WO2020027217A1 - Medicament for treating and/or preventing cancer - Google Patents

Abstract

The present invention relates to a medicament for treating and/or preventing a cancer, in particular, a non-treatment-resistant cancer which is used in combination with an immune checkpoint inhibitor, and contains, as an active ingredient, at least one selected from the group consisting of antibodies specific to IL-34, derivatives thereof, and inhibitory nucleic acids to IL-34 with or without an immune checkpoint inhibitor.

Description

Pharmaceutical for treating and / or preventing cancer

The present invention provides CSF-1R and IL-34 by inhibiting IL-34 expression, inhibiting IL-34 activity and / or binding to IL-34 for use in combination with an immune checkpoint inhibitor. A substance that inhibits binding to 34, or a combination of an immune checkpoint inhibitor and the substance as an active ingredient, for treating and / or preventing cancer, particularly a cancer that is not a treatment-resistant cancer. Pharmaceutical.

タ ン パ ク 質 Proteins called immune checkpoint proteins are attracting attention as biomolecules involved in suppressing the immune response in cancer. For example, one type of immune checkpoint protein, Programmed-cell Death 1 (PD-1), is expressed on the surface of activated T cells and expressed on the surface of antigen-presenting cells and cancer cells. -It is involved in down-regulation of T cell activation by binding to L2. PD-L1 is highly expressed in cancers such as lung, stomach, esophagus, colorectal, kidney, bladder, cervical, endometrial, head and neck, malignant Many cancers, including various solid tumors such as melanoma, are thought to bypass cancer immune surveillance and allow cancer cells to proliferate.

The concept of releasing T-cell immunosuppression and enhancing the cancer immune response by inhibiting cancer immune evasion is expected to provide a new cancer immunotherapy that can be applied to a wide variety of cancers. I have. Research and development of immune checkpoint inhibitors such as anti-PD-1 antibodies targeting an immune checkpoint protein, for example, PD-1, have been energetically conducted, and some of them have already been used in clinical practice.

On the other hand, it has been shown that tumor tissue itself suppresses the cancer immune response by tumor-associated macrophages (TAM) that have accumulated around it. TAM is thought to be involved in promoting the growth of cancer cells by suppressing the cancer immune response through the production of immunosuppressive cytokines, in addition to producing cell growth factors and inducing new blood vessels.

M-CSF (Macrophage Colony Stimulating Factor, also called CSF-1) is a kind of bone metabolism-related cytokine, is produced by monocytes and the like, and stimulates the formation of macrophage colonies. M-CSF is also produced in many types of cancer cells, and it is known that the expression level of M-CSF in a cancer tissue is correlated with the level of TAM accumulation in the tissue. Since the receptor that specifically binds to M-CSF is CSF-1R (Colony StimulatingactFactor-1 Receptor), anti-CSF-1R antibody can be used for various cancers such as leukemia, breast cancer, endometrial cancer, It has been proposed to use it for the treatment of prostate cancer, ovarian cancer, colorectal cancer, hepatocellular carcinoma, kidney cancer, multiple myeloma, etc. (Patent Document 1).

Our study reveals that another ligand for CSF-1R, Interleukin-34 (IL-34), is involved in cancer resistance through binding to CSF-1R Was done. Based on this finding, there has been provided a therapeutic resistance-reducing agent for a treatment-resistant cancer, which comprises a substance that inhibits IL-34 such as an siRNA against IL-34 or an anti-IL-34 antibody as an active ingredient (Patent Document 2).

JP 2013-529183 Gazette International publication WO2016 / 204216 pamphlet

An object of the present invention is to provide a medicine that can effectively treat cancer by using it in combination with an immune checkpoint inhibitor.

The present inventors have combined the inhibition of IL-34 with an immune checkpoint inhibitor to provide a therapeutic approach to cancers, including those that are not refractory to cancer, especially the IL-34 receptor CSF-1R It has been found that even for a cancer that does not express, a therapeutic effect superior to the therapeutic effect when an immune checkpoint inhibitor is used alone can be exerted, and the following inventions have been completed.

(1) containing, as an active ingredient, at least one selected from the group consisting of a specific antibody against IL-34 and a derivative thereof and an inhibitory nucleic acid against IL-34 for use in combination with an immune checkpoint inhibitor; For treating and / or preventing cancer.
(2) A cancer treatment comprising as an active ingredient a combination of an immune checkpoint inhibitor and at least one selected from the group consisting of a specific antibody to IL-34 and its derivative and an inhibitory nucleic acid to IL-34. And / or a medicament for prevention.
(3) the immune checkpoint inhibitor targets at least one selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, CD80 and CD86, (1) or (2) The medicament according to item 1.
(4) The medicament according to any one of (1) to (3), wherein the immune checkpoint inhibitor is at least one selected from the group consisting of an anti-PD-1 antibody and an anti-CTLA-4 antibody.
(5) The medicament according to any one of (1) to (4), wherein the cancer is not a treatment-resistant cancer.
(6) The medicament according to any one of (1) to (5), wherein the cancer is not a cancer having a therapeutic resistance to immune checkpoint inhibition therapy.
(7) The medicament according to any one of (1) to (6), wherein the cancer is a CSF-1R negative cancer.

