours. However, when the administration site was changed, each factor of the pharmacokinetic test results did not show a significant difference between groin and back administration.

[0247] Figure 9 presents the results of the 6th pharmacokinetic study of anti-Her2 monoclonal antibody-drug (MMAE) and anti-Her2 monoclonal antibody-drug (DM1) upon dorsal and lateral administration in minipigs. Figure 9(A) presents the PK profile for the ADC, Figure 9(B) presents the factors of the pharmacokinetic study results for the ADC, Figure 9(C) presents the PK profile for Total Ab, Figure 9(D) presents the factors of the pharmacokinetic study results for Total Ab, Figure 9(E) presents the PK profile for the drugs MMAE and DM1, and Figure 9(F) presents the factors of the pharmacokinetic study results for MMAE and DM1. Here, I-1 is the dorsal (left / right) subcutaneous administration group administered ALT-P7 4 mg / kg. I-2 is the dorsal (left / right) subcutaneous administration group of a mixture of ALT-P7 4 mg / kg and ALT-B4 (2,000 unit / mL). I-3 is the dorsal (left / right) subcutaneous administration group of Kadcyla 4 mg / kg. I-4 is the dorsal (left / right) subcutaneous administration group of a mixture of Kadcyla 4 mg / kg and ALT-B4 (6,000 unit / mL). In the case of Kadcyla, an increase in Cmax and AUC was observed when ALT-B4 was administered at 300 units / mg, whereas in the case of ALT-P7, although ALT-B4 was administered at 100 units / mg, the individual factors of the pharmacokinetic study results did not show significant differences.

[0248] Figures 10a and 10b present the correlation between the individual AUC of ALT-P7, an anti-Her2 monoclonal antibody-drug (MMAE) conjugate, and the units of ALT-B4 / ALT-P7 (units per mg) in a minipig inguinal administration pharmacokinetic study, as dot plots and box plots, respectively. The analysis results showed that as the ALT-B4 unit per mg of ALT-P7 increased, a significant increase in the ADC AUC was observed, and the variation in AUC was also observed to decrease. In particular, while the variation in AUC was large at 200 units / mg or less, it was observed to decrease significantly at 200 units / mg or more.

[0249] Figures 10c and 10d present the correlation between the individual AUC of ALT-P7, an anti-Her2 monoclonal antibody-drug (MMAE) conjugate, and the normalized concentration units (units per mg) of ALT-B4 / drug in a minipig inguinal administration pharmacokinetic study, as dot plots and box plots, respectively. The normalized concentration of the drug was calculated by multiplying the antibody-drug conjugate concentration by the drug-to-antibody ratio and the molecular weight of the drug, and then dividing the result by the total molecular weight of the antibody-drug conjugate. The analysis results showed that as the ALT-B4 unit per mg of normalized drug concentration increased, a significant increase in the ADC AUC was observed, and the variation in AUC was also observed to decrease. In particular, while the variation in AUC was large at concentrations below 20,000 units / mg, it was observed to decrease significantly at concentrations above 20,000 units / mg.

[0250] Figure 11a presents the results of general symptom observations regarding erythema, edema, and blisters up to day 10 in pharmacokinetic studies of anti-Her2 monoclonal antibody-drug (MMAE) and anti-Her2 monoclonal antibody-drug (DM1) conjugates in minipigs. The mean scores of each individual per group are presented. Analysis results showed that local toxicity was alleviated as the dose (units / mg) of ALT-B4 increased compared to ALT-P7, and symptoms were reduced by more than three times when the administration site was changed to dorsal-lateral administration (F-4 : H-3-2).

[0251] Figure 11b presents the correlation between the normalized concentration units (units per mg) of ALT-B4 / drug for ALT-P7 and Kadcyla and the results of general symptom observations by individual using a box plot. The analysis confirmed that the higher the normalized concentration units (units per mg) of ALT-B4 / drug, the lower the symptom score. A t-test was performed between the group with normalized concentration units (units per mg) of ALT-B4 / drug less than 20,000 units / mg and the group with greater than 20,000 units / mg, confirming a significant difference with a p-value of less than 0.01.

[0252] Figure 12 presents the results of histopathological observations in pharmacokinetic studies of anti-Her2 monoclonal antibody-drug (MMAE) and anti-Her2 monoclonal antibody-drug (DM1) conjugates in minipigs. Figure 12A presents the results based on the total score of the histopathological observations, and Figure 12B presents the results based on the total score divided by the number of slides per group. The histopathological results also showed that local toxicity was alleviated as the dose (units / mg) of ALT-B4 increased compared to ALT-P7.

[0253] Figure 13 shows the platelet, neutrophill percentage, and aspartate aminotransferase results from the pharmacokinetics study of the anti-Her2 monoclonal antibody-drug (MMAE) conjugate in minipigs.

[0254] Figure 13(A) shows the results of platelet, neutrophill percentage, and aspartate aminotransferase tests on blood collected from E-1, E-2, E-3, and E-4 336 hours after administration. Figure 13(B) shows the results of platelet, neutrophill percentage, and aspartate aminotransferase tests on blood collected from F-4 before administration and on days 4, 7, and 10 after administration. Figure 13(C) shows the results of platelet, neutrophill percentage, and aspartate aminotransferase tests on blood collected from G-1, G-2, and G-3 before administration and on days 3, 7, and 10 after administration. Figure 13(D) shows the results of platelet, neutrophil percentage, and aspartate aminotransferase tests on blood samples collected from H-1, H-2, and H-3-2 before administration and on days 4, 8, and 15 after administration. In Figure 13(A), it was observed that the platelet and neutrophil percentages were lowest in the IV administration group, while they were similar in the SC administration group. Additionally, regarding aspartate aminotransferase, it was highest in the IV administration group, whereas it was significantly lower in the ALT-B4 mixed administration group. Figures 13(B), 13(C), and 13(D) all show the SC administration group, where no significant decrease in platelet and neutrophil percentages was observed, indicating no hematotoxicity, and no significant increase in aspartate aminotransferase levels was observed either.

[0255]

[0256] Specific details for implementing the invention

[0257] The composition of the present invention will be explained in more detail below with reference to the following examples. However, it is obvious to those skilled in the art that the following examples are merely for illustrating the present invention and that the scope of the present invention is not limited to the descriptions in the examples, particularly specific ADCs, specific antibodies, specific linkers, and specific cytotoxic drugs.

[0258]

[0259] Example 1. Pharmacokinetics of Anti-Her2 Monoclonal Antibody in Rats

[0260] Example 1-1. Animal experiment

[0261] Sprague-Dawley rats were used as experimental animals, and four rats were assigned to each group: one receiving Herceptin subcutaneous injection mixed with hyaluronidase (Herceptin Hylecta, Roche) and the other receiving trastuzumab mixed with the hyaluronidase variant ALT-B4 (Alteogen, WO 2020-022791 A1, SEQ ID 99). Each sample was prepared by mixing with a buffer solution and administered intradermally to the left and right lower dorsal areas of each rat, for a total of 100 μL. Trastuzumab was administered intradermally at a dose of 5.7 mg per rat, and hyaluronidase was administered intradermally to achieve an activity of 100 units per rat. Blood was collected at 0 (before administration), 1, 2, 4, 8, 24, 48, 72, 96, and 120 hours, and serum was obtained after centrifugation and used for analysis.

[0262]

[0263] Example 1-2. Enzyme Immunoassay

[0264] The following enzyme immunoassay was performed to analyze trastuzumab in rat serum.

[0265] 100 μL of Human IgG Fc gamma-specific antibody, diluted to a concentration of 5 μg / mL in phosphate buffer, was dispensed into each well of a 96-well plate and incubated at room temperature for 14 to 18 hours. After removing unattached antibodies from each well, 240 μL of phosphate buffer solution containing 1% bovine serum albumin was dispensed and left at room temperature for 2 hours. The plates were washed three times with a wash solution (0.05% Tween 20, phosphate buffer) and the solution was removed. The sample was diluted with phosphate buffer solution containing 1% bovine serum albumin, added to each well, and incubated at room temperature for 2 hours. After washing each well five times with the wash solution, 100 μL of horseradish peroxidase-conjugated antibody (anti-human IgG-HRP, Fab specific) diluted in 0.05% Tween phosphate buffer was dispensed into each well and reacted at room temperature for 1 hour. After washing five times with the wash solution, 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) hydrogen peroxide chromogenic solution was added to each well and reacted in the dark for 30 minutes. The reaction was terminated by adding 100 μL of sulfuric acid solution to each well, and absorbance was measured using a SpectraMax Plus 384, excluding the 650 nm absorbance from the 450 nm absorbance. The quantitative values ​​of each sample were determined through regression analysis after constructing a standard curve for a standard substance including rat donor serum.

[0266]

[0267] Example 2. Pharmacokinetics of Anti-Her2 Monoclonal Antibody in Minipigs

[0268] Example 2-1. Animal Experiment

[0269] Minipigs were used as experimental animals, and 4 animals were assigned to the Herceptin subcutaneous injection group mixed with recombinant hyaluronidase (Herceptin Hylecta, Roche) and 3 animals were assigned to the Trastuzumab group mixed with a hyaluronidase variant (Alteogen). 1 mL of Herceptin subcutaneous injection stock solution and Trastuzumab containing hyaluronidase were administered subcutaneously into the lower abdomen of the minipigs. For each minipig, 120 mg of Herceptin subcutaneous injection stock solution and Trastuzumab were mixed to achieve a hyaluronidase activity of 2,000 units and administered subcutaneously. Blood was collected at 0 (before administration), 1, 2, 7, 24, 48, 72, 96, 168, 240, 336, and 501 hours, and serum was obtained after centrifugation and used for analysis.

[0270]

[0271] Example 2-2. Enzyme Immunoassay

[0272] The following enzyme immunoassay was performed to analyze trastuzumab in minipig serum.

[0273] 100 μL of Human IgG Fc gamma-specific antibody, diluted to a concentration of 5 μg / mL in phosphate buffer, was dispensed into each well of a 96-well plate and incubated at room temperature for 14 to 18 hours. After removing any unattached antibodies remaining in each well, 240 μL of phosphate buffer solution containing 1% bovine serum albumin was dispensed and left at room temperature for 2 hours. The plates were washed three times with a wash solution (0.05% Tween 20, phosphate buffer) and the solution was removed. The sample was diluted with phosphate buffer solution containing 1% bovine serum albumin, added to each well, and incubated at room temperature for 2 hours. After washing each well five times with the wash solution, 100 μL of horseradish peroxidase-conjugated antibody (anti-human IgG-HRP, Fab specific) diluted in 0.05% Tween phosphate buffer was dispensed into each well and incubated at room temperature for 1 hour. After washing five times with the wash solution, 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) hydrogen peroxide chromogenic solution was added to each well and incubated in the dark for 30 minutes. The reaction was terminated by adding 100 μL of sulfuric acid solution to each well, and absorbance was measured at 450 nm using a SpectraMax Plus 384. The quantitative values ​​of each sample were determined by regression analysis after constructing a standard curve for a standard substance including minipig donor serum.

[0274]

[0275] Example 3. Pharmacokinetics of Anti-Her2 Monoclonal Antibody-Drug (MMAE) Conjugate in Monkeys

[0276] Example 3-1. Animal Test

[0277] Cynomolgus monkeys were used as experimental animals, and three female monkeys were assigned to a single concentration of the antibody-drug conjugate ALT-P7 (Alteogen).

