WO2023176896A1 - Therapeutic drug for fungal infection and method for treating fungal infection - Google Patents

Abstract

Provided is a therapeutic drug for fungal infection, the drug containing human γδT cells as an active ingredient. The therapeutic agent for fungal infection has no adverse effects and tends not to be affected by drug resistance.

Description

Medications for treating filamentous fungal infections and methods for treating filamentous fungal infections

The present invention relates to a therapeutic agent for filamentous fungal infections and a method for treating filamentous fungal infections.
This application claims priority based on Japanese Patent Application No. 2022-043792 filed in Japan on March 18, 2022, the contents of which are incorporated herein.

Filamentous fungal infections, such as aspergillosis and mucormycosis, are diseases with a poor prognosis that are often seen in patients with weakened immune systems, and the standard therapy is the administration of low-molecular antifungal drugs, but cases with a poor prognosis There are many. Furthermore, in recent years, the number of filamentous fungi that are resistant to low-molecular-weight antifungal drugs has been on the rise. In addition, since the proteins that serve as drug discovery targets for filamentous fungi are structurally similar to proteins derived from human cells, it is extremely difficult to develop low-molecular-weight drugs that specifically act against filamentous fungi, resulting in nephrotoxicity. Side effects such as these are also notable. In fact, only one first-in-class drug has been marketed in the past 30 years. Therefore, it is essential to develop new drug modalities different from small molecules.

In recent years, the development of treatments using new approaches, such as immunotherapy and activation of host immunity, has been reported. Basically, fungal infections rarely occur in healthy people with normal immune function, and in patients who have received immunosuppressive drugs during transplantation, or viral infections that target the human immune system. It occurs in patients who have been affected by the disease, as well as in cases where immune function has declined due to aging. Therefore, it is expected that activating the immune system by some method will lead to treatment. In fact, there are reports on adoptive immunotherapy using natural killer (NK) cells whose proliferation is induced using interleukin-2 (IL-2) or the like (Non-Patent Documents 1 to 3).

Non-Patent Document 1 describes as follows. NK cells whose proliferation is induced using IL-2 or the like exhibit toxicity to tumor cells and cells infected with pathogenic microorganisms. Therefore, it is possible to develop therapeutic methods for fungal infections by utilizing the immune effector action or killing action of IL-2-induced NK cells. Specifically, it is possible to treat filamentous fungal infections by injecting IL-2-induced NK cells as adoptive immunotherapy into patients who have become susceptible to infection due to immunosuppressants during hematopoietic stem cell transplantation. There is. However, this literature does not explain how IL-2-induced NK cells control invasive fungal infections caused by fungi such as Aspergillus in patients receiving hematopoietic stem cell infusion therapy. has not been sufficiently clarified. One possibility is that in patients in whom immune tolerance has been induced by fungi such as Aspergillus, NK cells activate innate immune system cells derived from hematopoietic stem cells and their own innate immune system cells, increasing fungal phagocytosis. It is thought that it may enhance sterilization and bactericidal ability, but no scientific evidence has been shown for this. In addition, it is thought that NK cells activate B cells, which are cells of the adaptive immune system, and enhance their antibody production ability, increasing antibody-dependent cytotoxicity and cell phagocytosis, but there is no scientific evidence for this. Nor. Furthermore, NK cells enhance the activation of Th1 type CD4-positive helper T cells, which are cells of the adaptive immune system, and enhance the ability to produce cytokines such as interferon-γ (IFN-γ) and IL-2. It is thought that this may enhance the antifungal activity of the entire immune system, but there is no scientific evidence for this. In addition, NK cells enhance the activation of CD8-positive killer T cells, which are cells of the adaptive immune system, and enhance their ability to produce cytokines such as IFN-γ and tumor necrosis factor-α (TNF-α). , it is also possible that it induces antifungal effects, but no scientific evidence has been shown for this. As described above, there are many unknown points regarding the treatment of filamentous fungal infections using IL-2-induced NK cells.