According to the present invention, it is possible to obtain an excellent therapeutic effect exceeding the therapeutic effect of using an immune checkpoint inhibitor alone. Further, according to one embodiment of the present invention, effective treatment can be performed even on a cancer that does not express CSF-1R.

1 is a graph showing IL-34 and M-CSF concentrations in culture supernatants of an ovarian cancer cell line HM-1, its IL-34 knockout strain and an IL-34 overexpressing strain. 1 is a histogram showing the expression of CSF-1R in an ovarian cancer cell line HM-1, its IL-34 knockout strain and an IL-34 overexpressing strain. It is a graph which shows the survival rate of the mouse which transplanted the ovarian cancer cell line HM-1 or its IL-34 knockout strain, and administered anti-PD-1 antibody or control antibody. 1 is a graph showing the expression level of the IL-34 gene in a colon cancer cell line CT26, its IL-34 knockout strain, and an IL-34 overexpressing strain. 1 is a graph showing the M-CSF concentration in the culture supernatant of the colon cancer cell line CT26, its IL-34 knockout strain, and the IL-34 overexpressing strain. 1 is a histogram showing the expression of CSF-1R in a colorectal cancer cell line CT26, its IL-34 knockout strain, and an IL-34 overexpressing strain. It is a graph which shows the change of tumor volume in the mouse | mouth which transplanted the IL-34 knockout strain of the colon cancer cell line CT26 or its IL-34 overexpression strain, and administered the anti-PD-1 antibody or the control antibody. In the mouse transplanted with the IL-34 knockout strain of the colon cancer cell line CT26 or its IL-34 overexpressing strain, and administered with an anti-PD-1 antibody or a control antibody, represents the tumor volume on day 28 after the start of antibody administration. It is a graph. The ovarian cancer cell line HM-1, its IL-34 knockout strain or a strain in which IL-34 is overexpressed in the knockout strain is transplanted, and the abundance of M2 macrophages in mice administered with an anti-PD-1 antibody is shown. It is a graph. Transplant a colon cancer cell line CT26 IL-34 knockout strain or its IL-34 overexpressing strain, and administer a control antibody, an anti-PD-1 antibody, an anti-CTLA-4 antibody alone or an anti-PD-1 antibody and an anti-CTLA-4 antibody. It is a graph which shows the change of the tumor volume in the mouse which co-administered the CTLA-4 antibody. The notation on the right side of the graph lists the mouse groups in descending order of tumor volume on day 8 from top to bottom. Transplant a colon cancer cell line CT26 IL-34 knockout strain or its IL-34 overexpressing strain, and administer a control antibody, an anti-PD-1 antibody, an anti-CTLA-4 antibody alone or an anti-PD-1 antibody and an anti-CTLA-4 antibody. FIG. 9 is a graph showing the tumor volume on day 8 after the start of antibody administration in mice to which CTLA-4 antibody was concurrently administered. FIG. FIG. 2 is a view showing a control plan of a control antibody, an anti-IL-34 antibody, an anti-PD-1 antibody, and an anti-CTLA-4 antibody to mice transplanted with an IL-34 overexpressing strain of a colon cancer cell line CT26. Transplant a colon cancer cell line CT26 IL-34 overexpressing strain and administer control antibody, anti-IL-34 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody alone or anti-IL-34 antibody, anti-PD FIG. 9 is a graph showing changes in tumor volume in mice administered with a combination of the -1 antibody and the anti-CTLA-4 antibody. Transplant a colon cancer cell line CT26 IL-34 overexpressing strain and administer control antibody, anti-IL-34 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody alone or anti-IL-34 antibody, anti-PD FIG. 9 is a graph showing the tumor volume on day 13 after the start of antibody administration in mice administered with a combination of the -1 antibody and the anti-CTLA-4 antibody.

A first aspect of the present invention provides a CSF for inhibiting IL-34 expression, inhibiting IL-34 activity and / or binding to IL-34 for use in combination with an immune checkpoint inhibitor. The present invention relates to a medicament for treating and / or preventing cancer, comprising a substance that inhibits the binding between -1R and IL-34 as an active ingredient. Further, the second aspect of the present invention relates to an immune checkpoint inhibitor, which inhibits the expression of IL-34, inhibits the activity of IL-34, and / or binds to CSF-1R by binding to IL-34. The present invention relates to a medicament for treating and / or preventing cancer, comprising as an active ingredient a combination with a substance that inhibits binding to IL-34. Hereinafter, unless otherwise specified, the details of the present invention will be described using humans as representative examples, including IL-34 and other biological macromolecules and molecular biological mechanisms.