[0278] ALT-P7 is an antibody-drug conjugate in which MMAE is linked as a payload to an anti-Her2 antibody having the heavy and light chain amino acid sequences of SEQ ID NO. 7 and SEQ ID NO. 8 via an MC-vc-PAB linker.

[0279]

[0280] ALT-P7 was administered at a dose of 10 mg / kg via a single intravenous method at a rate of 1 mL / kg. Blood samples were collected at 1, 3, 6, 12, 24, 96, 168, 336, 504, and 672 hours after administration. The collected blood was centrifuged to obtain serum, which was analyzed for ALT-P7 antibody-drug conjugate (ADC) and total antibody forms. Additionally, plasma was used for drug (MMAE) analysis.

[0281]

[0282] Example 3-2. Enzymatic immunoassay of anti-Her2 monoclonal antibody-drug (MMAE) conjugate

[0283] Human Her2 / ErbB proteins were reacted on MSD (Meso Scale Discovery) high binding plates. After the reaction was complete, the plates were blocked using SuperBlock and washed. Diluted samples were added to the plates and reacted, followed by washing. Anti-MMAE specific antibodies were added to each well and reacted. After the reaction was complete and the plates were washed, MSD-Sulfo-TAG™ conjugated goat anti-mouse antibodies were added and reacted. Finally, after the reaction was complete and the plates were washed, 2x Read buffer T was added to the plates and analyzed within 20 minutes.

[0284]

[0285] Example 3-3. Enzyme immunoassay of anti-Her2 monoclonal antibody

[0286] Human Her2 / ErbB proteins were reacted into each well of a High binding plate (Meso Scale Discovery). After the reaction was complete, blocking was performed using SuperBlock blocking buffer (ThermoFisher), and each well was washed. Diluted samples were added to each well, reacted, and then washed. Anti-human IgG was added to each well and reacted. After the reaction was complete and each well was washed, MSD-Sulfo-TAG™-SA (Meso Scale Discovery) was added and reacted. After the reaction was complete and each well was washed, 2x Read buffer T (Meso Scale Discovery) was finally added to each well, and analysis was performed within 20 minutes.

[0287]

[0288] Example 3-4. Liquid Chromatography-Mass Spectrometry (LC / MS / MS) Method for Drug (MMAE)

[0289] Analysis was performed using MMAE as the standard and MMAE-d8 as the internal standard. 25 μL of the internal standard solution was mixed with 100 μL of monkey plasma. A solution of water and ammonium hydroxide mixed at a 100:1 volume ratio was added and thoroughly mixed, after which SLE+ (Supported Liquid Extraction, Biotage) solution was added to each well. 1 mL of methyl tertiary butyl ether was added to each well to elute the MMAE. After evaporating the eluent, it was redissolved in 40% methanol and injected into the LS / MS / MS. The liquid chromatography system used a C4 column at a flow rate of 0.8 mL / min and a gradient condition consisting of 5% methanol and 90% methanol containing 10 mM ammonium formate. Standard and internal standards were detected using a SCIEX API5000 LC / MS / MS system equipped with an ESI ionization source in cation mode. The standard was monitored using multiple reaction monitoring (MRM) transitions from m / z (mass-to-charge ratio)

[0719] + to

[0152] +, and the internal standard was monitored from m / z

[0727] + to

[0152] +.

[0290]

[0291] Example 4. Pharmacokinetics of Anti-Her2 Monoclonal Antibody-Drug (MMAE) Conjugate in Minipigs

[0292] Example 4-1. Primary animal test

[0293] Sus scrofa / Yucatan minipigs were used as experimental animals, and three male minipigs were assigned to each of the intravenous administration group, the subcutaneous administration group of the anti-Her2 monoclonal antibody-drug (MMAE) conjugate ALT-P7 (Alteogen), and the subcutaneous administration group of ALT-P7 mixed with the hyaluronidase variant ALT-B4 (Alteogen). ALT-P7 was administered at a dose of 4 mg / kg via single intravenous administration and 0.4 mL / kg via inguinal subcutaneous injection. For the ALT-P7 subcutaneous administration group mixed with ALT-B4, 0.4 mL / kg was administered by mixing ALT-P7 at 10 mg / mL with ALT-B4 at 2,000 unit / mL to achieve a total ALT-P7 dose of 4 mg / kg. Blood samples were collected at approximately 1, 6, 24, 48, 72, 96, 168, and 336 hours after predose (0 hr) administration. Serum was obtained after centrifuging the collected blood, and the ADC and Total Antibody forms of ALT-P7 were used for analysis. Additionally, plasma was obtained using an anticoagulant (K2 EDTA) and used for MMAE analysis. General symptoms in the experimental animals were observed and measured from the time of administration until the last blood collection. Histopathological examination of the administration site was performed after the last blood collection.

[0294]

[0295] Example 4-2. Secondary animal test

[0296] Sus scrofa / Yucatan minipigs were used as experimental animals, and three male minipigs were assigned to each of the intravenous administration group (Group 1), the subcutaneous administration group of ALT-P7 mixed with ALT-B4 (Groups 2 and 3), and the sequential administration group of ALT-B4 and ALT-P7 (Group 4). ALT-P7 was administered at a dose of 4 mg / kg via a single intravenous method at a rate of 0.08 mL / kg (Group 1). For the subcutaneous administration group of ALT-P7 mixed with ALT-B4, 52 mg / mL of ALT-P7 was mixed with ALT-B4 at doses of 2,000 unit / mL and 6,000 unit / mL, respectively, to achieve a total ALT-P7 dose of 4 mg / kg, and administered subcutaneously to the inguinal region at a rate of 0.08 mL / kg (Groups 2 and 3). The group administering ALT-B4 and ALT-P7 sequentially received ALT-B4 at 2,000 unit / mL first, followed by inguinal administration of ALT-P7 at 4 mg / kg (Group 4). Predose was performed (0 hr), and blood samples were collected at approximately 1, 6, 24, 48, 72, 96, 168, 192, 240, and 336 hours after administration. The collected blood was centrifuged to obtain serum, which was analyzed for the ALT-P7 ADC and Total Antibody forms. Plasma collected additionally was used for MMAE analysis. General symptoms in the experimental animals were observed and measured from the time of administration until the final blood collection. At the final collection (336 hours), blood was collected for clinical pathological examination, and hematological and blood biochemical tests were performed. Histopathological examinations of the administration site and the site of local toxicity were performed after the final blood collection.

[0297]

[0298] Example 4-3. Enzymatic immunoassay of anti-Her2 monoclonal antibody-drug (MMAE) conjugate

[0299] 100 μL of Human IgG Fc gamma-specific antibody, diluted to a concentration of 2.4 μg / mL in phosphate buffer, was dispensed into a 96-well plate and incubated at room temperature for 14 to 18 hours. After removing unattached antibodies from each well, 240 μL of phosphate buffer solution containing 1% bovine serum albumin was dispensed and left at room temperature for 2 hours. The plates were washed three times with a wash solution (0.05% Tween 20, phosphate buffer) and the solution was removed. The samples were diluted with phosphate buffer solution containing 1% bovine serum albumin and 0.1% Tween 20, added to each well, and incubated at room temperature for 2 hours. After washing each well 5 times with the washing solution, mouse anti-MMAE antibody was diluted in phosphate buffer solution containing 1% bovine serum albumin and 0.1% Tween 20, added to each well, and reacted at room temperature for 2 hours. After washing each well 5 times with the washing solution, horseradish peroxidase-conjugated anti-mouse IgG (H+L) was diluted in phosphate buffer solution containing 1% bovine serum albumin and 0.05% Tween, 100 μL was dispensed into each well, and reacted at room temperature for 1 hour. After washing 5 times with the washing solution, 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) hydrogen peroxide chromogenic solution was added to each well, and the reaction was carried out in the dark for 30 minutes. The reaction was terminated by adding 100 μL of sulfuric acid solution to each well, and the absorbance was measured using a SpectraMax M5, excluding the absorbance at 650 nm from the absorbance at 450 nm. The quantitative values ​​of each sample were obtained through regression analysis after constructing a standard curve for standard substances including minipig donor serum.

[0300]

[0301] Example 4-4. Enzyme immunoassay of anti-Her2 monoclonal antibody

[0302] 100 μL of Human IgG Fc gamma-specific antibody, diluted to a concentration of 2.4 μg / mL in phosphate buffer, was dispensed into a 96-well plate and incubated at room temperature for 14 to 18 hours. After removing any antibodies remaining unattached to the wells, 240 μL of phosphate buffer solution containing 1% bovine serum albumin was dispensed and left at room temperature for 2 hours. The plate was washed three times with a wash solution (0.05% Tween 20, phosphate buffer) and the solution was removed. The sample was diluted with phosphate buffer solution containing 1% bovine serum albumin and 0.1% Tween 20, added to a 96-well plate, and incubated at room temperature for 2 hours. After washing the 96-well plate five times with washing solution, horseradish peroxidase-conjugated antibody (anti-human IgG-HRP, Fab specific) was diluted in phosphate buffer containing 1% bovine serum albumin and 0.05% Tween, and 100 μL was dispensed into each well of the 96-well plate and incubated at room temperature for 1 hour. After washing five times with washing solution, 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) hydrogen peroxide chromogenic solution was added to each well, and the reaction was incubated in the dark for 30 minutes. The reaction was terminated by adding 100 μL of sulfuric acid solution to each well, and absorbance was measured using a SpectraMax M5, excluding the absorbance at 650 nm from the absorbance at 450 nm. The quantitative values ​​of each sample were determined through regression analysis after constructing a standard curve for the standard substances.

[0303]

[0304] Examples 4-5. Liquid Chromatography-Mass Spectrometry (LC / MS / MS) Method for Drug (MMAE)

[0305] Analysis was performed using MMAE as the standard and MMAE-d8 as the internal standard. 10 μL of the internal standard solution was mixed with 100 μL of minipig plasma. Additionally, 900 μL of methyl-tert-butyl ether was added, mixed, and centrifuged; the supernatant was stored in a freezer. The supernatant was transferred to a tube and dried using nitrogen gas. It was redissolved in 50% acetonitrile solution, centrifuged, and injected into an LC / MS / MS system. The liquid chromatography system utilized a phenylhexyl column at a flow rate of 0.3 mL / min and a gradient condition consisting of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. The standard and internal standard were detected using a SCIEX API6000 LC / MS / MS system equipped with an ESI ionization source in cation mode. The standard substance was monitored using the MRM (multiple reaction monitoring) transition from m / z (mass to charge ratio) [718.4]+ to [686.5]+, and the internal standard substance from m / z [726.4]+ to [694.4]+.

[0306]

[0307] Example 4-6. Method for observing general symptoms

[0308] Symptoms, including mortality, morbidity, and changes in appearance and behavior, were observed and recorded once a day during the pre-administration period and twice a day during the administration period, and the date, time, and duration were recorded. Based on the records, the symptoms were scored from 0 to 5 points according to their type and severity.

[0309]

[0310] Example 4-7. Hematological test method

[0311] 336 hours after drug administration, at least 0.5 mL of blood was collected from all animals, placed in a blood collection tube containing an anticoagulant (EDTA-K2), and items such as Total leukocyte count (WBC), Total red blood cell count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Reticulocyte count, WBC differential count (Absolute (#) and relative (%) differential counts: include neutrophils (NEU), eosinophils (EOS), basophils (BAS), monocytes (MON), lymphocytes (LYM), large unstained cells (LUC)), and Absolute (RETA) and relative (RET%) counts were measured. Measurements were performed using an automated hematology analyzer (ADVIA2120i hematology analyzer, Siemens, USA).