Non-Patent Document 2 describes as follows. The mechanism of immune action mediated by IL-2-induced NK cells against malignant tumors and viruses has been clarified, but IL-2 and filamentous fungi, especially Aspergillus fungi, which are the main cause of invasive aspergillosis. There is little knowledge regarding the -2-induced interaction with NK cells. Therefore, we investigated how NK cells exhibit antifungal effects. First, we investigated the in vitro interaction between human NK cells and Aspergillus fumigatus, and found that only germinated bacterial cells showed high immunogenicity and were able to induce Th1-like responses, including IFN-γ, TNF-α, etc. It has been shown that this enhances the production of cytokines. Furthermore, when NK cells were primed with human recombinant IL-2 and brought into direct contact with the pathogen, NK cells showed toxicity against A. fumigatus. The most interesting finding is that NK cells are A. When exhibiting a bactericidal effect on S. fumigutus cells, they did not degranulate and release the cytotoxic proteins perforin, granzyme, and granulysin, but through an alternative mechanism using another soluble factor. However, the mechanism is not completely clear. In this study, it was proposed that IFN-γ released by NK cells directly damages A. fumigatus, suggesting that human NK cells and IFN-γ may serve as a new therapeutic tool against fungal infections. . However, although IFN-γ acts on the IFN-γ receptor on human cells and exhibits effector action, it is unlikely that such a receptor exists in filamentous fungi. There are many unknowns regarding the details, such as whether it directly acts on filamentous fungi.

Non-Patent Document 3 describes as follows. Natural killer (NK) cells exhibit antifungal activity against A. fumigatus and, as a result, can enhance the host's defense function. However, little is known regarding the mechanism of interaction between NK cells and A. fumigatus. In this paper, human NK cells and A. fumigatus hyphae are brought into contact and gene expression and protein concentration of specific molecules are evaluated. The results revealed that when NK cells were exposed to A. fumigatus, the transcription and translation of inflammatory protein molecules within the NK cells was increased. However, the post-translational release of inflammatory proteins from NK cells was suppressed by some mechanism, and these molecules accumulated within NK cells and did not act outside the cells. On the other hand, A. fumigatus acted on NK cells and decreased the mRNA level of perforin, but on the contrary, increased its intracellular and extracellular protein concentrations. A. fumigatus also increased gene expression of stress-related molecules such as the heat shock protein HSP90 in human NK cells. These results are the first to reveal that A. fumigatus induces immunosuppression on NK cells, and will be useful for the development of new antifungal treatments. However, in order to effectively carry out the NK cell therapy suggested in this document, it is necessary to produce 1×10 ⁹ to 1×10 ¹⁰ or more NK cells using the GCTP standard, and the methodology has not been established. Therefore, it is currently extremely difficult to deploy it in actual clinical practice.

NK cells are cells that account for approximately 5% to 30% of mononuclear cells present in human peripheral blood, and are characterized by CD56 (+) and CD3 (-). The name NK derives from the fact that it exhibits toxicity to tumor cells and virus-infected cells both in vivo and in vitro without prior stimulation. In addition to acting as pure innate immune system cells, NK cells have also been reported to have properties similar to adaptive immune system cells, such as immunological memory, and have been shown to act as host immune cells against various pathogens ( Mainly responsible for antiviral action).
Based on previous knowledge, it is predicted that when NK cells are used as a cell preparation for antifungal therapy, the effect will be low unless the effector/target ratio is large. Therefore, it is thought that 1×10 ⁹ to 1×10 ¹⁰ or more NK cells are required for one treatment. However, with the conventional proliferation method using IL-2 as an activator, it is difficult to prepare 1×10 ⁹ to 1×10 ¹⁰ or more NK cells. Although methods for proliferating NK cells using genetically modified K562 cells and the like as feeder cells have been devised, a method for preparing NK cells that is compatible with Good Gene, Cellular, and Tissue-based Products Manufacturing Practice (GCTP) has not been established. Therefore, at present, it is extremely difficult to efficiently prepare NK cells as a cell preparation for antifungal therapy.

An object of the present invention is to provide a therapeutic agent for filamentous fungal infections and a method for treating filamentous fungal infections, which have no side effects and are less susceptible to drug resistance.