Immune checkpoint inhibitors Immune checkpoint inhibitors target a biological molecule called an immune checkpoint molecule that has the effect of suppressing the immune response to self or an excessive immune response, and inhibit the expression or biological activity of that molecule Or a substance that can be suppressed. As immune checkpoint molecules, PD-L1 / PD-L2 and its receptor PD-1, CD80 / CD86 and its receptor CTLA-4, galectin-9 and its receptor TIM-3, HVEM and its receptor BTLA and the like are known, and in the present invention, at least one selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, CD80 and CD86 Preferably, it is targeted for inhibition. Examples of immune checkpoint inhibitors include low molecular weight compounds that inhibit the physiological activity of immune checkpoint molecules, antibodies that specifically bind to immune checkpoint molecules, and inhibitory nucleic acids that suppress the expression of immune checkpoint molecules. Can be mentioned. Preferred immune checkpoint inhibitors in the present invention are specific antibodies against immune checkpoint molecules, preferably anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies and the like. Typical examples are already used in clinical practice such as nivolumab (anti-PD-1 antibody, trade name: Opdivo), ipilimumab (anti-CTLA-4 antibody, trade name: Yervoy), pembrolizumab (anti-PD-1 antibody, trade name: Keytruda) Antibody. One type of immune checkpoint inhibitor may be used alone, but two or more types are more preferably used in combination.

A substance that suppresses the expression of IL-34, inhibits the activity of IL-34, and / or inhibits the binding between CSF-1R and IL-34 by binding to IL-34. Contains a substance that suppresses the expression of 34, inhibits the activity of IL-34, and / or inhibits the binding of CSF-1R to IL-34 by binding to IL-34. These substances can be selected from the group consisting of nucleic acids, antibodies, antibody derivatives and compounds.

As a substance that suppresses the expression of IL-34, a substance that can suppress mRNA transcription from a gene encoding IL-34, a substance that can degrade the mRNA, and suppresses protein translation from the mRNA Substances that can be used. Typical examples of these substances can be designed and prepared by those skilled in the art based on the nucleotide sequences of mRNA translated into human IL-34 gene and protein registered in NCBI Gene as Gene ID: 146433. And inhibitory nucleic acids such as antisense RNA or siRNA. Examples of the inhibitory nucleic acid include siRNA commercially available from MyBioSource (IL34 siRNA (Human), Cat. #: MBS8239554) or an IL-34 mRNA that hybridizes to a base sequence on IL-34 mRNA targeted by the siRNA. Can be included.

In addition, shRNA containing the base sequence of siRNA, a DNA capable of inducing transcription of the antisense RNA or siRNA by being placed under the control of an appropriate expression promoter, and the base sequence of the antisense RNA or siRNA or the like RNA partially modified to have enhanced stability against degradation by nucleases is also included in the substance that suppresses expression according to the present invention, as a nucleic acid functionally equivalent to the antisense RNA or siRNA. Modifications for improving stability against degradation by nucleases include 2'O-methylation, 2'-F-formation, 4'-thiolation and the like.

{Furthermore, a chimeric RNA in which a part of the ribonucleotide of the above RNA is replaced with a corresponding deoxyribonucleotide or nucleotide analog is also included in the substance for suppressing expression according to the present invention. Examples of the nucleotide analog include 5-position-modified uridine or cytidine such as 5- (2-amino) propyluridine, 5-bromouridine and the like; 8-position-modified adenosine or guanosine such as 8-bromoguanosine and the like; deazanucleotides, Examples include 7-deaza-adenosine and the like; O- or N-alkylated nucleotides such as N6-methyladenosine.

種類 In the inhibitory nucleic acid, the type or number of the modified or substituted base is not particularly limited as long as the ability to suppress the expression of IL-34 is not lost.

The inhibitory nucleic acid can be artificially synthesized using a genetic recombination technique or a chemical synthesis technique. As a method for gene recombination, a method for chemically synthesizing a nucleic acid, a method for synthesizing a non-natural base, or a method for synthesizing a nucleic acid containing the same, a method known to those skilled in the art can be employed. The nucleic acid may be synthesized by using a device such as a so-called DNA synthesizer.

As a substance that inhibits the activity of IL-34 and a substance that inhibits the binding between CSF-1R and IL-34 by binding to IL-34, a low molecular compound that binds to IL-34 and inhibits its activity Or, a specific antibody or an antibody derivative, a low-molecular compound which binds to IL-34 to thereby inhibit the binding between CSF-1R and IL-34, or a specific antibody or an antibody derivative can be given. Examples of such substances are anti-IL-34 antibodies that block the binding of IL-34 to CSF-1R by blocking the CSF-1R binding site on IL-34, at a site different from the binding site. Anti-IL-34 antibodies that bind to IL-34 but sterically hinder the binding between IL-34 and CSF-1R, and derivatives thereof, and the like can be given.

The antibody can be a monoclonal antibody, a chimeric antibody, a humanized antibody or a human antibody, and the antibody derivative can be a Fab, Fab ', F (ab') 2, scFv or F (ab ') derived from the antibody. It may be an antibody fragment such as 2, a diabody, dsFv, a peptide containing CDR, or the like.

Antibody can be prepared by a general antibody preparation method including immunizing a suitable experimental animal such as rabbit, mouse, rat or the like, preferably using IL-34 produced by a genetic recombination technique as an antigen. Alternatively, anti-IL-34 antibodies described in known literature, for example, Tables 2015-510510 and WO2013 / 119716, which are incorporated herein by reference in their entireties, commercially available from R & D Systems. Human-IL-34 Antibody (Clone: 578416, Cat. #: MAB5265), IL-34 Monoclonal Antibody (Clone: 578416, Product #: MA5-24259) commercially available from Thermo Fisher Scientific, available from Millipore Anti-IL-34, clone 1D12 Antibody (Clone: 1D12, Cat. #: MABT493), commercially available from BioLegend, anti-human IL-34 Antibody (Clone: E033B8, Cat. #: 361401, 361402), and others An existing anti-IL-34 antibody or an antibody containing the same amino acid sequence as the CDR sequence of these antibodies as a CDR sequence may be used.