[0312]

[0313] Examples 4-8. Blood biochemical test method

[0314] After 336 hours of administration, at least 1.5 mL of blood was collected from all animals, placed in an anticoagulant-free tube, left at room temperature for at least 30 minutes, and then centrifuged (approx. 3,000 rpm, 10 minutes, room temperature) to separate the serum. Using isolated serum, parameters such as Glucose (GLU), Alanine aminotransferase (ALT), Blood urea nitrogen (BUN), Total bilirubin (TBIL), Creatinine (CREA), Alkaline phosphatase (ALP), Total protein (TP), Gamma glutamyl transpeptidase (GGT), Albumin (ALB), Creatine phosphokinase (CK), Albumin / globulin ratio (A / G), Calcium (Ca), Total cholesterol (TCHO), Inorganic phosphorus (IP), Triglyceride (TG), Sodium (Na), C-reactive Protein (CRP), Potassium (K), Aspartate aminotransferase (AST), and Chloride (Cl) were measured. Measurements were performed using an automated clinical chemistry analyzer (TBA 120FR chemistry analyzer, Toshiba Co., Japan).

[0315]

[0316] Examples 4-9 Histopathological examination

[0317] Slides of the administration site tissue were prepared, and histopathological examinations were performed after hematoxylin & eosin staining. Histopathological findings observed in each individual from the collected tissues were recorded.

[0318] Histopathological severity was classified into four levels—'very mild,' 'mild,' 'slightly severe,' and 'severe'—and quantified as a score ranging from 1 to 4 points for each level. The histopathological degree of local symptoms by group was evaluated by summing the values ​​calculated by multiplying the severity score for each lesion by the number of tissues in which the lesion was observed.

[0319]

[0320] Example 5. Pharmacokinetic study of anti-Her2 monoclonal antibody-drug (DM1) conjugate and anti-Her2 monoclonal antibody-drug (MMAE) in minipigs

[0321]

[0322] Example 5-1. Third animal test

[0323] Sus scrofa / Yucatan minipigs were used as experimental animals, and three male minipigs were assigned to each of the Kadcyla intravenous administration group (Group 1), the Kadcyla subcutaneous administration group mixed with ALT-B4 (Groups 2 and 3), and the ALT-B4 and ALT-P7 administration group (Group 4). Kadcyla was administered at a single intravenous dose of 0.2 mL / kg with a concentration of 4 mg / kg (Group 1). For the Kadcyla subcutaneous administration group mixed with ALT-B4, 20 mg / mL of Kadcyla was mixed with ALT-B4 at concentrations of 2,000 unit / mL and 6,000 unit / mL, respectively, and administered subcutaneously to the inguinal region at a concentration of 0.2 mL / kg to achieve a Kadcyla concentration of 4 mg / kg (Groups 2 and 3). The ALT-P7 subcutaneous injection group mixed with ALT-B4 was administered at a dose of 0.2 mL / kg, in which ALT-B4 was mixed with 20 mg / mL of ALT-P7 at 6,000 unit / mL to achieve a total ALT-P7 concentration of 4 mg / kg (Group 4). Blood samples were collected at predose (0 hr), and at approximately 0.67, 3, 6, 24, 72, 96, 120, 168, 192, and 240 hours after administration. The collected blood was centrifuged to obtain serum, which was analyzed in the form of Kadcyla and ALT-P7 as ADCs and Total Antibodies.

[0324]

[0325] Example 5-2. Enzymatic immunoassay of anti-Her2 monoclonal antibody-drug (DM1) conjugate

[0326] 100 μL of Human IgG Fc gamma-specific antibody, diluted to a concentration of 2.4 μg / mL in phosphate buffer, was dispensed into a 96-well plate and incubated at room temperature for 14 to 18 hours. After removing any antibodies remaining unattached to the wells, 240 μL of phosphate buffer solution containing 1% bovine serum albumin was dispensed and left at room temperature for 2 hours. The plate was washed three times with a wash solution (0.05% Tween 20, phosphate buffer) and the solution was removed. The sample was diluted with phosphate buffer solution containing 1% bovine serum albumin and 0.1% Tween 20, added to a 96-well plate, and incubated at room temperature for 2 hours. After washing the 96-well plate three times with washing solution, horseradish peroxidase-conjugated antibody (anti-DM1-HRP) was diluted in phosphate buffer containing 1% bovine serum albumin and 0.05% Tween, and 100 μL was dispensed into each well of the 96-well plate and incubated at room temperature for 1 hour. After washing three times with washing solution, 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) hydrogen peroxide chromogenic solution was added to each well, and the reaction was carried out in the dark for 30 minutes. The reaction was terminated by adding 100 μL of sulfuric acid solution to each well, and absorbance was measured using a SpectraMax M5, excluding the absorbance at 650 nm from the absorbance at 450 nm. The quantitative values ​​of each sample were determined through regression analysis after constructing a standard curve for the standard substances.

[0327]

[0328] Example 5-3. Enzymatic immunoassay of anti-Her2 monoclonal antibody-drug (MMAE) conjugate

[0329] 100 μL of Human IgG Fc gamma-specific antibody, diluted to a concentration of 2.4 μg / mL in phosphate buffer, was dispensed into a 96-well plate and incubated at room temperature for 14 to 18 hours. After removing any antibodies remaining unattached to the wells, 240 μL of phosphate buffer solution containing 1% bovine serum albumin was dispensed and left at room temperature for 2 hours. The plate was washed three times with a wash solution (0.05% Tween 20, phosphate buffer) and the solution was removed. The sample was diluted with phosphate buffer solution containing 1% bovine serum albumin and 0.1% Tween 20, added to a 96-well plate, and incubated at room temperature for 2 hours. After washing the 96-well plate three times with washing solution, horseradish peroxidase-conjugated antibody (anti-MMAE-HRP) was diluted in phosphate buffer containing 1% bovine serum albumin and 0.05% Tween, dispensed at 100 μL aliquots into each well, and incubated at room temperature for 1 hour. After washing three times with washing solution, 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) hydrogen peroxide chromogenic solution was added to each well, and the reaction was incubated in the dark for 30 minutes. The reaction was terminated by adding 100 μL of sulfuric acid solution to each well, and absorbance was measured using a SpectraMax M5, excluding the 650 nm absorbance from the 450 nm absorbance. The quantitative values ​​of each sample were determined through regression analysis after constructing a standard curve for the standard substances.

[0330]

[0331] Example 5-4. Enzyme immunoassay of anti-Her2 monoclonal antibody

[0332] 100 μL of Human IgG Fc gamma-specific antibody, diluted to a concentration of 2.4 μg / mL in phosphate buffer, was dispensed into a 96-well plate and incubated at room temperature for 14 to 18 hours. After removing any antibodies remaining unattached to the wells, 240 μL of phosphate buffer solution containing 1% bovine serum albumin was dispensed and left at room temperature for 2 hours. The plate was washed three times with a wash solution (0.05% Tween 20, phosphate buffer) and the solution was removed. The sample was diluted with phosphate buffer solution containing 1% bovine serum albumin and 0.1% Tween 20, added to a 96-well plate, and incubated at room temperature for 2 hours. After washing the 96-well plate five times with washing solution, horseradish peroxidase-conjugated antibody (anti-human IgG-HRP, Fab specific) was diluted in phosphate buffer containing 1% bovine serum albumin and 0.05% Tween, and 100 μL was dispensed into each well of the 96-well plate and incubated at room temperature for 1 hour. After washing five times with washing solution, 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) hydrogen peroxide chromogenic solution was added to each well, and the reaction was incubated in the dark for 30 minutes. The reaction was terminated by adding 100 μL of sulfuric acid solution to each well, and absorbance was measured using a SpectraMax M5, excluding the absorbance at 650 nm from the absorbance at 450 nm. The quantitative values ​​of each sample were determined through regression analysis after constructing a standard curve for the standard substances.

[0333]

[0334] Example 5-5. Liquid Chromatography-Mass Spectrometry (LC / MS / MS) Method of Drug (DM1)

[0335] Analysis was performed using DM1 (Mertansine) as the standard and Mertansine-13CD3 as the internal standard. 10 μL of the internal standard solution was mixed with 150 μL of minipig plasma. Additionally, 30 μL of Tris(2-carboxyethyl)phosphine hydrochloride (hereinafter 'TCEP') was added and mixed, followed by centrifugation and incubation in the dark at 37°C for 30 minutes. 30 μL of N-ethylmaleimide (hereinafter 'NEM') was added and mixed, followed by centrifugation and incubation in the dark at 37°C for 30 minutes. 150 μL of acetonitrile was added and mixed, followed by centrifugation. 325 μL of the supernatant was transferred to a tube, 575 μL of ultrapure water was added and mixed, followed by centrifugation. For solid phase extraction, activation and equilibration were performed using methanol and ultrapure water. 900 μL of a solution prepared by mixing 325 μL of the supernatant with 575 μL of ultrapure water was loaded into SPE wells for solid phase extraction. After washing with ultrapure water, 70 μL of MeOH containing 2% FA was added for extraction, and the supernatant was injected into the LC / MS / MS system. The liquid chromatography system used a C18 column at a flow rate of 0.4 mL / min and an isocratic condition consisting of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. Standards and internal standards were detected using a SCIEX TQ 7500+ LC / MS / MS system equipped with an ESI ionization source in cation mode. NEM-labeled standard substances have a mass-to-charge ratio (m / z) of [885.4]+ to [569.2]+, and NEM-labeled internal standard substances have a m / z of [889.5]+ to

[254] .Monitoring was performed using MRM (multiple reaction monitoring) transition to 1]+.

[0336]

[0337] Examples 5-6. Liquid Chromatography-Mass Spectrometry (LC / MS / MS) Method for Drug (MMAE)

[0338] Analysis was performed using MMAE as the standard and MMAE-d8 as the internal standard. 10 μL of the internal standard solution was mixed with 100 μL of minipig plasma. Additionally, 900 μL of methyl-tert-butyl ether was added, mixed, and centrifuged; the supernatant was stored in a freezer. The supernatant was transferred to a tube and dried using nitrogen gas. It was redissolved in 50% acetonitrile solution, centrifuged, and injected into an LC / MS / MS system. The liquid chromatography system utilized a phenylhexyl column at a flow rate of 0.3 mL / min and a gradient condition consisting of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. The standard and internal standard were detected using a SCIEX API6000 LC / MS / MS system equipped with an ESI ionization source in cation mode. The standard substance was monitored using the MRM (multiple reaction monitoring) transition from m / z (mass to charge ratio) [718.4]+ to [686.5]+, and the internal standard substance from m / z [726.4]+ to [694.4]+.

[0339]

[0340] Example 5-7. Method for observing general symptoms

[0341] During the administration period, symptoms including mortality, morbidity, and changes in appearance and behavior were observed and recorded twice daily, and the date, time, and duration were recorded. Based on the records, the symptoms were scored from 0 to 5 points according to their type and severity.

[0342]

[0343] Examples 5-8. Hematological test methods

[0344] A portion of blood collected before the administration of the test substance and on days 4, 7, and 10 after administration was injected into a CBC bottle containing an anticoagulant (EDTA-2K), and then an automated blood analyzer (ADVIA 2120, SIEMENS, USA) was used to test for items such as Red blood cell (RBC), Platelet count (PLT), Hematocrit (HCT), White blood cell (WBC), Red cell distribution width (RDW), Hb concentration distribution width (HDW), Hemoglobin concentration (HGB), Neutrophil (NEU), Mean corpuscular volume (MCV), Lymphocyte (LYM), Mean cell hemoglobin (MCH), Monocyte (MONO), Mean cell hemoglobin concentration (MCHC), Eosinophil (EOS), Mean platelet volume (MPV), Basophil (BASO), Reticulocytes (RET), and Large unstained cells (LUC).

