WO2019088176A1

WO2019088176A1 - Enhancer of effect of antibody-drug conjugate

Info

Publication number: WO2019088176A1
Application number: PCT/JP2018/040536
Authority: WO
Inventors: 広松岡
Original assignee: 国立大学法人神戸大学
Priority date: 2017-10-31
Filing date: 2018-10-31
Publication date: 2019-05-09

Abstract

Provided is a method for enhancing the effect of an antibody-drug conjugate such as GO (gemtuzumab ozogamicin). More specifically provided is an enhancer of the effect of an antibody-drug conjugate, which contains a lysosome activator.

Description

Antibody drug complex effect enhancer

The present invention relates to means etc. for enhancing the effect of antibody drug complex.

The results of chemotherapy for acute myeloid leukemia (AML) are not satisfactory, and the 5-year survival rate remains at less than 40%. The molecular target drug Gemtuzumab ozogamicin (Gemtuzumab Ozogamicin (GO)) is an antibody-drug complex (Antibody-Drug Conjugates: ADC) in which an anti-CD33 monoclonal antibody and calicheamicin having a potent cell-killing effect are combined. Since expression was observed in about 90% of AML cases, it was hoped to be a breakthrough molecular targeting drug for AML. In 2000, it was approved for relapsed / refractory elderly people AML in the United States, and in 2005 in Japan, GO administration was approved as a single agent for relapsed / refractory AML. However, the 5-year survival rate with single agent for relapsed / refractory AML remained at less than 30%. Therefore, a clinical trial (third phase clinical trial (SWOG S0106 trial) was conducted to enhance the effect by adding GO to the conventional standard treatment Daunorubicin + AraC for initial AML, but the effect on AML with GO addition group In 2010, the original publisher Pfizer voluntarily withdrew GO from the US market (in addition, after that, the GO was released by the FDA on September 14th, 2017, as it did not change, but rather caused fatal side effects). Re-approved). However, in Japan, approval has been continued and used.

WO 2008/026603 WO 2009/041643

An object of the present invention is to provide a method for enhancing the effect of GO (gemtuzumab ozogamicin).

The present inventors activate lysosomes by activating lysosomes (in particular, increasing the number of intracellular lysosomes) to effectively cleave the linker (hydrazone linker) that links GO gemtuzumab and ozogamicin. It has been found that mTOR inhibitors can be suitably used to convert (especially to increase the number of intracellular lysosomes). Then, after the antibody drug complex (Antibody-Drug Conjugate: ADC) is bound to the cells recognized by the antibody, it is usually taken up into the cell by endocytosis, and in lysosome, the linker that binds the antibody and the drug is cleaved. It is recognized that activating the lysosome can enhance the effect of not only GO, but also the antibody drug complex, since the separated drug exerts its drug effect in cells, and further improvement is completed to complete the present invention. It reached.

The present invention includes, for example, the subject matters described in the following sections.

Item A-1.
An antibody drug complex effect enhancer comprising a lysosomal activator.
Item A-2.
The enhancer according to Item A-1, wherein the lysosomal activator is an mTOR inhibitor.
Item A-3.
The enhancer according to item A-1, wherein the lysosomal activator is at least one selected from the group consisting of rapamycin, temsirolimus, everolimus, PP242, Torin1, AZD2014, and MLN0128.
Item A-4.
The drug of the antibody-drug complex is an anticancer drug (eg, calicheamicin, esperamycin, emtansine, auristatin F-HPA (F-hydroxypropylamide), a derivative of DX-8951 (DXd), and monomethyl auristatin E ( The enhancer according to any one of Items A-1 to A-3, which is at least one selected from the group consisting of MMAE).
Item A-5.
The antibody of the antibody-drug complex and the drug are linked by a linker that is cleaved in lysosome (preferably, a linker that is cleaved by an acidic cleavable linker or a linker that is cleaved by an intralysosomal enzyme) The enhancer according to any one of Items A-1 to A-4.
Item A-6.
Item A-1 to A, wherein the antibody of the antibody-drug complex and the drug are linked by at least one selected from the group consisting of a hydrazone linker, a maleimide linker, a thioether linker, a peptide linker, and a combination thereof The enhancer according to any one of -4.
Item A-7.
The antibody of the antibody drug complex is at least one selected from the group consisting of gemtuzumab, trastuzumab, brentuximab, inotuzumab, rituximab, ofatumumab, mogamulizumab, and alemtuzumab, at least one of items A-1 to A-4. An enhancer as described in any one.

Item B-1.
An antibody-drug complex composition used to be administered to a patient in a state where a lysosomal activator is or has been administered.
Item B-2.
The antibody / drug conjugate composition according to Item B-1 wherein the lysosomal activator is an mTOR inhibitor.
Item B-3.
The antibody-drug complex composition according to Item B-1, wherein the lysosomal activating agent is at least one selected from the group consisting of rapamycin, temsirolimus, everolimus, PP242, Torin1, AZD2014, and MLN0128.
Item B-4.
The drug of the antibody-drug complex is an anticancer drug (eg, calicheamicin, esperamycin, emtansine, auristatin F-HPA (F-hydroxypropylamide), a derivative of DX-8951 (DXd), and monomethyl auristatin E ( The antibody-drug complex composition according to any one of Items B-1 to B-3, which is at least one selected from the group consisting of MMAE).
Item B-5.
The antibody of the antibody-drug complex and the drug are linked by a linker that is cleaved in lysosome (preferably, a linker that is cleaved by an acidic cleavable linker or a linker that is cleaved by an intralysosomal enzyme) The antibody-drug complex composition according to any one of Items B-1 to B-4.
Item B-6.
Item B-1 to B, wherein the antibody of the antibody-drug complex and the drug are linked by at least one selected from the group consisting of a hydrazone linker, a maleimide linker, a thioether linker, a peptide linker, and a combination thereof The antibody-drug complex composition according to any one of -4.
Item B-7.
The antibody of the antibody-drug complex is at least one selected from the group consisting of gemtuzumab, trastuzumab, brentuximab, inotuzumab, rituximab, ofatumumab, mogamulizumab, and alemtuzumab, any one of paragraphs B-1 to B-4 The antibody-drug complex composition according to any one of the above.