The medicament of the present invention suppresses the expression of IL-34, inhibits the activity of IL-34, and / or inhibits the binding between CSF-1R and IL-34, other than the above nucleic acid, antibody or antibody derivative. It may contain substances that can be used. Such a substance can be screened for IL-34 expression suppression ability using an appropriate cell that expresses IL-34, or IL-34 activity inhibition ability or binding inhibition ability using CSF-1R and IL-34. Can be explored through screening.

In a preferred embodiment of the present invention, a substance that suppresses the expression of IL-34, inhibits the activity of IL-34, and / or inhibits the binding of CSF-1R to IL-34 by binding to IL-34 Is at least one selected from the group consisting of a specific antibody to IL-34 and a derivative thereof and an inhibitory nucleic acid to IL-34.

Pharmaceutical or Pharmaceutical Composition The pharmaceutical of the present invention suppresses the expression of IL-34, inhibits the activity of IL-34, and / or binds to IL-34 to bind CSF-1R to IL-34. A substance that inhibits binding or a combination of an immune checkpoint inhibitor and the above substance is contained as an active ingredient, and can be used as a medicament for treating and / or preventing cancer. Here, “containing as an active ingredient” means containing an effective amount of such a substance or a combination containing the substance.

The effective amount of the substance in the medicament of the present invention, when combined with an immune checkpoint inhibitor, exerts the effect of treating and / or preventing cancer exerted when the immune checkpoint inhibitor is used alone. Means an amount that exerts an effect of greater strength, and is appropriately determined according to the type of the substance and the combined immune checkpoint inhibitor, usage, age, sex, body weight, type of cancer and other conditions, etc. You.

用語 As used herein, the term “treatment” encompasses all types of medically acceptable therapeutic intervention for the cure, temporary remission, etc. of a disease. Further, the term "prevention" includes all types of medically acceptable preventive intervention for the purpose of preventing or suppressing the onset or development of a disease. That is, the treatment and / or prevention of cancer refers to medically acceptable interventions for various purposes, including delaying or stopping the progression of cancer, regression or elimination of lesions, prevention of onset or prevention of recurrence, etc. Is included.

The medicament of the present invention may be a subject suffering from or possibly suffering from cancer, for example, rodents including mice, rats, hamsters, guinea pigs, humans, chimpanzees, primates including rhesus monkeys, pigs, cows, goats and horses. Administered to mammals such as domestic animals including sheep, pets including dogs and cats. Preferred subjects are humans.

The cancer that can be treated and / or prevented by the medicament of the present invention can be any type of cancer, and examples include lung cancer, stomach cancer, esophagus cancer, colon cancer, kidney cancer, and bladder. Cancer, ovarian cancer, breast cancer, cervical cancer, endometrial cancer, head and neck cancer, malignant melanoma and the like. In addition, cancer that can be treated and / or prevented by the medicament of the present invention can be cancer at any stage.

In one embodiment of the present invention, the cancer that can be treated and / or prevented does not include multiple myeloma having myeloma cells positive for IL34 and negative for M-CSF. Thus, this embodiment provides for inhibiting the expression of IL-34, inhibiting the activity of IL-34 and / or binding to IL-34 for use in combination with an immune checkpoint inhibitor. A substance that inhibits the binding between IL-34 and IL-34, or a combination of an immune checkpoint inhibitor and the substance as an active ingredient, wherein cancer (however, myeloma cells positive for IL34 expression and negative for M-CSF expression) (Excluding multiple myeloma which has).

In one embodiment of the present invention, the cancer that can be treated and / or prevented is not a treatment-resistant cancer that has treatment resistance to chemotherapy, radiation therapy, immunotherapy and the like. Thus, this embodiment provides for inhibiting the expression of IL-34, inhibiting the activity of IL-34 and / or binding to IL-34 for use in combination with an immune checkpoint inhibitor. And / or prophylaxis of cancer (excluding treatment-resistant cancer), which contains, as an active ingredient, a substance that inhibits the binding between IL-2 and IL-34, or a combination of an immune checkpoint inhibitor and the substance. It can also be expressed as a medicine for In a further specific embodiment, there is also provided a medicament for treating and / or preventing non-therapeutic cancer, comprising the substance or the combination as an active ingredient. In these embodiments, the treatment resistance is preferably treatment resistance to immune checkpoint inhibition therapy and acquired treatment resistance.

The resistance to cancer treatment can be confirmed by methods known to those skilled in the art. For example, cancer cells collected from a patient can be treated with appropriate treatment, such as treatment-resistant cancer that does not respond to treatment-resistant cancer. For cancer that is not cancer, it can be confirmed by irradiating an anticancer agent or an immunotherapeutic agent or radiation at a dose that can be expected to be effective, and observing the degree of survival or proliferation of the cancer cells.