[0345]

[0346] Examples 5-9. Blood biochemical test method

[0347] A portion of blood collected before the administration of the test substance and on days 4, 7, and 10 after administration was injected into a vacutainer tube containing a clot activator and left at room temperature for approximately 15-20 minutes to coagulate. The serum obtained by centrifuging at 3,000 rpm for 10 minutes was then used to measure Aspartate aminotransferase (AST), Triglyceride (TG), Alanine aminotransferase (ALT), Total protein (TP), Alkaline phosphatase (ALP), Albumin (ALB), Gamma-glutamyl transferase (GGT), Albumin / Globulin ratio (A / G ratio), Creatine phosphokinase (CPK), Blood urea nitrogen (BUN), Total bilirubin (TBIL), Creatinine (CRE), Glucose (GLU), Inorganic phosphorus (IP), Total cholesterol (TCHO), and Calcium (Ca). Sodium (Na), Potassium (K), and Chloride (Cl) were measured using an automatic electrolyte analyzer (AVL 9180 Roche, Switzerland).

[0348]

[0349] Examples 5-10 Histopathological examination

[0350] Slides of the administration site tissue were prepared, and histopathological examinations were performed after hematoxylin & eosin staining. Histopathological findings observed in each individual from the collected tissues were recorded.

[0351] Histopathological severity was classified into four levels—'very mild,' 'mild,' 'slightly severe,' and 'severe'—and quantified as a score ranging from 1 to 4 points for each level. The histopathological degree of local symptoms by group was evaluated by summing the values ​​calculated by multiplying the severity score for each lesion by the number of tissues in which the lesion was observed.

[0352]

[0353] Example 6. Pharmacokinetics Study of Sequential Administration of Anti-Her2 Monoclonal Antibody-Drug (MMAE) in Minipigs

[0354] Example 6-1. 4th animal experiment

[0355] Sus scrofa / Yucatan minipigs were used as experimental animals, and three male minipigs were assigned to each group: a group receiving sequential subcutaneous administration of ALT-B4 and ALT-P7 into the inguinal region (Group 1), and groups receiving sequential subcutaneous administration of ALT-B4 and ALT-P7 mixed with ALT-B4 into the inguinal region (Groups 2 and 3). In the group receiving sequential administration of ALT-B4 and ALT-P7, ALT-B4 was administered first at 3,000 unit / mL, followed by ALT-P7 at 4 mg / kg (Group 1). In the group receiving sequential subcutaneous administration of ALT-B4 and ALT-P7 mixed with ALT-B4, ALT-B4 is administered first at 3,000 unit / mL, followed by the administration of 0.2 mL / kg of ALT-P7 mixed with 2,000 unit / mL and 4,000 unit / mL of ALT-B4 to achieve a concentration of 4 mg / kg (Groups 2 and 3). Blood samples are collected at predose (0 hr) and at approximately 0.67, 3, 6, 24, 48, 72, 120, 168, 192, and 240 hours after administration. The collected blood is centrifuged to obtain serum, which is analyzed for ALT-P7 in both ADC and Total Antibody forms. Additionally, plasma is collected for MMAE analysis. Observation and measurement of general symptoms in experimental animals are performed from the time of drug administration until the final blood collection.

[0356]

[0357] Example 6-2. Enzymatic immunoassay of anti-Her2 monoclonal antibody-drug (MMAE) conjugate

[0358] Dilute the Human IgG Fc gamma-specific antibody in phosphate buffer to a concentration of 2.4 μg / mL, dispense 100 μL into a 96-well plate, and incubate at room temperature for 14 to 18 hours. After removing any antibodies remaining unattached to the wells, dispense 240 μL of phosphate buffer solution containing 1% bovine serum albumin and leave at room temperature for 2 hours. Wash three times with a wash solution (0.05% Tween 20, phosphate buffer) and remove the solution. Dilute the sample with phosphate buffer solution containing 1% bovine serum albumin and 0.1% Tween 20, add it to a 96-well plate, and incubate at room temperature for 2 hours. After washing the 96-well plate three times with washing solution, the horseradish peroxidase-conjugated antibody (anti-MMAE-HRP) is diluted in phosphate buffer containing 1% bovine serum albumin and 0.05% Tween, and 100 μL is dispensed into each well of the 96-well plate and incubated at room temperature for 1 hour. After washing three times with washing solution, 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) hydrogen peroxide chromogenic solution is added to each well, and the reaction is allowed in the dark for 30 minutes. The reaction is terminated by adding 100 μL of sulfuric acid solution to each well, and the absorbance is measured using a SpectraMax M5, excluding the absorbance at 650 nm from the absorbance at 450 nm. The quantitative value of each sample is determined by regression analysis after constructing a standard curve for the standard substance.

[0359]

[0360] Example 6-3. Enzyme immunoassay of anti-Her2 monoclonal antibody

[0361] 100 μL of Human IgG Fc gamma-specific antibody, diluted to a concentration of 2.4 μg / mL in phosphate buffer, was dispensed into a 96-well plate and incubated at room temperature for 14 to 18 hours. After removing any antibodies remaining unattached to the wells, 240 μL of phosphate buffer solution containing 1% bovine serum albumin was dispensed and left at room temperature for 2 hours. The plate was washed three times with a wash solution (0.05% Tween 20, phosphate buffer) and the solution was removed. The sample was diluted with phosphate buffer solution containing 1% bovine serum albumin and 0.1% Tween 20, added to a 96-well plate, and incubated at room temperature for 2 hours. After washing the 96-well plate five times with the washing solution, the horseradish peroxidase-conjugated antibody (anti-human IgG-HRP, Fab specific) is diluted in phosphate buffer containing 1% bovine serum albumin and 0.05% Tween, and 100 μL is dispensed into each well of the 96-well plate and incubated at room temperature for 1 hour. After washing five times with the washing solution, 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) hydrogen peroxide chromogenic solution is added to each well, and the reaction is allowed in the dark for 30 minutes. The reaction is terminated by adding 100 μL of sulfuric acid solution to each well, and the absorbance is measured using a SpectraMax M5, subtracting the 650 nm absorbance from the 450 nm absorbance. The quantitative value of each sample is determined by regression analysis after constructing a standard curve for the standard substance.

[0362]

[0363] Example 6-4. Liquid Chromatography-Mass Spectrometry (LC / MS / MS) Method for Drug (MMAE)

[0364] Analysis is performed using MMAE as the standard and MMAE-d8 as the internal standard. 10 μL of the internal standard solution is mixed with 100 μL of minipig plasma. Additionally, 900 μL of methyl-tert-butyl ether is added, mixed, and centrifuged; the supernatant is stored in a freezer. The supernatant is transferred to a tube and dried using nitrogen gas. It is redissolved in 50% acetonitrile solution, centrifuged, and injected into the LC / MS / MS system. The liquid chromatography system uses a phenylhexyl column at a flow rate of 0.3 mL / min and a gradient condition consisting of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. The standard and internal standard are detected using a SCIEX API6000 LC / MS / MS system equipped with an ESI ionization source in cation mode. Standard substances are monitored using MRM (multiple reaction monitoring) transitions from m / z (mass to charge ratio) [718.4]+ to [686.5]+, and internal standards from m / z [726.4]+ to [694.4]+.

[0365]

[0366] Example 6-5. Method for observing general symptoms

[0367] During the administration period, symptoms including mortality, morbidity, and changes in appearance and behavior were observed and recorded twice daily, and the date, time, and duration were recorded. Based on the records, the symptoms were scored from 0 to 5 points according to their type and severity.

[0368]

[0369] Example 6-6. Hematological test method

[0370] A portion of blood collected before the administration of the test substance and on days 3, 7, and 10 after administration was injected into a CBC bottle containing an anticoagulant (EDTA-2K), and then an automated blood analyzer (ADVIA 2120, SIEMENS, USA) was used to test for items such as Red blood cell (RBC), Platelet count (PLT), Hematocrit (HCT), White blood cell (WBC), Red cell distribution width (RDW), Hb concentration distribution width (HDW), Hemoglobin concentration (HGB), Neutrophil (NEU), Mean corpuscular volume (MCV), Lymphocyte (LYM), Mean cell hemoglobin (MCH), Monocyte (MONO), Mean cell hemoglobin concentration (MCHC), Eosinophil (EOS), Mean platelet volume (MPV), Basophil (BASO), Reticulocytes (RET), and Large unstained cells (LUC).

[0371]

[0372] Examples 6-7. Blood biochemical test method

[0373] A portion of blood collected before the administration of the test substance and on days 3, 7, and 10 after administration was injected into a vacutainer tube containing a clot activator and left at room temperature for approximately 15-20 minutes to coagulate. The serum obtained by centrifuging at 3,000 rpm for 10 minutes was used to measure Aspartate aminotransferase (AST), Triglyceride (TG), Alanine aminotransferase (ALT), Total protein (TP), Alkaline phosphatase (ALP), Albumin (ALB), Gamma-glutamyl transferase (GGT), Albumin / Globulin ratio (A / G ratio), Creatine phosphokinase (CPK), Blood urea nitrogen (BUN), Total bilirubin (TBIL), Creatinine (CRE), Glucose (GLU), Inorganic phosphorus (IP), Total cholesterol (TCHO), and Calcium (Ca). Sodium (Na), Potassium (K), and Chloride (Cl) were measured using an automatic electrolyte analyzer (AVL 9180 Roche, Switzerland).

[0374]

[0375] Examples 6-8 Histopathological examination

[0376] Slides of tissue from the injection site, the site of severe symptoms, and the fixation site were prepared, and histopathological examinations were performed after hematoxylin & eosin staining. Histopathological findings observed in each individual from the collected tissues were recorded.

[0377] Histopathological severity was classified into four levels—'very mild,' 'mild,' 'slightly severe,' and 'severe'—and quantified as a score ranging from 1 to 4 points. For each lesion, the severity score was multiplied by the number of tissues in which the lesion was observed, the values ​​were summed, and then divided by the number of tissues in each group to equalize the results, thereby evaluating the histopathological degree of local symptoms in each group.