Item C.
An antibody-drug complex according to any one of Items A-1 to A-7, comprising: the enhancer according to any one of Items A-1 to A-7; and the antibody-drug complex composition according to any one of Items B-1 to B-7. A composition or treatment kit for treating a disease to be treated by a drug.

Item D-1.
A leukemia therapeutic composition containing an anti-CD33 antibody-DNA cleaving anticancer drug complex, which is used to be administered to a patient in a state in which a lysosomal activator is or has been administered.
Item D-2.
The leukemia therapeutic composition according to Item D-1, wherein the lysosomal activator is an mTOR inhibitor.
Item D-3.
The leukemia therapeutic composition according to Item D-1 or D-2, wherein the mTOR inhibitor is at least one selected from the group consisting of rapamycin, temsirolimus, everolimus, PP242, Torin1, AZD2014, and MLN0128.
Item D-4.
The leukemia therapeutic composition according to any of paragraphs D-1 to D-3, wherein the leukemia is acute myelogenous leukemia (AML).
Item D-5.
The leukemia therapeutic composition according to any of paragraphs D-1 to D-4, wherein the DNA cleaving anticancer agent is calicheamicin or esperamycin.
Item D-6.
The leukemia therapeutic composition according to any one of Items D-1 to D-5, wherein the anti-CD33 antibody-DNA cleaving anticancer drug complex is gemtuzumab ozogamicin.
Item D-7.
An anti-CD33 antibody-DNA cleavage anti-cancer agent complex effect enhancing composition comprising an anti-CD33 antibody-DNA cleavage anti-cancer agent complex and a lysosomal activator.
Item D-8.
A kit for treating leukemia, comprising an anti-CD33 antibody-DNA cleavage anticancer drug complex and a lysosomal activator.
Item D-9.
An anti-CD33 antibody-DNA cleavage anti-cancer drug complex effect enhancer comprising a lysosomal activator.

According to the present invention, the therapeutic effect of GO (gemtuzumab ozogamicin) as well as the therapeutic effect of an antibody-drug complex other than GO can be enhanced, and there are few side effects.

The percentage of dead cells (specific apoptosis) when treated with cells at each concentration of PP242 is shown. The result of having analyzed the extent of the phosphorylation of AKT protein and S6K protein at the time of a cell process by each density | concentration PP242 analyzed by a western blot is shown. The result of having evaluated the dead cell percentage (specific apoptosis)% is shown, when PP242 single treatment, GO (gemtuzumab ozogamicin) single treatment, or a combination treatment of PP242 and GO is performed on each AML cell line. The results of staining and analysis of each AML cell line (without treatment, treatment with PP242 alone, treatment with GO alone, or treatment with PP242 and GO in combination) with the fluorescent dye LysoTracker (registered trademark) are shown. The mechanism of action of GO (gemtuzumab ozogamicin) is shown. Treatment of acute myeloid leukemia cell line (U937) with AZD2014 alone, GO (gemtuzumab ozogamicin) alone, or combination treatment with AZD2014 and GO, and after 36 hours of culture, Annexin V-PI (propidium iodide) staining It shows the result of having evaluated the dead cell rate (specific apoptosis)% using the flow cytometry. Acute myeloid leukemia cell line (SKM-1) is treated with AZD2014 alone, GO (gemtuzumab ozogamicin) alone, or combined treatment with AZD2014 and GO, and after culture for 36 hours, Annexin V-PI (propidium iodide) It shows the result of performing staining and evaluating dead cell percentage (specific apoptosis)% using flow cytometry. The results of staining and analysis of the U937 cell line (without treatment, treatment with AZD2014 alone, treatment with GO alone, or treatment with AZD2014 and a combination of GOD) using the fluorescent dye LysoTracker (registered trademark) are shown. Primary cultured cells (primary cells) were prepared from 6 patients with AML, and using these AML primary cultured cells (AML cells 1 to 6), it was examined whether the GO effect was enhanced by the combination of AZD2014 and GO. (Percentage of dead cells) is shown. The results of examining whether enhancement of GO effect is observed by combining INK128 and GO by performing Annexin V-PI (propidium iodide) staining after culturing for 48 hours using SKM-1 cells (percentage of dead cells) Show. Using Daudi cells, the mTOR inhibitor PP242 400 nM and the antibody / drug complex inotuzumab ozogamicin (IO) were used alone or in combination at 2 ng / ml. (Percentage of dead cells) is shown.

Hereinafter, each embodiment of the present invention will be described in more detail.

The present invention encompasses antibody drug complex effect enhancers containing lysosomal activators. (It may be referred to as “the enhancer of the present invention”.) Antibody-Drug Conjugate (ADC) is bound to cells recognized by antibody, then taken up into the cell usually by endocytosis, and further in lysosomes. The linker that binds the antibody and the drug is cleaved, and the separated drug exerts its drug effect in cells. Therefore, activating the lysosome can enhance the effect of the antibody-drug complex.

As the lysosome activating agent, one which increases the number of lysosomes per cell (lysosome number increasing agent) is particularly preferable. As such a lysosomal activator, for example, an mTOR inhibitor is preferably mentioned. The above non-patent document 3 suggests that some mTOR inhibitors have the effect of increasing the number of lysosomes per cell. mTOR is a mammalian target of rapamycin in mammals and is one of protein kinases (serine and threonine kinases) involved in intracellular signal transduction. mTOR forms a complex with multiple proteins, and the complex is called mTORC. mTOR forms two functionally different complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). The mTOR inhibitor may be an mTORC1 inhibitor, an mTORC2 inhibitor, or an mTORC dual inhibitor (ie an inhibitor that inhibits both mTORC1 and mTORC2), with the mTORC dual inhibitor being preferred.