In another embodiment, the cancer that can be treated and / or prevented by the medicament of the present invention is a CSF-1R-negative cancer. Here, a CSF-1R-negative cancer is a cancer in which CSF-1R is not substantially expressed.For example, expression of CSF-1R is determined by a molecular biological technique such as RT-PCR or immunological detection. A cancer that cannot be detected or whose expression level is low.

In yet another embodiment, the cancer that can be treated and / or prevented by the medicament of the present invention is an M-CSF positive cancer. Here, an M-CSF-positive cancer is a cancer in which M-CSF is substantially expressed.For example, expression of M-CSF is determined by a molecular biological technique such as RT-PCR or immunological detection. A cancer that can be detected.

The medicament of the present invention comprises, in addition to the above-mentioned active ingredients, drugs or buffers other than the active ingredients, antioxidants, preservatives, proteins, hydrophilic polymers, amino acids, chelating agents, nonionic surfactants, A pharmaceutical composition can be formed or formulated with pharmaceutically acceptable components such as excipients, stabilizers and carriers, and used. Pharmaceutically acceptable components are well-known to those skilled in the art, and those skilled in the art can convert the components according to the form of the preparation from the components described in, for example, the Japanese Pharmacopoeia (17th Edition) and other standards within the scope of ordinary practicing ability. Can be appropriately selected for use.

剤 The dosage form of the medicament of the present invention or a pharmaceutical composition containing the same is arbitrary and can be appropriately selected depending on the target site, the type of cancer, and the like. The dosage form is generally preferably a parenteral preparation, and may be, for example, an injection, a transdermal preparation, an enteral preparation, a drip, and the like. The administration route of the medicament of the present invention is not particularly limited. In the case of a parenteral preparation, for example, intravascular administration (preferably intravenous administration), intraperitoneal administration, intestinal administration, subcutaneous administration, administration to a target site, or the like. Local administration and the like can be mentioned. In one of the preferred embodiments, the medicament of the present invention is administered to a living body by intravenous administration or local administration. The dose of the medicament of the present invention or a pharmaceutical composition containing the same is appropriately selected depending on the usage, age, sex, body weight, type of cancer and other conditions of the subject.

医 薬 The medicament of the first aspect of the present invention or a pharmaceutical composition containing the medicament is for use in combination with an immune checkpoint inhibitor. A medicament "for use in combination with an immune checkpoint inhibitor" may be a medicament in a state before it is actually combined, i.e., a combination medicament, as long as it is intended to be used in combination with an immune checkpoint inhibitor. Pharmaceuticals used for the production of, and pharmaceuticals manufactured or sold for use in combination are also within the scope of the present invention. The expression “used in combination with an immune checkpoint inhibitor” as used herein means that it is necessary to use it, that is, for a patient in whom treatment and / or prevention of cancer is desired, together with or separately from the immune checkpoint inhibitor. At the same time or sequentially.

医 薬 The medicament of the second aspect of the present invention or a pharmaceutical composition containing the same contains, as an active ingredient, a combination of an immune checkpoint inhibitor and the above substance. A medicament "containing a combination as an active ingredient" is a medicament prepared for the purpose of administering an immune checkpoint inhibitor and said substance together or separately, simultaneously or sequentially to a subject in need thereof. As long as it is a combination preparation containing the immune checkpoint inhibitor and the substance independently formulated, or a combination preparation containing the immune checkpoint inhibitor and the substance as long as it can be formulated. Good.

In the present invention, the amount of the immune checkpoint inhibitor may be an amount capable of treating and / or preventing cancer when combined with the above substance, and is generally equivalent to the amount when used alone. Or less. The use of an immune checkpoint inhibitor in an amount equivalent to that used alone resulted in a stronger effect on cancer, and no effect could be confirmed in a shorter time or when used alone In a subject, cancer can be treated and / or prevented. Also, using an amount of an immune checkpoint inhibitor that is less than that used alone has the advantage that the amount of the immune checkpoint inhibitor can be reduced while maintaining its effect on cancer.

に お い て In the medicament of the present invention, one type of immune checkpoint inhibitor may be used alone, or two or more types may be used in combination. “Use in combination” means that two or more immune checkpoint inhibitors are administered together or separately, simultaneously or sequentially to a patient in need thereof, ie, in whom treatment and / or prevention of cancer is desired. To be administered.

Methods for the Treatment and / or Prevention of Cancer The present invention also provides immune checkpoint inhibitors, and suppresses IL-34 expression, inhibits IL-34 activity, and / or binds to IL-34 Comprising administering to the subject in need thereof, together or separately, simultaneously or sequentially, an effective amount of each of the substances that inhibit the binding of CSF-1R to IL-34. A method for treatment and / or prevention is provided as another aspect.

The present invention further provides an immune checkpoint inhibitor, which binds CSF-1R to IL-34 by inhibiting the expression of IL-34, inhibiting the activity of IL-34, and / or binding to IL-34. In yet another aspect, a method for treating and / or preventing cancer, comprising administering to a subject in need thereof an effective amount of a combination with a substance that inhibits cancer.