[0378]

[0379] Example 7. Pharmacokinetic study of anti-Her2 monoclonal antibody-drug (MMAE) and anti-Her2 monoclonal antibody-drug (DM1) upon dorsal administration in minipigs

[0380] Example 7-1. 5th Animal Test

[0381] Sus scrofa / Yucatan minipigs were used as experimental animals, and three female minipigs were assigned to each group: Group 1, which received sequential subcutaneous administration of ALT-B4 and ALT-P7 mixed with ALT-B4, and Group 2, which received subcutaneous administration of ALT-P7 mixed with ALT-B4. For the group receiving subcutaneous administration of ALT-P7 mixed with ALT-B4 at two separate sites (Group 3), two female minipigs were assigned to Group 3-1 and three female minipigs were assigned to Group 3-2. The group receiving sequential subcutaneous administration of ALT-B4 and ALT-P7 mixed with ALT-B4 first administered ALT-B4 at 3,000 unit / mL to the right dorsal side, and then administered 0.2 mL / kg to the same site after mixing ALT-P7 at 20 mg / mL with ALT-B4 at 6,000 unit / mL to make ALT-P7 4 mg / kg (Group 1). The group receiving subcutaneous administration of ALT-P7 mixed with ALT-B4 first administered ALT-P7 at 20 mg / mL with ALT-B4 at 6,000 unit / mL to make ALT-P7 4 mg / kg to the right dorsal side at 0.2 mL / kg (Group 2). In the group administered subcutaneously with ALT-P7 mixed with ALT-B4 at two divided injection sites, 2 animals were administered 0.2 mL / kg of a mixture of ALT-P7 20 mg / mL and ALT-B4 6,000 unit / mL to make ALT-P7 4 mg / kg, respectively, to the right dorsal side and left dorsal side, resulting in a total dose of 8 mg / mL and a total solution volume of 0.4 mL / kg per animal (Group 3-1). In the group administered subcutaneously with ALT-P7 mixed with ALT-B4 at two divided injection sites, 3 animals were administered 0.2 mL / kg of a mixture of ALT-P7 20 mg / mL and ALT-B4 6,000 unit / mL to make ALT-P7 2 mg / kg.1 mL / kg was administered to the right and left dorsal sides, respectively, resulting in a total dose of 4 mg / mL and a total solution volume of 0.2 mL / kg per animal (Group 3-2). Predose was performed (0 hr), and blood samples were collected at approximately 0.67, 3, 6, 24, 72, 120, 168, 192, 240, and 336 hours after administration. The collected blood was centrifuged to obtain serum, which was analyzed for the ALT-P7 ADC and Total Antibody forms. Additionally, plasma was collected for MMAE analysis. General symptoms in the experimental animals were observed and measured from the time of administration until the last blood collection.

[0382]

[0383] Example 7-2. 6th Animal Test

[0384] Sus scrofa / Yucatan minipigs were used as experimental animals, and three female minipigs were assigned to each of the groups: the group receiving subcutaneous administration of ALT-P7 (Group 1), the group receiving subcutaneous administration of ALT-P7 mixed with ALT-B4 (Group 2), the group receiving subcutaneous administration of Kadcyla (Group 3), and the group receiving subcutaneous administration of Kadcyla mixed with ALT-B4 (Group 4). For the group receiving subcutaneous administration of ALT-P7, 20 mg / mL of ALT-P7 was administered to the right and left dorsal sides at a dose of 0.1 mL / kg to achieve a concentration of 2 mg / kg, with a total dose of 4 mg / mL and a total administration volume of 0.2 mL / kg per animal (Group 1). The group receiving subcutaneous administration of ALT-P7 mixed with ALT-B4 was administered 0.1 mL / kg to the right and left dorsal sides, respectively, by mixing ALT-P7 20 mg / mL with ALT-B4 at 2,000 unit / mL to make ALT-P7 2 mg / kg, with a total dose of 4 mg / mL and a total volume of 0.2 mL / kg per animal (Group 2). The group receiving subcutaneous administration of Kadcyla was administered 0.1 mL / kg to the right and left dorsal sides, respectively, by mixing Kadcyla 20 mg / mL to make Kadcyla 2 mg / kg, with a total dose of 4 mg / mL and a total volume of 0.2 mL / kg per animal (Group 3). In the group administered subcutaneously with Kadcyla mixed with ALT-B4, 20 mg / mL of Kadcyla was mixed with 6,000 unit / mL of ALT-B4 to make Kadcyla 2 mg / kg, and 0.1 mL / kg was administered to the right and left dorsal sides, respectively, resulting in a total dose of 4 mg / mL and a total administration volume of 0.2 mL / kg per animal (Group 4). Predose (0 hr), and blood samples were collected at approximately 0.67, 3, 6, 24, 72, 120, 168, 192, 240, and 336 hours after administration.Serum was obtained after centrifuging the collected blood and analyzed in the form of Kadcyla and ALT-P7 ADCs and Total Antibody. Additionally, plasma was used for MMAE and DM1 analysis. General symptoms in the experimental animals were observed and measured from the time of drug administration until the final blood collection.

[0385]

[0386] Example 7-3. Enzymatic immunoassay of anti-Her2 monoclonal antibody-drug (MMAE) conjugate

[0387] 100 μL of Human IgG Fc gamma-specific antibody, diluted to a concentration of 2.4 μg / mL in phosphate buffer, was dispensed into a 96-well plate and incubated at room temperature for 14 to 18 hours. After removing any antibodies remaining unattached to the wells, 240 μL of phosphate buffer solution containing 1% bovine serum albumin was dispensed and left at room temperature for 2 hours. The plate was washed three times with a wash solution (0.05% Tween 20, phosphate buffer) and the solution was removed. The sample was diluted with phosphate buffer solution containing 1% bovine serum albumin and 0.1% Tween 20, added to a 96-well plate, and incubated at room temperature for 2 hours. After washing the 96-well plate three times with washing solution, horseradish peroxidase-conjugated antibody (anti-MMAE-HRP) was diluted in phosphate buffer containing 1% bovine serum albumin and 0.05% Tween, and 100 μL was dispensed into each well of the 96-well plate and incubated at room temperature for 1 hour. After washing three times with washing solution, 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) hydrogen peroxide chromogenic solution was added to each well, and the reaction was carried out in the dark for 30 minutes. The reaction was terminated by adding 100 μL of sulfuric acid solution to each well, and absorbance was measured using a SpectraMax M5, excluding the absorbance at 650 nm from the absorbance at 450 nm. The quantitative values ​​of each sample were determined through regression analysis after constructing a standard curve for the standard substances.

[0388]

[0389] Example 7-4. Enzymatic immunoassay of anti-Her2 monoclonal antibody-drug (DM1) conjugate

[0390] 100 μL of Human IgG Fc gamma-specific antibody, diluted to a concentration of 2.4 μg / mL in phosphate buffer, was dispensed into a 96-well plate and incubated at room temperature for 14 to 18 hours. After removing any antibodies remaining unattached to the wells, 240 μL of phosphate buffer solution containing 1% bovine serum albumin was dispensed and left at room temperature for 2 hours. The plate was washed three times with a wash solution (0.05% Tween 20, phosphate buffer) and the solution was removed. The sample was diluted with phosphate buffer solution containing 1% bovine serum albumin and 0.1% Tween 20, added to a 96-well plate, and incubated at room temperature for 2 hours. After washing the 96-well plate three times with washing solution, horseradish peroxidase-conjugated antibody (anti-DM1-HRP) was diluted in phosphate buffer containing 1% bovine serum albumin and 0.05% Tween, and 100 μL was dispensed into each well of the 96-well plate and incubated at room temperature for 1 hour. After washing three times with washing solution, 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) hydrogen peroxide chromogenic solution was added to each well, and the reaction was carried out in the dark for 30 minutes. The reaction was terminated by adding 100 μL of sulfuric acid solution to each well, and absorbance was measured using a SpectraMax M5, excluding the absorbance at 650 nm from the absorbance at 450 nm. The quantitative values ​​of each sample were determined through regression analysis after constructing a standard curve for the standard substances.

[0391]

[0392] Example 7-5. Enzyme immunoassay of anti-Her2 monoclonal antibody

[0393] 100 μL of Human IgG Fc gamma-specific antibody, diluted to a concentration of 2.4 μg / mL in phosphate buffer, was dispensed into a 96-well plate and incubated at room temperature for 14 to 18 hours. After removing any antibodies remaining unattached to the wells, 240 μL of phosphate buffer solution containing 1% bovine serum albumin was dispensed and left at room temperature for 2 hours. The plate was washed three times with a wash solution (0.05% Tween 20, phosphate buffer) and the solution was removed. The sample was diluted with phosphate buffer solution containing 1% bovine serum albumin and 0.1% Tween 20, added to a 96-well plate, and incubated at room temperature for 2 hours. After washing the 96-well plate five times with washing solution, horseradish peroxidase-conjugated antibody (anti-human IgG-HRP, Fab specific) was diluted in phosphate buffer containing 1% bovine serum albumin and 0.05% Tween, and 100 μL was dispensed into each well of the 96-well plate and incubated at room temperature for 1 hour. After washing five times with washing solution, 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) hydrogen peroxide chromogenic solution was added to each well, and the reaction was incubated in the dark for 30 minutes. The reaction was terminated by adding 100 μL of sulfuric acid solution to each well, and absorbance was measured using a SpectraMax M5, excluding the absorbance at 650 nm from the absorbance at 450 nm. The quantitative values ​​of each sample were determined through regression analysis after constructing a standard curve for the standard substances.

[0394]

[0395] Example 7-6. Liquid Chromatography-Mass Spectrometry (LC / MS / MS) Method for Drug (MMAE)

[0396] Analysis was performed using MMAE as the standard and MMAE-d8 as the internal standard. 10 μL of the internal standard solution was mixed with 100 μL of minipig plasma. Additionally, 900 μL of methyl-tert-butyl ether was added, mixed, and centrifuged; the supernatant was stored in a freezer. The supernatant was transferred to a tube and dried using nitrogen gas. It was redissolved in 50% acetonitrile solution, centrifuged, and injected into the LC / MS / MS system. The liquid chromatography system was operated at a flow rate of 0.3 mL / min using a phenylhexyl column and a gradient condition consisting of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. The standard and internal standard were detected using a SCIEX TQ 6500+ LC / MS / MS system equipped with an ESI ionization source in cation mode. The standard substance was monitored using the MRM (multiple reaction monitoring) transition from m / z (mass to charge ratio) [718.4]+ to [686.5]+, and the internal standard substance from m / z [726.4]+ to [694.4]+.

[0397]

[0398] Example 7-7. Liquid Chromatography-Mass Spectrometry (LC / MS / MS) Method of Drug (DM1)

[0399] Analysis was performed using DM1 (Mertansine) as the standard and Mertansine-13CD3 as the internal standard. 10 μL of the internal standard solution was mixed with 150 μL of minipig plasma. Additionally, 30 μL of Tris(2-carboxyethyl)phosphine hydrochloride (hereinafter 'TCEP') was added and mixed, followed by centrifugation and incubation in the dark at 37°C for 30 minutes. 30 μL of N-ethylmaleimide (hereinafter 'NEM') was added and mixed, followed by centrifugation and incubation in the dark at 37°C for 30 minutes. 150 μL of acetonitrile was added and mixed, followed by centrifugation. 325 μL of the supernatant was transferred to a tube, 575 μL of ultrapure water was added and mixed, followed by centrifugation. For solid phase extraction, activation and equilibration were performed using methanol and ultrapure water. 900 μL of a solution prepared by mixing 325 μL of the supernatant with 575 μL of ultrapure water was loaded into SPE wells for solid phase extraction. After washing with ultrapure water, 70 μL of MeOH containing 2% FA was added for extraction, and the supernatant was injected into the LC / MS / MS system. The liquid chromatography system used a C18 column at a flow rate of 0.4 mL / min and an isocratic condition consisting of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. Standards and internal standards were detected using a SCIEX TQ 7500+ LC / MS / MS system equipped with an ESI ionization source in cation mode. NEM-labeled standard substances have a mass-to-charge ratio (m / z) of [885.4]+ to [569.2]+, and NEM-labeled internal standard substances have a m / z of [889.5]+ to

[569] .Monitoring was performed using MRM (multiple reaction monitoring) transition to 2]+.

[0400] Examples 7-8. Method for observing general symptoms

[0401] Symptoms, including mortality, morbidity, and changes in appearance and behavior, were observed and recorded once a day before administration and twice a day during administration, and the date, time, and duration were recorded. Based on the records, the symptoms were scored from 0 to 5 points according to their type and severity.