As mTOR inhibitors, known mTOR inhibitors can be used. More specifically, for example, rapamycin, temsirolimus, everolimus, PP242 (torquinib), Torin1, AZD2014, MLN0128, ductorisive (BEZ235), AZD8055, SF2523, CZ415, PI-103, KU-0063794, tacrolimus, ridaforolimus, Voxtalisib (SAR 245409, XL 765) Analogue, Omiparisib (GSK 2126458, GSK 458), OSI-027, PF-04691502, Apitrisive (GDC-0980, RG 7422), GSK 1059615, Gedatrisive, WYE-354, Torin 2, WYE-125132 (WYE-132) , BGT226 (NVP-BGT226), P529, PP1 1, WYE-687, WAY-600, ETP-46464, GDC-0349, zotarolimus (ABT-578), Voxtalisib (XL765, SAR245409), chrysophonic acid, CC-223, LY3023414, 3BDO, MHY1485 etc. Can. Rapamycin, temsirolimus and everolimus are mTORC1 inhibitors, and PP242, Torin1, AZD2014 and MLN0128 are mTORC dual inhibitors. mTOR inhibitors can be purchased commercially and used.

The lysosomal activator can be used singly or in combination of two or more.

There is no particular limitation on the antibody-drug conjugate (ADC), and for example, known antibody-drug conjugates can be used. In antibody-drug conjugates, the antibody and the drug are linked by a linker. As the linker, for example, a linker that is cleaved in lysosome is preferable. The linker that is cleaved in lysosome includes, for example, a linker that is linked by a linker that is cleaved in an acidic state or that is cleaved by an enzyme in lysosome. As such a linker, a linker known in the art (particularly in the antibody drug complex field) or a linker under development can be used. As such linkers, more specifically, for example, hydrazone linker (eg, semicarbazone linker), maleimide linker, thioether linker, peptide linker (eg, valine-citrulline (vc) dipeptide linker, valine-alanine (valine) And the like) and the like, and the like) and the like) and the like. Moreover, the linker comprised by these combination is also preferable. For example, a peptide linker and a maleimide linker can be combined and used as one linker (for example, a maleimide linker can be disposed between an antibody and a peptide linker). In addition, these linkers have a structure including the structures (semicarbazone, maleimide, hydrazone, thioether, valine-citrulline dipeptide, valine-alanine dipeptide, glycyn-glycyn-phenylalanyn-glycyn (GGFG) peptide etc.) described in each of them. Is a linker having The hydrazone linker is stable in serum and, after internalization into cells, is hydrolyzed in the acidic environment within the lysosome. The peptide linker is degraded by lysosomal proteases (eg cathepsin B etc). Thioether linkers are not easily cleaved, have excellent stability in serum, and are difficult to release drugs in serum, and thus are considered to be less likely to cause side effects. After being taken up by tumor cells, the whole antibody is degraded in lysosome to release toxin. Alternatively, as the linker, a linker such as Fleximer linker (Mersana Therapeutics) can also be used.

Also, known linkers listed in, for example, Int J Mol Sci. 2016 Apr; 17 (4): 561 or linkers that can be easily conceived from the known linkers can be used.
The linker contained in the antibody drug complex may be one kind alone, or two or more kinds may be combined. One or more of the above linkers may be disposed in one linker. In addition, in one antibody-drug complex, all of the linkers that bind the antibody and the drug may be the same, or two or more types of linkers may be used.

Further, the drug (payload) of the antibody-drug complex is not particularly limited, but an anticancer drug is preferably exemplified. As the anticancer agent, for example, one which exerts an anticancer effect by cleaving DNA (a DNA cleaving anticancer agent) is preferably mentioned, but there is no particular limitation. Furthermore, as anticancer agents, more specifically, for example, calicheamicin, esperamycin, emtansine, auristatin F-HPA (F-hydroxypropylamide), a derivative of DX-8951 (DXd), monomethyl auristatin E ( MMAE) and the like can be mentioned.

The antibody may be a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody. When the present invention is applied to human, the antibody is preferably a chimeric antibody, a humanized antibody or a fully humanized antibody (human antibody). These antibodies can be produced by known methods or methods easily conceived from known methods. Although not particularly limited, the contents described in, for example, WO2008 / 026603, WO2009 / 041643 and the like can be referred to.

Preferably, the antibody and the drug that make up the antibody drug complex are related to each other. That is, it is preferable that the antibody recognizes a site where the drug effect is particularly required. For example, when the drug of the antibody drug complex is an anticancer drug, the antibody of the antibody drug complex is preferably an antibody that recognizes a cancer cell, and more preferably an antibody that specifically binds to a cancer cell. An antibody that recognizes such cancer cells can be appropriately prepared using known cancer cell markers as antigens. Although not particularly limited, for example, anti-CD33 antibody, anti-CD30 antibody, anti-CD22 antibody, anti-Her2 antibody and the like can be mentioned. When the cancer is leukemia, anti-CD33 antibody, anti-CD30 antibody, anti-CD22 antibody and the like are particularly preferable, and when the cancer is breast cancer and the like, the anti-Her2 antibody and the like are particularly preferable.

Although not particularly limited, as the antibody, more specifically, gemtuzumab, trastuzumab, brentuximab, inotuzumab, rituximab, ofatumumab, mogamulizumab, alemtuzumab and the like are preferably mentioned.