Enhancing the effects of immune checkpoint inhibitors In one embodiment of the present invention, the expression of CSF-1R and IL is inhibited by inhibiting the expression of IL-34, inhibiting the activity of IL-34 and / or binding to IL-34. Substances that inhibit binding to -34 are considered to have an effect of enhancing the effect of the combined immune checkpoint inhibitor. Therefore, the present invention provides a substance that inhibits the expression of IL-34, inhibits the activity of IL-34, and / or effectively inhibits the binding of CSF-1R to IL-34 by binding to IL-34. An enhancer of an immune checkpoint inhibitor contained as a component; and CSF-1R and IL-34 by suppressing the expression of IL-34, inhibiting the activity of IL-34 and / or binding to IL-34. Administering an effective amount of a substance that inhibits binding to a subject to which an immune checkpoint inhibitor has been or is expected to be administered. Methods for enhancing the effects are provided as separate embodiments. Here, "enhancing the effect of an immune checkpoint inhibitor" and "enhancing agent of an immune checkpoint inhibitor" refer to cancer treatment and / or cancer exerted when the immune checkpoint inhibitor is used alone. Or to exert an effect of a strength exceeding the effect of prevention, and refers to an agent therefor, the effective amount of which is the type of the substance and the combined immune checkpoint inhibitor, usage, age, sex, body weight of the subject, It is determined as appropriate according to the type of the nozzle and other conditions. The meaning of each of the other terms in this embodiment is as described in another preceding embodiment.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1
1) IL34-gene knockout kit via CRISPR, Mouse (-) (SantaCruz, SC-429354), in which a guide RNA for knocking out mouse IL-34 gene was incorporated into pCas-guide vector, was used for ovarian cancer cell line HM -1 (obtained from RIKEN RBC) was transfected using FuGENE (registered trademark) 6 Transfection Reagent (Promega) to prepare an ovarian cancer cell line (HM1 / IL34KO) in which the IL-34 gene was knocked out. In addition, an IL-34 expression vector prepared by incorporating the mouse IL-34 gene (Gene ID: 76527) into a plasmid vector pLenti-EF1a-C-Myc-DDK-IRES-PURO (Origene, PS100085) was added to HM1 / IL34KO. The ovarian cancer cell line (HM1 / IL34OE) overexpressing IL-34 was produced.

HM-1 (hereinafter, also referred to as WT HM-1), HM1 / IL34KO and HM1 / IL34OE were treated with 10% FBS supplemented Mem-alpha (Wako, 135-15175) at 37 ° C. under 5% CO ₂ . Cultured. The supernatant after the culture was collected, and IL-34 and IL-34 in the supernatant were collected using an ELISA kit (Legend MAX mouse IL-34 ELISA kit, catalog no. 439107) and a Mouse HSC Panel (13-plex) (Legendplex 740677). The concentration of M-CSF was quantified. The IL-34 production observed in the WT HM-1 culture supernatant was not observed in the HM1 / IL34KO culture supernatant, but the IL-34 expression level was significantly increased in the HM1 / IL34OE culture supernatant. In addition, knockout of IL-34 in HM1 / IL34KO and overexpression of IL-34 in HM1 / IL34OE were confirmed. In addition, all cell lines produced M-CSF in excess of the detection limit (FIG. 1).

Further, the cultured cells were stained with an APC-labeled anti-CSF-1R antibody (Biolegend, 135510) and an isotype control antibody (Rat IgG2a, 400512), and analyzed by flow cytometry (FACS Aria II, Beckman Coulter). All cell lines were negative for CSF-1R expression (FIG. 2).

2) B6C3F1 mice (female, 6-8 weeks old) were divided into 4 groups (WT IgG (n = 7), KO IgG (n = 6), WT αPD-1 (n = 7), KO αPD-1 (n = 8)), WT HM-1 was assigned to two groups (WT IgG, WT αPD-1), and HM-1 / IL34KO was assigned to the remaining two groups (KO IgG, KO αPD-1) at 1 × each. It was transplanted into 10 ⁵ cells / mouse by intraperitoneal. From the second day after transplantation, control IgG (ChromePure Rat IgG, whole molecule, Jackson ImmunoResearch) was applied to two groups (WT IgG, KO IgG), and the remaining two groups (WT αPD-1, KO αPD-1). PD-1 antibody (RMP1-14, distributed by Dr. Hideo Yagita, Juntendo University School of Medicine) was intraperitoneally administered twice a week at 100 μg / 200 μl / mouse each and bred in a normal breeding environment. FIG. 3 shows the survival rates of the mice in each group when the animals were bred up to 55 days after the transplantation of the cancer cells.

In the group to which HM-1 / IL34KO was transplanted and administered anti-PD-1 antibody (KO αPD-1 mAb), the group to which HM-1 was transplanted and administered control IgG (WT Control IgG) and HM-1 / IL34KO were administered Compared to both the transplanted and control IgG-treated group (KO Control IgG) and the HM-1 transplanted and anti-PD-1 antibody treated group (WT-αPD-1 mAb), a significant increase in survival time was observed. An excellent antitumor effect was confirmed by the combination of the inhibition of IL-34 and the anti-PD-1 antibody.