[0402]

[0403] Examples 7-9. Hematological test methods

[0404] Before administration of the test substance and on days 4, 8, and 15 after administration, at least 0.5 mL of blood was collected from all animals, placed in a blood collection tube containing an anticoagulant (EDTA-K2), and items such as Total leukocyte count (WBC), Total red blood cell count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Reticulocyte count, WBC differential count (Absolute (#) and relative (%) differential counts: include neutrophils (NEU), eosinophils (EOS), basophils (BAS), monocytes (MON), lymphocytes (LYM), large unstained cells (LUC)), and Absolute (RETA) and relative (RET%) counts were measured. Measurements were performed using an automated hematology analyzer (ADVIA2120i hematology analyzer, Siemens, USA).

[0405]

[0406] Examples 7-10. Blood biochemical test method

[0407] At least 1.5 mL of blood was collected from all animals before administration of the test substance and on days 4, 8, and 15 after administration, placed in a tube without anticoagulant, left at room temperature for at least 30 minutes, and then centrifuged (approx. 3,000 rpm, 10 minutes, room temperature) to separate the serum. Using isolated serum, parameters such as Glucose (GLU), Alanine aminotransferase (ALT), Blood urea nitrogen (BUN), Total bilirubin (TBIL), Creatinine (CREA), Alkaline phosphatase (ALP), Total protein (TP), Gamma glutamyl transpeptidase (GGT), Albumin (ALB), Creatine phosphokinase (CK), Albumin / globulin ratio (A / G), Calcium (Ca), Total cholesterol (TCHO), Inorganic phosphorus (IP), Triglyceride (TG), Sodium (Na), C-reactive Protein (CRP), Potassium (K), Aspartate aminotransferase (AST), and Chloride (Cl) were measured. Measurements were performed using an automated clinical chemistry analyzer (TBA 120FR chemistry analyzer, Toshiba Co., Japan).

[0408]

[0409] Examples 7-11 Histopathological examination

[0410] Slides of the administration site tissue were prepared, and histopathological examinations were performed after hematoxylin & eosin staining. Histopathological findings observed in each individual from the collected tissues were recorded.

[0411] Histopathological severity was classified into four levels—'very mild,' 'mild,' 'slightly severe,' and 'severe'—and quantified as a score ranging from 1 to 4 points for each level. The histopathological degree of local symptoms by group was evaluated by summing the values ​​calculated by multiplying the severity score for each lesion by the number of tissues in which the lesion was observed.

[0412]

[0413] I have attached the electronic file.

Claims

1. A subcutaneous administration formulation comprising a protein having hyaluronan degradation activity used in combination with an antibody-drug conjugate (ADC) comprising one or more drug portions conjugated to an antibody or an antigen-binding antibody fragment, wherein the subcutaneous administration injectable formulation comprises: (a) comprising a protein having hyaluronan degradation activity of 4,000–16,000 unit / mL; and / or (b) a subcutaneous administration formulation characterized by administering a protein having hyaluronan degradation activity in an activity range of 10 to 470,000 units per 1 mg of drug.

2. The subcutaneous administration formulation according to claim 1, wherein the subcutaneous administration formulation containing a protein having hyaluronan degradation activity is administered simultaneously or sequentially with an antibody-drug conjugate (ADC).

3. A subcutaneous administration formulation according to paragraph 2, wherein the sequential administration is characterized in that a subcutaneous administration formulation containing a protein having hyaluronan degradation activity is administered first, and then, after a certain period of time has elapsed, a formulation containing an antibody-drug conjugate (ADC) is administered subcutaneously to the same site.

4. A subcutaneous administration formulation according to claim 3, characterized in that a subcutaneous administration formulation containing a protein having hyaluronan degradation activity is administered first, and then a formulation containing an antibody-drug conjugate (ADC) is administered secondarily after 30 seconds to 10 hours have elapsed.

5. A subcutaneous administration formulation characterized in that, in any one of claims 2 to 4, the antibody-drug conjugate (ADC) is administered subcutaneously either alone or together with a protein having hyaluronan degradation activity during subsequent administration.

6. A subcutaneous administration formulation according to any one of claims 2 to 4, wherein the subcutaneous administration formulation containing the protein having hyaluronan degradation activity is characterized in that, when administered concurrently with an antibody-drug conjugate (ADC), the active range of the protein having hyaluronan degradation activity is administered at 150 to 470,000 units per 1 mg of drug, and when administered sequentially, the active range of the protein having hyaluronan degradation activity is administered at 10 to 50,000 units per 1 mg of drug.

7. A subcutaneous administration formulation according to claim 2, characterized in that, when the antibody-drug conjugate (ADC) and the protein having hyaluronan degradation activity are administered subcutaneously as individual formulations or as a single mixed formulation, respectively, in the case of simultaneous or sequential administration of the subcutaneous administration injectable formulation containing the protein having hyaluronan degradation activity and the antibody-drug conjugate (ADC).

8. A subcutaneous administration formulation characterized in that, in any one of claims 1 to 7, the protein having hyaluronan degradation activity is hyaluronidase.

9. A subcutaneous administration formulation according to claim 8, characterized in that the hyaluronidase is of bacterial, bovine, sheep, or human origin.

10. A subcutaneous administration formulation according to claim 9, characterized in that the hyaluronidase is a human-derived PH20 or a variant thereof.

11. A subcutaneous administration formulation according to claim 10, wherein the human-derived hyaluronidase comprises a substitution of an amino acid residue, a deletion at the N-terminus, and / or a deletion at the C-terminus in the amino acid sequence of SEQ ID NO.

1.

12. In claim 11, the human-derived hyaluronidase in the amino acid sequence of SEQ ID NO. 1, (1) Includes L36 to S490, (2) a) T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T; (b) L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (c) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, and N363G; (d) T341G, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (e) T341A, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (f) T341C, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (g) T341D, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (h) I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T; and (i) S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T; comprising one or more amino acid residue substitutions selected from; A subcutaneous administration formulation characterized by doing so.

13. A subcutaneous administration formulation according to claim 12, characterized in that the human-derived hyaluronidase has the amino acid sequence of SEQ ID NO.

6.

14. In any one of claims 1 to 7, the drug (payload) included in the antibody-drug conjugate (ADC) is a microtubule (tubulin) inhibitor, metansinoid or its derivative, anthracycline, topoisomerase inhibitor I or II inhibitor, camptothecin or its derivative, calicemycin, auristatin, nitrogen mustard, ethyleneimine derivative, alkyl sulfonate, nitrosourea, triagen, folic acid analog, taxane, COX-2 inhibitor, pyrimidine analog, purine analog, antibiotic, enzyme inhibitor, epipodophyllotoxin, platinum coordination complex, vinca alkaloid, substituted urea, methylhydrazine derivative, adrenocorticosteroid inhibitor, hormone antagonist, anmetatallite, alkylating agent, antimitotic agent, anti-angiogenic agent, tyrosine kinase inhibitor, mTOR inhibitor, heat shock A subcutaneous administration formulation characterized by being one or more selected from the group consisting of protein (HSP90) inhibitors, proteosome inhibitors, HDAC inhibitors, and proapotogenic agents.

15. In any one of claims 1 to 7, the antibody-drug conjugate (AAD) is AM9, AG7, ALCAM, ALPG, AXL, CCR2, CCR7, CD7, CD19, CD22, CD25, CD33, CD37, CD38, CD46, CD48, CD70, CD74, CD79B, CD248, CD274, CD276, CDH3, CDH6, CEACAM5, CLDN6, CLDN18.2, cMET, DLL3, DPEP3, DUX4, EGFR, EPCAM, FAP, FGFR2, FN1, FOLH1, FOLR1, GCC, Globo H, GPNMB, GPR20, GPRC5D, HAVCR1, HER2, HER3, ICAM1, IGF1R, IL3RA, ITGB6, KAAG1, A subcutaneous administration formulation comprising an antibody or antigen-binding antibody fragment against KIT, KLK3, LGALS3BP, LRRC15, LY6E, LY75, LYPD3, MELTF, MS4A1, MSLN, MUC1, NCAM1, NECTIN4, NT5E, PRLR, PROM1, PTK7, RNF43, ROR1, ROR2, S. aureus, SDC1, SEZ6, SLC34A2, SLC39A6, SLITRK6, ST8SIA1, TACSTD2, TDGF1, TFRC, TM4SF1, TNF, TNFRSF1A, TNFRSF8, TNFRSF17, TPBG, TRV6, VEGFA, VTCN1, or tissue factor.

16. In any one of claims 1 to 7, the antibody-drug conjugate is IMGC-936, AbGn-107, Pralusatamab Lavtansine, SGN-ALPV, Enapotamab Vedotin, Mekbotamab Vedotin, Mifacetamab Uzoftyrin, LCB17-0877, TAK-500, JBH-492, Grisnilimab Cetaritox, Roncastuximab Tesirin, Coltuximab Lavtansine, ABBV-319, IKS-03, Inotuzumab Ozogamicin, Moxetumomab Pasodotox, Efratuzumab-SN38, ADCT-602, TAC-001, BAY-1862864, TRPH-222, RM-1995, Kamidanlumab Tesirin, Gemtuzumab Ozogamicin, Lintuzumab-Ac-225, BL-M11D1, Naratuximab Emtansine, AGS-67E, STI-6129, CD38 ADC, FOR-46, SGN-CD48A, PRO-1160, ARX-305, STRO-001, Folatuzumab Vedotin, Iladatuzumab Vedotin, NBT-508, SHR-A1912, MP-ENDOS-ADC, SGN-PDL1V, Vobramitamab Duocamazine, Ifinatamab Deruxtecan, Mirzotamab Clezutoclax, HS-20093, IBI129, MHB088C, BAT-8009, YL201, BC3195, DS-6000, Tusamitamab Lavtansine, Lavetuzumab Govitecan, EBC-129, M-9140, DS-9606, TORL-1-23, LM-302, RC-118, SHR-A1904, SOT-102, XNW-27011, ATG-022, BA1301, CMG-901, CPO-102, IBI343, JS107, SKB-315, SYSA-1801, TORL-2-307-ADC, TQB2103, Telisotuzumab Vedotin, RC-108, REGN5093-M114, BYON-3521, MYTX-011, SHR-A1403, TR1801-ADC, Rovalfituzumab Tesyrin, SC-002, Tamlintamab Famozirin, AOC-1020, Cetuximab Sarotalocan, Departuxizumab Mapodotin, Losatuxizumab Vedotin, Cerclutamab Talirin, MRG-003, AVID-100, BB-1705,EGFR-EDV-RRM1, Oportuzumab Monatox, OMTX-705, Aprutumab Exadotin, PYX-201, MEDI-3726, Mirvetuximab Sorabtansine, Paletuzumab Ecteribulin, Rubeltamab Tazebibulin, AZD-5335, PRO-1184, AMT-151, BAT-8006, IMGN151, TAK-164, OBI-999, Glembatumumab Vedotin, DS-6157a, LM-305, CDX-014, Dicitamab Vedotin, Trastuzumab Deruxtecan, Trastuzumab Emtansine, Trastuzumab Duocamazine, Pertuzumab Zubotulimod, A-166, ARX-788, DP-303c, LCB14-0110, MRG-002, SHR-A1811, B-003, BAT-8001, BB-1701, BDC-1001, DB-1303, DX126-262, FDA-022, GQ-1001, IBI-354, ALT-P7, BAT-8010, BI-CON-02, BL-M07D1, FDA022-BB05, GB-251, GQ-1005, GQ-1007, HS630, MT-5111, NJH-395, PF-06804103, SHR-A1201, TQB2102, ZV0203, Patritumab Deruxtecan, BL-B01D1, SHR-A2009, Enrimomab Pegol, Ronigutamab Ugodotin, IGF-MTX, Pibekimab Sunirin, SGN-B6A, ADCT-901, MGTA-117, ARX-517, MP-LGS-ADC, Samrotamab Vedotin, RG-7841, 16A5-MCC-DM1, MEN-1309, Rupartumab Amadintin, SGN-CD228A, CON-4619, MRG-001, TRS-005, Anetumab Lavtansine, BMS-986148, RC-88, DAC-005, DXC-005, Lorbotuzumab Mertansine, Enfortumab Vedotin, 9MW-2821, BAT-8007, SHR-A2102, SKB-410, SYS6002, BB-1709, Rolinsatamab Talirin, OXS-1650, Cofetuzumab Felidotin, SC-006, Zilovertamab Vedotin, NBE-002, ABL-202, LCB-71,Ozuriftamab Vedotin, RG-7861, Indatuximab Lavtansine, SC-011, Upipitamab Rilsodotin, XMT-1592, Radiratuzumab Vedotin, Sirtratumab Vedotin, PF-06688992, Sacituzumab Gobitecan, Datopotamab Deruxtecan, SKB-264, BIO-106, DB-1305, ESG-401, MHB036C, SHR-A1921, BAT-8008, BL-M02D1, DAC-002, FDA-018, YL202, BIIB-015, CX-2029, AGX101, ABBV-154, ABBV-3373, Brentuzumab Vedotin, F0002-ADC, Belantamab mafodotin, espectamab devotansin, HDP-101, AMG-224, MEDI-2228, ASN-004, SYD-1875, AB-160, AZD-8205, HS-20089, SGN-B7H4V, XMT-1660, tisotumab vedotin, MRG-004A, XB-002, CBP1008 for FOLR1 and TRV6, AZD-9592 for EGFR and cMET, M-1231 for EGFR and MUC1, zanidamab zobodotin, JSKN-003, KM-501, CBX-12, CPO-204, DAN-222, DB-1202, DS001, DXC-009, DXC004A, Subcutaneous administration formulations comprising MG1901, MG2001, MG2002A, SGN-STNV, SHR-4602, SYS6010, TORL-3-600, TORL-4-500, or TUB-030.