Also, although not particularly limited, as the antibody-drug complex, more specifically, for example, Gemtuzumab ozogamicin (product name Myrotag (registered trademark)), Trastuzumab emtansine (product name Kadthyra (registered trademark)), XMT-1522, DS-8201a, Brentuximab bedotin (product name: Adcetris (registered trademark)), inotuzumab ozogamicin (product name: Besponsa), and the like. However, there is no particular limitation on these. For example,

A known antibody drug conjugate can be searched, and a known antibody drug conjugate obtained as a search result can also be preferably used in the present invention. For example,

The known antibody-drug conjugates are grouped together, and these can also be preferably used in the present invention.

The potentiator of the present invention may consist of only the lysosomal activator, or may contain other components other than the lysosomal activator. The other ingredients are not particularly limited, and pharmaceutically acceptable bases, carriers, additives (eg, excipients, binders, disintegrants, lubricants, solvents, sweeteners, coloring agents, flavoring agents, odorants) Agents, surfactants, moisturizers, preservatives, pH adjusters, thickening agents, and the like. Such base materials, carriers, additives and the like are specifically described, for example, in the Pharmaceutical Additives Dictionary 2016 (Yakuji Nipponsha Co., Ltd.) and the like, and for example, those described therein can be used. The form of the preparation is also not particularly limited, and the active ingredient and other ingredients are mixed in a conventional manner, for example, tablets, coated tablets, powders, granules, fine granules, capsules, pills, solutions, suspensions, emulsions It can be prepared into a formulation such as a jelly, a chewable, a soft tablet and the like. As a route of administration of the enhancer of the present invention, for example, oral administration or intravenous administration is preferable.

The enhancer of the present invention is preferably administered to a patient in an amount capable of activating (particularly increasing the number of lysosomes) to the patient by the lysosomal activator contained, and various lysosomal activators based on the degree of activation. The dose and route of administration can be set as appropriate. And according to this, the dose and administration route of the enhancer of this invention can also be set suitably. In particular, with regard to lysosomal activators for which human administration studies have already been conducted, the dose and administration route used in the administration studies can also be appropriately set by reference. For example, AZD2014 is an orally administrable drug in clinical trials by AstraZeneca, and Phase 1 test results including PK results have already been published in the literature (above-mentioned non-patent document 2). The recommended dose (recommended dose, RD) is 50 mg twice a day, which can be used as a reference. For example, it can be about 50 to 200 mg, 60 to 180 mg, 70 to 160 mg, 80 to 140 mg, or 90 to 120 mg per day for adults. In addition, the daily dose may be administered at once, or may be divided into two, three, or four times a day.

The present invention also relates to an antibody-drug complex composition (composition containing an antibody-drug complex), which is used to be administered to a patient to which a lysosomal activating agent has been or has been administered. Include. The antibody drug complex composition may be a composition consisting only of the antibody drug complex, or may further contain other components. The other components are not particularly limited, and, for example, the same components as the other components in the lysosomal activator described above can be used. In addition, as a pharmaceutically acceptable carrier, for example, sterile water, physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, binder Etc. In particular, sterile compositions for injection can be formulated according to the usual formulation practices using vehicles such as distilled water for injection. More specifically, the aqueous solution for injection includes, for example, physiological saline, glucose, and isotonic solutions containing other adjuvants (eg, D-sorbitol, D-mannose, D-mannitol, sodium chloride). Suitable solubilizers may be used in combination with, for example, alcohols (ethanol etc.), polyalcohols (propylene glycol, polyethylene glycol etc.), nonionic surfactants (polysorbate 80 (TM), HCO-50 etc.). Further, examples of the oily liquid include sesame oil, soybean oil and the like, and benzyl benzoate and / or benzyl alcohol may be used in combination as a solubilizing agent. They may also be formulated with buffers (eg, phosphate buffer and sodium acetate buffer), soothing agents (eg, procaine hydrochloride), stabilizers (eg, benzyl alcohol and phenol), and antioxidants.

The lysosomal active agent and antibody-drug complex used in the antibody-drug complex composition are the same as described above.

Hereinafter, a composition containing an anti-CD33 antibody-DNA cleaving anticancer drug complex (that is, a complex in which an anti-CD33 antibody and a DNA cleaving anticancer drug are bound) is particularly preferable as the antibody drug complex composition. This will be described in detail as an example. By way of explanation using the example, those skilled in the art can understand the entire antibody-drug complex composition used to be administered to a patient in a state in which a lysosomal activator is or has been administered. That is, in the description using the example, the applicable portion of not only the example but also the other antibody drug complex composition applies to the other antibody drug complex composition.

A composition having a leukemia cell recognition antibody (preferably a leukemia cell specific antibody) -anticancer drug complex is useful as a leukemia treatment composition. Hereinafter, such a composition may be referred to as "the leukemia therapeutic composition of the present invention". Above all, a composition containing an anti-CD33 antibody-DNA cleaving anticancer drug complex is particularly useful as a leukemia therapeutic composition. Hereinafter, although a composition containing an anti-CD33 antibody-DNA cleaving anticancer drug complex is typically described in detail, the leukemia composition of the present invention is not limited thereto, and those skilled in the art are not For example, it can be understood that the applicable portion of the antibody-drug complex composition using different antibodies and / or drugs is also applicable to the antibody-drug complex composition using different antibodies and / or drugs. For example, in the description, the description “anti-CD33 antibody-DNA cleavage anticancer drug complex” means “cancer cell recognition antibody (preferably cancer cell specific antibody) -anticancer drug complex”, “leukemic cell recognition” It can be read as an antibody (preferably leukemia cell specific antibody) -anticancer drug complex "," leukemia cell recognition antibody (preferably leukemia cell specific antibody) -DNA cleavage anticancer drug complex "and the like.