The present invention includes the following aspects.
[1] A therapeutic agent for filamentous fungal infections containing human γδT cells as the main active ingredient.
[2] The drug for treating filamentous fungal infections according to [1], which is administered to patients with reduced immunity.
[3] The therapeutic agent for filamentous fungal infections according to [2], wherein the patient with decreased immunity is a patient with cellular immunodeficiency.
[4] The drug for treating filamentous fungal infections according to [3], wherein the cellular immunodeficiency patient is an adult T-cell leukemia/lymphoma patient.
[5] The therapeutic agent for a filamentous fungal infection according to any one of [1] to [4], wherein the human γδT cells are cultured human γδT cells.
[6] Use of human γδT cells for producing a therapeutic agent for filamentous fungal infections.
[7] The use according to [6], wherein the human γδT cells are cultured human γδT cells.
[8] Human γδ T cells for use in the treatment of fungal infections.
[9] Cultured human γδT cells for use in the treatment of fungal infections.
[10] A method for treating a filamentous fungal infection, comprising administering a therapeutically effective amount of human γδT cells to a patient in need of treatment.

According to the present invention, it is possible to provide a therapeutic drug for filamentous fungal infections and a method for treating filamentous fungal infections, which have no side effects and are less susceptible to drug resistance.

FIG. 1 is a graph showing the purity and number of γδT cells in the peripheral blood of a healthy person before culture and after 10 days of culture. FIG. 2 is a graph showing the results of co-culture of Af293 and γδT cells.

Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the embodiments described below, and various modifications can be made without departing from the gist of the present invention.

In the present invention, the filamentous fungal infection is a human filamentous fungal infection. The fungal infections to be treated by the present invention are particularly deep-seated fungal infections, such as aspergillosis and mucormycosis.

Aspergillosis is an opportunistic infection caused by inhaling the spores of Aspergillus, a filamentous fungus that is ubiquitous in the environment.The spores germinate, grow, become hyphae, enter blood vessels, and become invasive. The disease causes hemorrhagic necrosis and infarction. They may present with symptoms of asthma, pneumonia, sinusitis, or rapidly progressive systemic disease. Diagnosis is mainly made clinically, but image tests, histopathological tests, and specimen staining and culture may be helpful. Treatment is with voriconazole, amphotericin B (or its lipid formulation), caspofungin, micafungin, or itraconazole. Fungal balls may require surgical removal.
Major risk factors for aspergillosis include long-term neutropenia (typically >7 days), long-term high-dose steroid therapy, and organ transplantation (particularly bone marrow transplantation with graft-versus-host disease [GVHD]). , and genetic disorders related to neutrophil function (eg, chronic granulomatous disease). Aspergillus fungi tend to infect open spaces such as lung cavities, sinuses, and ear canals (e.g., ear canal mycosis) caused by previous lung disease (eg, bronchiectasis, tumors, tuberculosis). Such infections tend to be locally invasive and destructive, but can sometimes lead to systemic dissemination, especially in immunocompromised patients with neutropenia or corticosteroid immunosuppression. Often seen in patients.
Invasive aspergillosis is a fatal disease that occurs in severely immunocompromised hosts, and the mortality rate remains high despite the development of new antifungal drugs and improved treatment strategies. In recent years, bacteria resistant to azole antifungal agents have been on the rise.

Mucormycosis refers to infectious diseases caused by various fungal species belonging to the order Mucorales, such as the genera Rhizopus, Rhizomucor, and Mucor. Symptoms most frequently result from aggressive necrotic lesions of the nose and palate, resulting in pain, fever, orbital cellulitis, proptosis, and purulent nasal discharge. This may be followed by central nervous system symptoms. Pulmonary symptoms are severe and include a productive cough, high fever, and difficulty breathing. Disseminated infection may occur in severely immunocompromised patients. Diagnosis is mainly made clinically, but it is necessary to strongly suspect this condition, and it is confirmed by histopathological examination and culture. Treatment consists of intravenous amphotericin B and surgery to remove necrotic tissue. Despite aggressive treatment, mortality is high. Mucormycosis is most common in immunocompromised individuals, patients with poorly controlled diabetes (particularly those with ketoacidosis), and patients receiving the iron chelator deferoxamine.