Example 2
1) In the same manner as in 1) of Example 1, a colorectal cancer cell line (CT26 / CT26) in which the IL-34 gene was knocked out was obtained from a colorectal cancer cell line CT26 (distributed by Dr. Hidemitsu Kitamura). IL-34 gene overexpression strains (CT26 / IL34OE) were prepared by incorporating the IL-34 gene into IL34KO) and CT26 / IL34KO.

CT26, CT26 / IL34KO and CT26 / IL34OE were cultured at 37 ° C. under 5% CO ₂ using 10% FBS supplemented RPMI-1640 (Wako, 189-02025). Collect cells after culture, extract RNA using Tripure Isolation Reagent (Roche Life Science), synthesize cDNA using ReverTra Ace qPCR RT Master Mix (TOYOBO), and use KAPA SYBR Fast qPCR Kit to RT-PCR was performed with the primers.
Mouse Il-34
Forward primer 5'-CTTTGGGAAACCAGAATTTGGAG -3 '(SEQ ID NO: 1)
Reverse primer 5'- GCAATCCTGTAGTTGATGGGGAA -3 '(SEQ ID NO: 2)

While weak IL-34 expression was observed in wild-type CT26, IL-34 expression was abolished in CT26 / IL34KO, and a large increase in IL-34 expression was observed in CT26 / IL34OE, and CT26 / IL34KO Knockout of IL-34 and overexpression of IL-34 in CT26 / IL34OE were confirmed (FIG. 4).

Further, the concentration of M-CSF in the culture supernatant was quantified in the same manner as 1) in Example 1. All cell lines produced M-CSF at or above the detection limit (FIG. 5). Furthermore, the expression of CSF-1R in the cultured cells was analyzed by flow cytometry in the same manner as in 1) of Example 1. In all cell lines, CSF-1R expression was negative (FIG. 6).

2) BALB / c mice (female, 7 weeks old) were divided into 4 groups (KO IgG, OE IgG, KO αPD-1, OE αPD-1; n = 5), and two groups (KO IgG, KO αPD-1) was subcutaneously transplanted with CT26 / IL34KO, and the remaining two groups (OE IgG, OE αPD-1) were subcutaneously transplanted with CT26 / IL34OE at 2 × 10 ⁵ cells / animal. From day 7 after transplantation, control IgG (ChromePure Rat IgG, whole molecule, Jackson ImmunoResearch) was applied to two groups (KO IgG, OE IgG) and anti-control IgG to the remaining two groups (KO αPD-1, OE αPD-1). PD-1 antibody (RMP1-14, distributed by Juntendo University School of Medicine, Dr. Hideo Yagida) was administered intraperitoneally at a dose of 250 μg / mouse each week, and bred in a normal breeding environment. FIG. 7 shows the change in the tumor volume (average value) of the mice in each group up to 28 days after the start of the antibody administration, and FIG. 8 shows the tumor volume of each group on the 28th day after the start of the antibody administration.

In the group that received CT26 / IL34KO and received anti-PD-1 antibody (KO-1αPD-1 mAb), the group that received CT26 / IL34OE and received control IgG (OE Control IgG), and the group that received CT26 / IL34KO and received control IgG The tumor volume was significantly smaller than that of the group administered with KO Control IgG (CT) / CT26 / IL34OE and the group treated with anti-PD-1 antibody (OE αPD-1 mAb). Excellent antitumor effect was confirmed by the combination of inhibition and anti-PD-1 antibody.

Test Example B6C3F1 mice were intraperitoneally administered with HM1, HM1 / IL34KO and HM1 / IL34OE, each at 1 × 10 ⁶ mice / animal. From day 7 after the administration, the intraperitoneal washings of the mice were collected with PBS (including heparin) to prepare a single cell suspension. This was stained with anti-CD11b (Biolegend), anti-F4 / 80 (Biolegend) and anti-CD206 (Biolegend), and the content of M2 macrophages was analyzed by flow cytometry analysis (CD206 positive for CD11b positive and F4 / 80 positive cells). Cell percentage) was measured.

The results are shown in FIG. M2 macrophage content of mice administered HM1 / IL34KO was slightly more than half that of mice administered wild-type HM1, while mice administered HM1 / IL34OE overexpressing IL-34 contained M2 macrophages. The content was equivalent to that of HM1-treated mice.

Both HM-1 cell line and CT26 cell line were M-CSF positive and CSF-1R negative, and the knockout of IL-34 did not dramatically reduce M2 macrophages. There are unknown mechanisms for enhancing the antitumor effect of the anti-PD-1 antibody, other than the inhibition of the interaction between CSF-1R expressed by cancer cells and IL-34 and the inhibition of IL-34-induced TAM accumulation It was speculated that

Example 3
BALB / c mice (female, 7 weeks old, Sankyo Lab Service) in 10 groups (KO-NT, OE-NT, KO IgG, OE IgG, KO αPD-1, OE αPD-1, KO αCTLA-4, OE αCTLA-4, KO αPD-1 & αCTLA-4, OE αPD-1 &αCTLA-4; n = 5). KO-NT, KO IgG, KO αPD-1, KO αCTLA-4, and KO αPD-1 & αCTLA-4 were subcutaneously transplanted with 2 × 10 ⁵ CT26 / IL34KO cells / matrigel, and OE- CT26 / IL34OE suspended in Matrigel was subcutaneously transplanted to NT, OE IgG, OE αPD-1, OE αCTLA-4, and OE αPD-1 & αCTLA-4 at 2 × 10 ⁵ cells / animal.