The anti-CD33 antibody may be any antibody that recognizes the CD33 antigen, and is preferably an antibody that specifically recognizes the CD33 antigen. Also, the antibody may be a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody. When the present invention is applied to human, the antibody is preferably a chimeric antibody, a humanized antibody or a fully humanized antibody (human antibody). These antibodies can be produced by known methods or methods easily conceived from known methods. Although not particularly limited, the contents described in, for example, WO2008 / 026603, WO2009 / 041643 and the like can be referred to.

DNA cleaving anticancer agents are anticancer agents that exhibit an anticancer effect by cleaving DNA. As such an anticancer agent, a known one can be preferably used. Among them, calicheamicin or esperamycin is preferred, and calicheamicin (especially ozogamicin) is more preferred.

The anti-CD33 antibody and the DNA cleaving anticancer agent are preferably linked by a linker that is cleaved (hydrolyzed) in an acidic state, or linked by a linker that is cleaved by an enzyme in lysosome. Examples of such a linker are as described above, and examples thereof include a maleimide linker, a hydrazone linker (for example, a semicarbazone linker), a thioether linker and the like, and among them, a hydrazone bond is preferable.

As the anti-CD33 antibody-DNA cleaving anticancer drug complex, for example, a complex having a structure in which the anti-CD33 antibody is bound to the DNA cleaving anticancer drug by the binding can be preferably used. In particular, Gemtuzumab ozogamicin (GO: trade name “Mylotag” (registered trademark)) is preferable.

The leukemia to be treated with the composition for treating leukemia of the present invention is preferably leukemia (CD33 positive leukemia) in which leukemia cells expressing anti-CD33 antigen are present (CD33 positive leukemia in acute myeloid leukemia (AML)). It is particularly preferred for treatment because of the high proportion of cells.

The anti-CD33 antibody-DNA cleaving anticancer drug complex can be used singly or in combination of two or more.

The leukemia therapeutic composition of the present invention is used to be administered to a patient in a (i) state or (ii) a state in which a lysosomal activator is administered. That is, the leukemia therapeutic composition of the present invention may be used to (i) be administered to a patient to which a lysosomal activator has already been administered, or (ii) a lysosomal activator has not been administered yet. May be used to be administered to a patient scheduled to be administered. In any case, the administration interval between the lysosomal activator and the leukemia therapeutic composition of the present invention is not particularly limited as long as the effects of the present invention are not impaired. For example, the administration interval may be about two weeks, 1.5 weeks, one week, 6, 5, 4, 3, 2, or 1 day. In addition, the anti-CD33 antibody-DNA cleaving anticancer drug complex contained in the leukemia therapeutic composition of the present invention in the state where the lysosome in leukemia cells is activated (particularly, the number of lysosomes is increased) by the lysosomal activator. The interval may be such that the body can reach leukemia cells. For example, the administration interval between the lysosomal activator and the leukemia therapeutic composition of the present invention is preferably within 6, 5, 4, 3, 2, or 1 hour, and more preferably within 30 minutes. It is further preferred that it be within 15 minutes, and even more preferred that it be administered sequentially or simultaneously within 3 minutes. The use of (ii) above includes co-administration with a lysosomal activator.

The dosage of the leukemia therapeutic composition of the present invention can be appropriately set based on the amount of the anti-CD33 antibody-DNA cleavage anticancer drug complex contained. That is, the dose of the leukemia therapeutic composition of the present invention may be adjusted so that the dose of the anti-CD33 antibody-DNA cleaving anticancer drug complex contained can exert an anticancer effect. In addition, intravenous administration is preferred as the administration route. In particular, when the anti-CD33 antibody-DNA cleaving anticancer drug complex is GO, for example, the dose of GO is preferably 1 to 20 mg / m ² per adult day, 2, 3, 4, 5 6, 7, 8, 9, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg / m ² is more preferable. Alternatively, multiple administrations (for example, 2, 3, 4 or 5 administrations) may be carried out, but in such a case, administration 2 to 4 weeks after the previous administration is preferred.

In addition, it is preferable to administer to the patient an amount capable of activating lysosome (in particular, increase the number of lysosomes) to the lysosomal activating agent, and the dosage and administration route of various lysosomal activating agents are appropriately determined It can be set. In particular, with regard to lysosomal activators for which human administration studies have already been conducted, the dose and administration route used in the administration studies can also be appropriately set by reference. For example, AZD2014 is an orally administrable drug in clinical trials by AstraZeneca, and Phase 1 test results including PK results have already been published in the literature (above-mentioned non-patent document 2). The recommended dose (recommended dose, RD) is 50 mg twice a day, which can be used as a reference. For example, it can be about 50 to 200 mg, 60 to 180 mg, 70 to 160 mg, 80 to 140 mg, or 90 to 120 mg per day for adults. In addition, the daily dose may be administered at once, or may be divided into two, three, or four times a day. As a route of administration, oral administration or intravenous administration is preferred.

A composition containing GO, which is used to be administered to a patient to which AZD 2014 has been administered or to be administered, is given as a particularly preferred embodiment of the leukemia therapeutic composition of the present invention. In this case, it is preferable that the dose of AZD 2014 be the above dose and the number of doses, and the dose of GO be also the above dose and the number of doses. For example, a preferred example is a dosing schedule in which GOD is administered 2 or 3 times in 2 weeks (9 to 10 mg in a single dose), and AZD 2014 is dosed twice daily at around 50 mg twice daily. Can.