17. A mixed subcutaneous administration formulation comprising an antibody-drug conjugate (ADC) comprising one or more drug portions conjugated to an antibody or an antigen-binding antibody fragment, and a protein having hyaluronan degradation activity, wherein the mixed subcutaneous administration injectable formulation comprises: (a) comprising a protein having hyaluronan degradation activity of 4,000–16,000 unit / mL; and / or (b) a mixed subcutaneous administration formulation characterized by containing a protein with an activity range of 10 to 470,000 units having hyaluronan degradation activity per 1 mg of drug.

18. An antibody-drug conjugate comprising one or more drug portions conjugated to an antibody or an antigen-binding antibody fragment, a hyaluronidase having hyaluronan degradation activity, and instructions, In the above instruction manual, subcutaneous administration of the antibody-drug conjugate is performed in a pre-administration - post-administration manner, and The above pre-administration is the subcutaneous administration of hyaluronidase alone at the site of subcutaneous administration of the antibody-drug conjugate-hyaluronidase mixed formulation prior to subcutaneous administration of the antibody-drug conjugate-hyaluronidase mixed formulation, and A kit for subcutaneous administration of an antibody-drug conjugate, wherein the above-mentioned subsequent administration involves administering a mixed formulation of the antibody-drug conjugate and hyaluronidase, or the antibody-drug conjugate and hyaluronidase, respectively, to the hyaluronidase administration site within 30 seconds to 10 hours of the above-mentioned prior administration.

19. In Paragraph 18, In the above instruction manual The above pre-administration is administered at a dose of 1,000 to 16,000 unit / mL based on hyaluronidase activity, and A kit for subcutaneous administration of an antibody-drug conjugate, wherein the above administration is described as administering 1,000 to 16,000 unit / mL based on hyaluronidase activity when administering a mixed formulation of the antibody-drug conjugate and hyaluronidase, or when administering the antibody-drug conjugate and hyaluronidase separately.

20. In Paragraph 18, A kit for subcutaneous administration of an antibody-drug conjugate, wherein the above instruction manual describes that subcutaneous administration of the antibody-drug conjugate according to the above pre-administration-post-administration method includes an initiation administration performed daily for 1 week or 2 to 5 times per week for 2 weeks, and a maintenance administration performed at least once every 2 weeks to 6 months after the initiation administration.

21. In Paragraph 18, The above kit is a kit for subcutaneous administration of an antibody-drug conjugate, comprising a mixed formulation of the antibody-drug conjugate and hyaluronidase and a hyaluronidase monotherapy formulation.

22. In Paragraph 18, The above kit is a kit for subcutaneous administration of an antibody-drug conjugate, comprising an antibody-drug conjugate monoformulation and a hyaluronidase monoformulation.

23. In Paragraph 18, A kit for subcutaneous administration of an antibody-drug conjugate in which the above instruction manual describes the above pre-dose and / or above post-dose as effective doses for producing therapeutic effects.

24. In claim 17 or 18, the drug (payload) included in the antibody-drug conjugate (ADC) is a microtubule (tubulin) inhibitor, metansinoid or its derivative, anthracycline, topoisomerase inhibitor I or II inhibitor, camptothecin or its derivative, calicemycin, auristatin, nitrogen mustard, ethyleneimine derivative, alkyl sulfonate, nitrosourea, triagen, folate analog, taxane, COX-2 inhibitor, pyrimidine analog, purine analog, antibiotic, enzyme inhibitor, epipodophyllotoxin, platinum coordination complex, vinca alkaloid, substituted urea, methylhydrazine derivative, adrenocorticosteroid inhibitor, hormone antagonist, anmetata, alkylating agent, antimitotic agent, anti-angiogenic agent, tyrosine kinase inhibitor, mTOR inhibitor, heat shock A kit for subcutaneous administration of an antibody-drug conjugate characterized by being one or more selected from the group consisting of protein (HSP90) inhibitors, proteosome inhibitors, HDAC inhibitors, and proapotogenic agents.

25. In Paragraph 17 or 18, The antibody-drug conjugates include ADAM9, AG7, ALCAM, ALPG, AXL, CCR2, CCR7, CD7, CD19, CD22, CD25, CD33, CD37, CD38, CD46, CD48, CD70, CD74, CD79B, CD248, CD274, CD276, CDH3, CDH6, CEACAM5, CLDN6, CLDN18.2, cMET, DLL3, DPEP3, DUX4, EGFR, EPCAM, FAP, FGFR2, FN1, FOLH1, FOLR1, GCC, Globo H, GPNMB, GPR20, GPRC5D, HAVCR1, HER2, HER3, ICAM1, IGF1R, IL3RA, ITGB6, KAAG1, KIT, KLK3, LGALS3BP, LRRC15, A kit for subcutaneous administration of an antibody-drug conjugate comprising an antibody or antigen-binding antibody fragment against LY6E, LY75, LYPD3, MELTF, MS4A1, MSLN, MUC1, NCAM1, NECTIN4, NT5E, PRLR, PROM1, PTK7, RNF43, ROR1, ROR2, S. aureus, SDC1, SEZ6, SLC34A2, SLC39A6, SLITRK6, ST8SIA1, TACSTD2, TDGF1, TFRC, TM4SF1, TNF, TNFRSF1A, TNFRSF8, TNFRSF17, TPBG, TRV6, VEGFA, VTCN1, or tissue factor.

26. In Paragraph 17 or 18, The above antibody-drug conjugates are IMGC-936, AbGn-107, Pralusatamab Lavtansine, SGN-ALPV, Enapotamab Vedotin, Megbotamab Vedotin, Mifacetamab Uzoftyrin, LCB17-0877, TAK-500, JBH-492, Grisnilimab Cetaritox, Roncastuximab Tesirin, Coltuximab Lavtansine, ABBV-319, IKS-03, Inotuzumab Ozogamicin, Moxetumomab Pasodotox, Efratuzumab-SN38, ADCT-602, TAC-001, BAY-1862864, TRPH-222, RM-1995, Kamidanlumab Tesirin, Gemtuzumab Ozogamicin, Lintuzumab-Ac-225, BL-M11D1, Naratuximab Emtansine, AGS-67E, STI-6129, CD38 ADC, FOR-46, SGN-CD48A, PRO-1160, ARX-305, STRO-001, Folatuzumab Vedotin, Iladatuzumab Vedotin, NBT-508, SHR-A1912, MP-ENDOS-ADC, SGN-PDL1V, Vobramitamab Duocamazine, Ifinatamab Deruxtecan, Mirzotamab Clezutoclax, HS-20093, IBI129, MHB088C, BAT-8009, YL201, BC3195, DS-6000, Tusamitamab Lavtansine, Lavetuzumab Goritecan, EBC-129, M-9140, DS-9606, TORL-1-23, LM-302, RC-118, SHR-A1904, SOT-102, XNW-27011, ATG-022, BA1301, CMG-901, CPO-102, IBI343, JS107, SKB-315, SYSA-1801, TORL-2-307-ADC, TQB2103, Telisotuzumab Vedotin, RC-108, REGN5093-M114, BYON-3521, MYTX-011, SHR-A1403, TR1801-ADC, Rovalfituzumab Tesyrin, SC-002, Tamlintamab Famozirin, AOC-1020, Cetuximab Sarotalocan, Departuxizumab Mapodotin, Losatuxizumab Vedotin, Cerclutamab Talirin, MRG-003, AVID-100, BB-1705, EGFR-EDV-RRM1, Oportuzumab Monatox,OMTX-705, Aftumab ixadotin, PYX-201, MEDI-3726, Mirvetuximab sorabtansine, Paletuzumab ecteribulin, Rubeltamab tazebibulin, AZD-5335, PRO-1184, AMT-151, BAT-8006, IMGN151, TAK-164, OBI-999, Glembatumumab vedotin, DS-6157a, LM-305, CDX-014, Dicitamab vedotin, Trastuzumab deruxtecan, Trastuzumab emtansine, Trastuzumab duocamazine, Pertuzumab jubotulimod, A-166, ARX-788, DP-303c, LCB14-0110, MRG-002, SHR-A1811, B-003, BAT-8001, BB-1701, BDC-1001, DB-1303, DX126-262, FDA-022, GQ-1001, IBI-354, ALT-P7, BAT-8010, BI-CON-02, BL-M07D1, FDA022-BB05, GB-251, GQ-1005, GQ-1007, HS630, MT-5111, NJH-395, PF-06804103, SHR-A1201, TQB2102, ZV0203, Patritumab Deruxtecan, BL-B01D1, SHR-A2009, Enrimomab Pegol, Ronigutamab Ugodotine, IGF-MTX, Pibekimab Sunirin, SGN-B6A, ADCT-901, MGTA-117, ARX-517, MP-LGS-ADC, Samrotamab Vedotin, RG-7841, 16A5-MCC-DM1, MEN-1309, Rupartumab Amadintin, SGN-CD228A, CON-4619, MRG-001, TRS-005, Anetumab Lavtansine, BMS-986148, RC-88, DAC-005, DXC-005, Lorbotuzumab Mertansine, Enfortumab Vedotin, 9MW-2821, BAT-8007, SHR-A2102, SKB-410, SYS6002, BB-1709, Rolinsatamab Talirin, OXS-1650, Cofetuzumab Felidotine, SC-006, Zilovertamab Vedotine, NBE-002, ABL-202, LCB-71, Ozuriftamab Vedotine, RG-7861, Indatuximab Labtansine,SC-011, Upipitamab rilsodotin, XMT-1592, Radiratuzumab vedotin, Sirtratumab vedotin, PF-06688992, Sacituzumab govitecan, Datopotamab deruxtecan, SKB-264, BIO-106, DB-1305, ESG-401, MHB036C, SHR-A1921, BAT-8008, BL-M02D1, DAC-002, FDA-018, YL202, BIIB-015, CX-2029, AGX101, ABBV-154, ABBV-3373, Brentuzumab vedotin, F0002-ADC, Belantamab mafodotin, Espectamab devotansin, HDP-101, AMG-224, MEDI-2228, ASN-004, SYD-1875, AB-160, AZD-8205, HS-20089, SGN-B7H4V, XMT-1660, Tisotumab Vedotin, MRG-004A, XB-002, CBP1008 for FOLR1 and TRV6, AZD-9592 for EGFR and cMET, M-1231 for EGFR and MUC1, Zanitamab Zobodotin, JSKN-003, KM-501, CBX-12, CPO-204, DAN-222, DB-1202, DS001, DXC-009, DXC004A, MG1901, MG2001, MG2002A, SGN-STNV, A kit for subcutaneous administration of an antibody-drug conjugate comprising SHR-4602, SYS6010, TORL-3-600, TORL-4-500, or TUB-030.