On the day when the average major axis of the tumor is just under 5 mm (5-7 days after transplantation), the first administration day is KO-IgG and OE-IgG and control IgG (ChromePure-Rat-IgG, whole-molecule, Jackson-ImmunoResearch) once a week. KO αPD-1 and OE αPD-1 with anti-PD-1 antibody (RMP1-14), and KO αCTLA-4 and OE αCTLA-4 with anti-CTLA-4 antibody (UC10-4F10, Juntendo University School of Medicine, KO よ り αPD-1 & αCTLA-4 and OE αPD-1 & αCTLA-4, anti-PD-1 antibody and anti-CTLA-4 antibody, both of which were 250 μg / mouse Each of them was intraperitoneally administered and bred under a normal breeding environment. FIG. 10 shows the change in the tumor volume (average value) of the mice in each group up to 8 days after the start of the antibody administration, and FIG. 11 shows the tumor volume of each group on the 8th day after the start of the antibody administration. The notation on the right side of the graph in FIG. 10 lists the groups of mice in descending order of tumor volume on day 8 from top to bottom.

In the group to which CT26 / IL34OE was transplanted, tumor growth was hardly suppressed even when anti-PD-1 antibody or anti-CTLA-4 antibody was administered alone, and the combined administration of anti-PD-1 antibody and anti-CTLA-4 antibody Tumor growth was suppressed. On the other hand, in the group to which CT26 / IL34KO was transplanted, tumor growth was suppressed by the administration of the anti-PD-1 antibody or the anti-CTLA-4 antibody alone, and particularly, the tumor growth was suppressed by the combined administration of the anti-PD-1 antibody and the anti-CTLA-4 antibody. Was significantly suppressed. From the above, the excellent antitumor effect of the combination of the inhibition of IL-34 and anti-PD-1 antibody or anti-CTLA-4 antibody, and the inhibition of IL-34 and anti-PD-1 antibody and anti-CTLA-4 antibody An even better antitumor effect of the combination was confirmed.

Example 4
BALB / c mice (female, 7 weeks old, Sankyo Lab Service) were divided into eight groups, and CT26 / IL34OE suspended in Matrigel was subcutaneously transplanted at 2 × 10 ⁵ mice / animal. The day when the average major axis of the tumor was just under 5 mm (5 to 7 days after transplantation) was set as the first administration day, and control IgG (ChromePure Rat IgG, whole molecule, Jackson ImmunoResearch), anti-IL- 34 antibody (Millipore, MABT493), anti-PD-1 antibody (RMP1-14), anti-CTLA-4 antibody (UC10-4F10, distributed by Dr. Hideo Yagida, Juntendo University School of Medicine) Bred under the rearing environment. FIG. 13 shows the change in the tumor volume (average value) of the mice in each group up to 13 days after the start of the antibody administration, and FIG. 14 shows the tumor volume of each group on the 13th day after the start of the antibody administration. The notation on the right side of the graph in FIG. 13 lists the mouse groups in descending order of tumor volume on day 13 from top to bottom.

も Also in this example, suppression of tumor growth was observed by co-administering anti-IL-34 antibody with anti-PD-1 antibody or anti-CTLA-4 antibody. In particular, tumor growth was remarkably suppressed by using three kinds of antibodies, an anti-IL-34 antibody, an anti-PD-1 antibody and an anti-CTLA-4 antibody in combination. From the above, the excellent antitumor effect of the combination of the inhibition of IL-34 and anti-PD-1 antibody or anti-CTLA-4 antibody, and the inhibition of IL-34 and anti-PD-1 antibody and anti-CTLA-4 antibody An even better antitumor effect of the combination was confirmed.

Claims (7)

A cancer treatment comprising as an active ingredient at least one selected from the group consisting of a specific antibody against IL-34 and a derivative thereof and an inhibitory nucleic acid against IL-34 for use in combination with an immune checkpoint inhibitor. And / or a medicament for prevention. A cancer treatment and / or, comprising, as an active ingredient, a combination of an immune checkpoint inhibitor and at least one selected from the group consisting of a specific antibody to IL-34 and a derivative thereof and an inhibitory nucleic acid to IL-34. Medications for prevention. The pharmaceutical according to claim 1 or 2, wherein the immune checkpoint inhibitor targets at least one selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, CD80 and CD86. 4. The medicament according to claim 1, wherein the immune checkpoint inhibitor is at least one selected from the group consisting of an anti-PD-1 antibody and an anti-CTLA-4 antibody. The pharmaceutical according to any one of claims 1 to 4, wherein the cancer is not a treatment-resistant cancer. The medicament according to any one of claims 1 to 5, wherein the cancer is not a cancer resistant to treatment for immune checkpoint inhibition therapy. The medicament according to any one of claims 1 to 6, wherein the cancer is a CSF-1R-negative cancer.

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