The leukemia therapeutic composition of the present invention may contain, in addition to the anti-CD33 antibody-DNA cleavage anticancer drug complex, a pharmaceutically acceptable carrier and the like. Examples of the carrier include sterile water and physiological saline, vegetable oils, emulsifiers, suspensions, surfactants, stabilizers, flavors, excipients, vehicles, preservatives, binders, and the like. In particular, sterile compositions for injection can be formulated according to the usual formulation practices using vehicles such as distilled water for injection. More specifically, the aqueous solution for injection includes, for example, physiological saline, glucose, and isotonic solutions containing other adjuvants (eg, D-sorbitol, D-mannose, D-mannitol, sodium chloride). Suitable solubilizers may be used in combination with, for example, alcohols (ethanol etc.), polyalcohols (propylene glycol, polyethylene glycol etc.), nonionic surfactants (polysorbate 80 (TM), HCO-50 etc.). Further, examples of the oily liquid include sesame oil, soybean oil and the like, and benzyl benzoate and / or benzyl alcohol may be used in combination as a solubilizing agent. They may also be formulated with buffers (eg, phosphate buffer and sodium acetate buffer), soothing agents (eg, procaine hydrochloride), stabilizers (eg, benzyl alcohol and phenol), and antioxidants.

Furthermore, the present invention provides an anti-CD33 antibody-DNA cleavage anticancer drug complex effect enhancing composition comprising an anti-CD33 antibody-DNA cleavage anticancer drug complex and a lysosomal activator (ie, an anti-CD33 antibody— Also included are compositions that have an enhanced effect compared to administration of a DNA cleaving anticancer drug complex alone. The anti-CD33 antibody-DNA cleavage anticancer drug complex and the lysosomal activator included in the effect enhancing composition are the same as described above. In addition, the effect-enhancing composition may contain other components other than the anti-CD33 antibody-DNA cleavage anticancer drug complex and the lysosomal activator. As such other components, for example, the same pharmaceutically acceptable carrier in the above-mentioned leukemia therapeutic composition can be used. The administration route of the effect enhancing composition is preferably intravenous administration.
Furthermore, as described above, the description is also valid for antibody-drug complex compositions using different antibodies and / or drugs. That is, the present invention also encompasses the antibody drug conjugate effect enhancing composition, which comprises an antibody drug conjugate and a lysosomal activator.

The present invention also encompasses a kit for treating leukemia, which comprises an anti-CD33 antibody-DNA cleavage anticancer drug complex and a lysosomal activator. The anti-CD33 antibody-DNA cleaving anticancer drug complex and the lysosomal activator provided in the kit are the same as described above. The kit may also contain equipment other than the anti-CD33 antibody-DNA cleavage anticancer drug complex and the lysosomal activator. Such equipment is not particularly limited, and examples thereof include a drip needle and a disinfectant (for example, ethanol).
Furthermore, as described above, the description is also valid for antibody-drug complex compositions using different antibodies and / or drugs. That is, the present invention also encompasses a kit for treating a disease to be treated by a drug of antibody drug complex, comprising an antibody drug complex and a lysosomal activator.

The present invention also encompasses an anti-CD33 antibody-DNA cleavage anti-cancer drug complex effect enhancer comprising a lysosomal activator. The lysosomal activator is the same as described above. In addition, the anti-CD33 antibody-DNA cleaving anticancer drug complex to be enhanced is the same as described above. The effect enhancer may include other components other than the lysosomal activator. As such other components, for example, the same pharmaceutically acceptable carrier in the above-mentioned leukemia therapeutic composition can be used.
Furthermore, as described above, the description is also valid for antibody-drug complex compositions using different antibodies and / or drugs. That is, the present invention also encompasses antibody drug complex effect enhancers, including lysosomal activators.

In the present specification, the term "comprising" also includes "consisting essentially of" and "consisting of" (The term "comprising" includes "consisting essentially of" and "consisting of."). Also, the contents described in the documents and web pages listed in the present specification are incorporated herein by reference. In addition, various characteristics (properties, structures, functions, etc.) described for each embodiment of the present invention described above may be combined in any way in specifying the subject matter included in the present invention. That is, the present invention includes all the subject matter consisting of all combinations of the combinable features described in the present specification.

Hereinafter, the present invention will be described more specifically, but the present invention is not limited to the following examples. The sources of various AML cells used in the study are described below. SKNO-1 (JCRB Cell Bank, Tokyo, Japan), U937 (ATCC, Manassas, VA, USA), THP-1 (ECACC, PD, UK), SKM-1 (established by the inventor).

Investigation of mTOR Inhibitor Use Concentration It is suggested in the above-mentioned Non-Patent Document 3 that some mTOR inhibitors have the effect of increasing the number of lysosomes per cell. The mTOR inhibitor PP242 used in the literature was selected as the mTOR inhibitor used in the present invention, etc., and the PP242 concentration that did not cause cell damage and exhibits the mTOR inhibitory action was examined. PP242 purchased and used a commercial item. The cells were cultured at 37 ° C., 5% CO ₂ and passaged every 48 hours using RPMI 1640 as a culture medium, and used for examination.

Specifically, 100-600 nM of PP242 is added to culture medium of acute myeloid leukemia cell line (U937), and culture is performed for 24 hours, and the concentration which does not cause cell damage as compared with the case of not adding PP242 (control) investigated. The degree of cell damage was determined by comparing the% specific apoptosis. The Annexin V-FITC Apoptosis Detection Kit (Nacalai Tesque, Inc., Japan) was used to examine the percentage of dead cells. Specific apoptosis is the percentage of dead cells calculated by subtracting the natural cell death in the control (normally 4 to 5% of cell death is usually observed naturally in cell line culture) from the cell death measurement value by flow cytometry. It is. More specifically, cells were double-stained with Annexin V-FITC PI (Propidium Iodide), and dead cells were determined by flow cytometry. The examination was performed using a kit: Annexin V-FITC Apoptosis Detection Kit (Nacalai Tesque, Inc., Japan).