27. In Paragraph 17, A kit for subcutaneous administration of an antibody-drug conjugate, the above instruction manual states that subcutaneous administration of the antibody-drug conjugate can be performed at multiple sites.

28. A method for treating a disease by subcutaneously administering an antibody-drug conjugate comprising one or more drug portions conjugated to an antibody or an antigen-binding antibody fragment, wherein Subcutaneous administration of antibody-drug conjugates is performed in a pre-administration - post-administration manner, and The above pre-administration is the subcutaneous administration of hyaluronidase alone at the site of subcutaneous administration of the antibody-drug conjugate-hyaluronidase mixed formulation prior to subcutaneous administration of the antibody-drug conjugate-hyaluronidase mixed formulation, and A method for treating a disease by subcutaneous administration of an antibody-drug conjugate, wherein the above subsequent administration involves administering a mixed formulation of the antibody-drug conjugate and hyaluronidase, or the antibody-drug conjugate and hyaluronidase, respectively, to the site of administration of the hyaluronidase after the above prior administration.

29. In Paragraph 28, A method for treating a disease by subcutaneous administration of an antibody-drug conjugate, wherein the above subsequent administration is performed within 30 seconds to 10 hours after the above prior administration.

30. In Paragraph 28, The above pre-administration is administered at a dose of 1,000 to 16,000 unit / mL based on hyaluronidase activity, and A method for treating a disease by subcutaneous administration of an antibody-drug conjugate, wherein the above post-administration involves administering a hyaluronidase activity of 1,000 to 16,000 unit / mL when administering a mixed formulation of the antibody-drug conjugate and hyaluronidase, or when administering the antibody-drug conjugate and hyaluronidase separately.

31. In Paragraph 28, A method for treating a disease by subcutaneous administration of an antibody-drug conjugate according to the above pre-administration - post-administration method, comprising an initiation administration performed daily for 1 week or 2 to 5 times per week for 2 to 3 weeks, and a maintenance administration performed at least once every 2 weeks to 6 months after the initiation administration.

32. In Paragraph 28, A method for treating a disease by subcutaneous administration of an antibody-drug conjugate, wherein the dose administered at the aforementioned pre-administration and / or the dose administered at the aforementioned post-administration is an effective dose for producing a therapeutic effect.

33. In Paragraph 28, The drug (payload) included in the above antibody-drug conjugate (ADC) is a microtubule (tubulin) inhibitor, metansinoid or its derivative, anthracycline, topoisomerase inhibitor I or II inhibitor, camptothecin or its derivative, calicemycin, auristatin, nitrogen mustard, ethyleneimine derivative, alkyl sulfonate, nitrosourea, triagen, folate analog, taxane, COX-2 inhibitor, pyrimidine analog, purine analog, antibiotic, enzyme inhibitor, epipodophyllotoxin, platinum coordination complex, vinca alkaloid, substituted urea, methylhydrazine derivative, adrenocorticosteroid inhibitor, hormone antagonist, anmetatars, alkylating agents, antimitotic agents, anti-angiogenic agents, tyrosine kinase inhibitor, mTOR inhibitor, heat shock protein (HSP90) inhibitor, proteosome inhibitor, HDAC inhibitor, and A method for treating a disease by subcutaneous administration of an antibody-drug conjugate characterized by being one or more selected from a group of proapoptotic agents.

34. In Paragraph 28, The antibody-drug conjugates include ADAM9, AG7, ALCAM, ALPG, AXL, CCR2, CCR7, CD7, CD19, CD22, CD25, CD33, CD37, CD38, CD46, CD48, CD70, CD74, CD79B, CD248, CD274, CD276, CDH3, CDH6, CEACAM5, CLDN6, CLDN18.2, cMET, DLL3, DPEP3, DUX4, EGFR, EPCAM, FAP, FGFR2, FN1, FOLH1, FOLR1, GCC, Globo H, GPNMB, GPR20, GPRC5D, HAVCR1, HER2, HER3, ICAM1, IGF1R, IL3RA, ITGB6, KAAG1, KIT, KLK3, LGALS3BP, LRRC15, A method for treating a disease by subcutaneous administration of an antibody-drug conjugate comprising an antibody or antigen-binding antibody fragment against LY6E, LY75, LYPD3, MELTF, MS4A1, MSLN, MUC1, NCAM1, NECTIN4, NT5E, PRLR, PROM1, PTK7, RNF43, ROR1, ROR2, S. aureus, SDC1, SEZ6, SLC34A2, SLC39A6, SLITRK6, ST8SIA1, TACSTD2, TDGF1, TFRC, TM4SF1, TNF, TNFRSF1A, TNFRSF8, TNFRSF17, TPBG, TRV6, VEGFA, VTCN1, or tissue factor.

35. In Paragraph 28, The above antibody-drug conjugates are IMGC-936, AbGn-107, Pralusatamab Lavtansine, SGN-ALPV, Enapotamab Vedotin, Megbotamab Vedotin, Mifacetamab Uzoftyrin, LCB17-0877, TAK-500, JBH-492, Grisnilimab Cetaritox, Roncastuximab Tesirin, Coltuximab Lavtansine, ABBV-319, IKS-03, Inotuzumab Ozogamicin, Moxetumomab Pasodotox, Efratuzumab-SN38, ADCT-602, TAC-001, BAY-1862864, TRPH-222, RM-1995, Kamidanlumab Tesirin, Gemtuzumab Ozogamicin, Lintuzumab-Ac-225, BL-M11D1, Naratuximab Emtansine, AGS-67E, STI-6129, CD38 ADC, FOR-46, SGN-CD48A, PRO-1160, ARX-305, STRO-001, Folatuzumab Vedotin, Iladatuzumab Vedotin, NBT-508, SHR-A1912, MP-ENDOS-ADC, SGN-PDL1V, Vobramitamab Duocamazine, Ifinatamab Deruxtecan, Mirzotamab Clezutoclax, HS-20093, IBI129, MHB088C, BAT-8009, YL201, BC3195, DS-6000, Tusamitamab Lavtansine, Lavetuzumab Goritecan, EBC-129, M-9140, DS-9606, TORL-1-23, LM-302, RC-118, SHR-A1904, SOT-102, XNW-27011, ATG-022, BA1301, CMG-901, CPO-102, IBI343, JS107, SKB-315, SYSA-1801, TORL-2-307-ADC, TQB2103, Telisotuzumab Vedotin, RC-108, REGN5093-M114, BYON-3521, MYTX-011, SHR-A1403, TR1801-ADC, Rovalfituzumab Tesyrin, SC-002, Tamlintamab Famozirin, AOC-1020, Cetuximab Sarotalocan, Departuxizumab Mapodotin, Losatuxizumab Vedotin, Cerclutamab Talirin, MRG-003, AVID-100, BB-1705, EGFR-EDV-RRM1, Oportuzumab Monatox,OMTX-705, Aftumab ixadotin, PYX-201, MEDI-3726, Mirvetuximab sorabtansine, Paletuzumab ecteribulin, Rubeltamab tazebibulin, AZD-5335, PRO-1184, AMT-151, BAT-8006, IMGN151, TAK-164, OBI-999, Glembatumumab vedotin, DS-6157a, LM-305, CDX-014, Dicitamab vedotin, Trastuzumab deruxtecan, Trastuzumab emtansine, Trastuzumab duocamazine, Pertuzumab jubotulimod, A-166, ARX-788, DP-303c, LCB14-0110, MRG-002, SHR-A1811, B-003, BAT-8001, BB-1701, BDC-1001, DB-1303, DX126-262, FDA-022, GQ-1001, IBI-354, ALT-P7, BAT-8010, BI-CON-02, BL-M07D1, FDA022-BB05, GB-251, GQ-1005, GQ-1007, HS630, MT-5111, NJH-395, PF-06804103, SHR-A1201, TQB2102, ZV0203, Patritumab Deruxtecan, BL-B01D1, SHR-A2009, Enrimomab Pegol, Ronigutamab Ugodotine, IGF-MTX, Pibekimab Sunirin, SGN-B6A, ADCT-901, MGTA-117, ARX-517, MP-LGS-ADC, Samrotamab Vedotin, RG-7841, 16A5-MCC-DM1, MEN-1309, Rupartumab Amadintin, SGN-CD228A, CON-4619, MRG-001, TRS-005, Anetumab Lavtansine, BMS-986148, RC-88, DAC-005, DXC-005, Lorbotuzumab Mertansine, Enfortumab Vedotin, 9MW-2821, BAT-8007, SHR-A2102, SKB-410, SYS6002, BB-1709, Rolinsatamab Talirin, OXS-1650, Cofetuzumab Felidotine, SC-006, Zilovertamab Vedotine, NBE-002, ABL-202, LCB-71, Ozuriftamab Vedotine, RG-7861, Indatuximab Labtansine,SC-011, Upipitamab rilsodotin, XMT-1592, Radiratuzumab vedotin, Sirtratumab vedotin, PF-06688992, Sacituzumab govitecan, Datopotamab deruxtecan, SKB-264, BIO-106, DB-1305, ESG-401, MHB036C, SHR-A1921, BAT-8008, BL-M02D1, DAC-002, FDA-018, YL202, BIIB-015, CX-2029, AGX101, ABBV-154, ABBV-3373, Brentuzumab vedotin, F0002-ADC, Belantamab mafodotin, Espectamab devotansin, HDP-101, AMG-224, MEDI-2228, ASN-004, SYD-1875, AB-160, AZD-8205, HS-20089, SGN-B7H4V, XMT-1660, Tisotumab Vedotin, MRG-004A, XB-002, CBP1008 for FOLR1 and TRV6, AZD-9592 for EGFR and cMET, M-1231 for EGFR and MUC1, Zanitamab Zobodotin, JSKN-003, KM-501, CBX-12, CPO-204, DAN-222, DB-1202, DS001, DXC-009, DXC004A, MG1901, MG2001, MG2002A, SGN-STNV, A method for treating a disease by subcutaneous administration of an antibody-drug conjugate comprising SHR-4602, SYS6010, TORL-3-600, TORL-4-500, or TUB-030.

36. In Paragraph 28, A method for treating a disease by subcutaneous administration of an antibody-drug conjugate, wherein the above-mentioned subcutaneous administration of the antibody-drug conjugate is performed at multiple sites.

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