Furthermore, in order to confirm whether the mTOR inhibitory effect was obtained in cells cultured at each PP242 concentration, the degree of phosphorylation of AKT protein and S6K protein was analyzed by Western blot. It is well known that inhibition of mTOR reduces phosphorylation of AKT protein and S6K protein, and the phosphorylation is widely used for mTOR inhibition studies. A decrease in phosphorylated S6K indicates mTORC1 inhibition and a decrease in phosphorylated AKT indicates mTORC2 inhibition. The source of the antibody used for Western blot analysis is described below. Phospho-Akt (Ser473) Antibody (CST, # 4060), Akt Antibody (CST, # 9272), Phospho-p70 S6 Kinase (Thr389) Antibody (CST, # 9205), p70 S6K Antibodies (CST, # 9202).

FIG. 1 shows the specific apoptosis% (FIG. 1a) and the result of Western blot analysis (FIG. 1b) in the examination. In FIG. 1a, "Ctr" indicates a control, "P100" indicates PP242 100 nM, and so forth. Also, in FIG. 1 b, “p-AKT” indicates phosphorylated AKT protein, and “(p-) 70S6k” indicates (phosphorylated) S6K protein. From the results, it was confirmed that almost no cell damage occurred at 100 to 500 nM of PP242 (FIG. 1a), and an mTOR inhibitory effect was obtained at 200 to 500 nM (FIG. 1b). Based on the results, PP242 treatment was performed at a concentration of 500 nM unless otherwise specified in the following study.

Examination of treatment with PP242 and GO for each acute myeloid leukemia (AML) cell line PP242 alone treatment, GO for 4 acute myeloid leukemia cell lines (SKNO-1, U937, THP-1, SKM-1) (Gemtuzumab ozogamicin) treatment alone or combined treatment with PP242 and GO, and after 36 hours of culture, annexin V-PI (propidium iodide) staining is performed, and the percentage of dead cells is measured using flow cytometry (specific apoptosis) Evaluated%. The results are shown in FIG. Synergistic effects were observed in the two drug combinations in four cell lines. In FIG. 2, * indicates p <0.001.

The GO treatment concentration was 0.5 μg / ml for THP-1 and SKNO-1, and 2.5 μg / ml for other cell lines. This concentration setting is based on the following circumstances. The blood concentration (Pharmacokinetics, PK) under administration of an approved dose of GO (9 mg / m 2 twice a day for 14 days or more) has been reported (MIROTAGG® Interview Form). According to the PK data, it is preferable to administer 2.5 μg / ml of GO to the AML cell line, since the average minimum blood concentration maintained for 8 hours to 24 hours or more after human administration is about 2.5 μg / ml. did. However, GO was administered at 0.5 μg / ml in a cell line in which the effect was strongly observed. The GO treatment concentration for each AML cell line was the same in the subsequent studies unless otherwise stated.

Analysis of Lysosomes in Acute Myeloid Leukemia (AML) Cell Line The AML cells were stained with the fluorescent dye LysoTracker (registered trademark) (Invitrogen) to examine lysosomes. The fluorescent dye functions to stain an acidic structure in cells and is considered to stain normal acidic lysosome. Specifically, lysosomes of U937 and THP-1 were stained by the fluorescence expression. The results are shown in FIG. In the left panel of FIG. 3, cell corners are blue and lysosomes are red. Further, the right side of FIG. 3 is a graph of the intensity of red fluorescence. From the results, it was considered that the LysoTracke fluorescent spot in AML cells was very small and thus the number of lysosomes was small. In addition, it was also found that, by the PP242 treatment, LysoTracke fluorescent spots were enhanced about 1.5 times in the U937 cell line, and with the PP242 and GO combined treatment, the LysoTracke fluorescence remained enhanced. Similar results were obtained with the THP-1 cell line.

The action mechanism of GO, which is an antibody-drug conjugate (ADC), is digestion of a linker in lysosome. The linker of GO is a hydrazone linker which is hydrolyzed in an acidic environment, and the linker links the anti-CD33 monoclonal antibody and calicheamicin (ozogamicin). When GO binds to leukemia cells to form a GO-CD33 antigen complex, the natural function of the cell is that the GO-CD33 antigen complex is taken up into the cell and the GO-CD33 antigen complex exists in the phagocytes. It becomes (internalization). The phagocytes fuse with lysosomes to become secondary phagocytes, and the linker is hydrolyzed in the acidic environment in lysosomes to release calicheamicin to become active form. Free and active calicheamicin binds to DNA in the nucleus, causing DNA double strand breaks, leading to cell death (FIG. 4). Thus, retention of lysosomal function in leukemia cells is important for GO effect expression.

However, from the results of FIG. 2 and FIG. 3, it is found that the number of lysosomes in AML cells is very small, and that the number of lysosomes in AML cells can be increased by the mTOR inhibitor (specifically, PP242 in this case). It was found that the combination of mTOR inhibitor and GO can induce cell death more effectively (synergistically). Furthermore, the effect is a result of efficiently cleaving the hydrazone linker of GO by increasing the number of lysosomes in AML cells by the mTOR inhibitor, and contributing more calicheamicin to DNA double strand breaks. It was guessed.

Examination of treatment with AZD2014 and GO for each acute myeloid leukemia (AML) cell line Since enhancement of GO effect was observed when PP242 was used in combination with GO, we focused on AZD2014, which is a mTORC dual inhibitor similar to PP242. AZD2014 is a drug under investigation by AstraZeneca, and Phase 1 test results including PK results have already been published in the literature (above Non-Patent Document 2), and can be purchased from a reagent company. The recommended dose (recommended dose, RD) for humans defined in the Phase 1 study of AZD 2014 is 50 mg twice daily. The blood concentration achievable as an average trough value (= minimum of average) 12 hours after 50 mg administration was about 250 nM. In addition, administration of 250 nM AZD2014 alone did not show a strong cell killing effect on AML cell lines (the representative cell line U937 cell line showed no cell killing effect up to 500 nM). Furthermore, when mTORC1 inhibitory effect was examined by Western blot analysis in the same manner as described above, a decrease in phosphorylated S6K (indicating mTORC1 inhibition) and a decrease in phosphorylated AKT (indicating mTORC2 inhibition) were observed with AZD2014 alone treatment with 250 nM. recognized. Therefore, it was decided to administer AZD2014 250 nM to the AML cell line. (GO was administered 2.5 μg / ml to the U937 cell line as described above.)
Acute myelogenous leukemia cell line (U937 or SKM-1) is treated with AZD2014 alone, GO (gemtuzumab ozogamicin) alone, or combined treatment with AZD2014 and GO, and cultured for 36 hours after Annexin V-PI (propidium) Iodide) staining was performed to assess the% specific apoptosis using flow cytometry. The results are shown in Figure 5a (U937) and Figure 5b (SKM-1).

Furthermore, in the same manner as described above, lysosomes are stained by staining each U937 cell (without treatment, treatment with AZD2014 alone, treatment with GO alone, or treatment with AZD2014 in combination with AZD2014) with the fluorescent dye LysoTracker (registered trademark) (Invitrogen) went. The results are shown in FIG. In the left panel of FIG. 6, cell nuclei are blue and lysosomes are red. Further, the right side of FIG. 6 is a graph of the intensity of red fluorescence (a relative value with the control being 1).

From the results of FIG. 5 and FIG. 6, it can be confirmed that the GO effect is enhanced by the combined use with GO also when AZD2014 is used as the mTOR inhibitor. Furthermore, the enhancement of the effect is the result that the hydrazone linker of GO was efficiently cleaved by increasing the number of lysosomes in AML cells by the mTOR inhibitor, and more calicheamicin contributed to DNA double strand cleavage. It was corroborated to be

Furthermore, primary cultured cells (primary cells) are prepared from 6 patients with AML, and using the AML primary cultured cells (AML cells 1 to 6), the GO effect is enhanced by the combination of AZD2014 and GO in the same manner as above. We examined whether it was recognized. The results (percentage of dead cells) are graphed and shown in FIG. Also from the results, it can be confirmed that the GO effect is enhanced by the combined use of the mTOR inhibitor (AZD2014) and GO. The preparation of primary culture cells was performed according to the method described in the above-mentioned supplementary materials and methods of Non-Patent Document 1 (Leukemia (2013) 27, 233-235).

Confirmation of combined effect with various mTOR inhibitors and antibody drug complexes As MLOR 128, instead of PP242, MLN 0128 (also referred to as INK128 or TAK-228) is used to obtain the above "acute myeloid leukemia (AML) cells" The LysoTracke fluorescent spot is enhanced in the U937 cell line by INK128 treatment (concentration 50 nM) when examined in the same manner as the method described in “Analytical analysis of lysosomes in strains” (cells using U937), even with INK128 and GO combination treatment Almost unchanged, it was confirmed that LysoTracke fluorescence remained enhanced.

Furthermore, using SKM-1 cells (myelodysplastic syndrome, ie, cells derived from a preleukemic state due to an abnormality in bone marrow function), the above procedure was repeated except that Annexin V-PI (propidium iodide) staining was performed after 48 hours of culture. In the same manner as in the above, it was examined whether the combination of INK128 and GO could enhance the GO effect. The results (percentage of dead cells) are graphed and shown in FIG. 8 (** P <0.01, *** P <0.001; compared with GO alone). Also from the results, it can be confirmed that the GO effect is enhanced by the combined use of the mTOR inhibitor (INK128) and GO.

Furthermore, using Daudi cells (B-cell tumor Burkitt's lymphoma-derived cells), the mTOR inhibitor PP242 400 nM and the antibody drug complex inotuzumab ozogamicin (IO) 2 ng / ml alone or in combination In the same manner as described above, it was examined whether these combined use could enhance the IO effect. The results (percentage of dead cells) are graphed and shown in FIG. 9 (left: PP242, center: IO, right: PP242 + IO). Also from the results, it can be confirmed that the antibody-drug complex effect is enhanced by the combined use of the mTOR inhibitor (PP 242) and the antibody drug complex (IO).

From the above results, it has been confirmed that the lysosomal activator (in particular, the mTOR inhibitor) exerts an effect of enhancing the effect of the antibody-drug complex.

Claims

An antibody drug complex effect enhancer comprising a lysosomal activator.
The enhancer according to claim 1, wherein the lysosomal activator is an mTOR inhibitor.
The enhancer according to claim 1, wherein the lysosomal activator is at least one selected from the group consisting of rapamycin, temsirolimus, everolimus, PP242, Torin1, AZD2014, and MLN0128.
The enhancer according to any one of claims 1 to 3, wherein the drug of the antibody drug complex is an anticancer drug.
The enhancer according to any one of claims 1 to 4, wherein the antibody of the antibody drug complex and the drug are linked by a linker that is cleaved in lysosome.
An antibody-drug complex composition used to be administered to a patient in a state where a lysosomal activator is or has been administered.
The antibody-drug complex composition according to claim 6, wherein the lysosomal activator is an mTOR inhibitor.
The antibody-drug complex composition according to claim 6, wherein the lysosomal activating agent is at least one selected from the group consisting of rapamycin, temsirolimus, everolimus, PP242, Torin1, AZD2014, and MLN0128.
The antibody drug complex composition according to any one of claims 6 to 8, wherein the drug of the antibody drug complex is an anticancer drug.
The antibody drug conjugate composition according to any one of claims 6 to 8, wherein the antibody of the antibody drug conjugate and the drug are linked by a linker that is cleaved in lysosome.
A drug of the antibody-drug complex, comprising the enhancer according to any one of claims 1 to 5 and the antibody-drug complex composition according to any one of claims 6 to 10, is a treatment target A composition or treatment kit for treating a disease.