[Treatment for filamentous fungal infections]
One embodiment of the present invention is a therapeutic agent for filamentous fungal infections containing human γδT cells as the main active ingredient.

(Human γδT cells)
Human γδT cells, which are the active ingredients of the therapeutic agent of this embodiment, are γδT cells isolated from human peripheral blood or cultured cells thereof. Human γδT cells may be autologous or allogeneic. In other words, as human γδT cells, both γδT cells and cultured cells thereof isolated from a subject to whom the therapeutic drug is administered, as well as γδT cells and cultured cells thereof isolated from a donor other than the subject to whom the therapeutic drug is administered, may be used. I can do it.

Human γδT cells have the following characteristics.
・Accounts for 1-5% of T cells in peripheral blood.
-Characterized by CD4 (-), CD8 (-) (some γδ T cells express CD8 α chain dimer and therefore become weakly positive for CD8), and CD3 (+).
- Expresses the T cell receptor (TCR), which is composed of γ and δ chains.
-Unlike human αβT cells, they are not restricted by major histocompatibility complex (MHC).
- As described below, ex vivo expansion culture is possible using human peripheral blood (proliferation/activation by recognition of phosphoantigen).
・The results of a phase II study in the field of human solid tumors confirmed that there are no major safety concerns.

As human γδT cells, human γδT cells expressing a Vγ9Vδ2-positive T cell receptor (there are two nomenclatures for Vγ, so Vγ9 is also written as Vγ2) are preferred.

When human γδT cells isolated from human peripheral blood are used as cultured cells grown, the culture method is not particularly limited, but the nitrogen-containing bisphosphonic acid prodrug tetrakis-pivaloyloxymethyl 2-(thiazole-2- A method of producing highly enriched human γδT cells using ylamino)ethylidene-1,1-bisphosphonate (PTA) and IL-2 is preferred. Furthermore, it is also possible to enhance the proliferation of γδT cells by adding IL-18 or mutant IL-18 to the expansion culture system. As a specific cell proliferation method, for example, Tanaka et al. Vol., No. 3, p.587-599), Okuno et al. (2020) (Okuno, D., and 13 others, Comparison of a Novel Bisphosphonate Prodrug and Zoledronic Acid in the Induction of Cytotoxicity in Human Vγ2Vδ2 T Cells, Frontiers in Immunology, July 2020, Volume 21, Article 1405) can be used. By these methods, peripheral blood γδT cells can be efficiently proliferated to about 500 to 10,000 times the number of cells with a purity of 90% to 99% or more in 8 to 11 days of culture. Alternatively, one or more of pyrophosphate monoester, phosphoric acid monoester, nitrogen-containing bisphosphonic acid, nitrogen-containing bisphosphonic acid prodrug, alkyl amine, or alkenyl amine is selected instead of PTA, and IL-2 or A combination of one or more of IL-15, IL-18, or their mutants having equivalent effects may be added and cultured. For example, IL-2 variants are disclosed in US 2014/0046026 A1, IL-18 variants are disclosed in EP 0 692 536 A2, EP 0 712 931 A2, EP 0 767 178 A1, and WO 1997/002441 A1, etc. Alternatively, it is disclosed in WO 2022/172944 A1 (PCT/JP2022/005051), but is not limited thereto.

(Target for administration)
The subject to whom the therapeutic agent of this embodiment is administered is not particularly limited as long as it is a patient who has developed a filamentous fungal infection, but patients with weakened immunity are preferred.
The patient with decreased immunity is preferably a patient with cellular immunodeficiency.
The cell-mediated immunodeficiency patient is preferably an adult T-cell leukemia/lymphoma patient.

(Administration method)
The method for administering the therapeutic agent of this embodiment is not particularly limited, but it is preferable to intravenously infuse a cell suspension containing human γδT cells. Although the detailed mechanism of intravenously infused γδT cells is unknown, they tend to accumulate in the lungs and are particularly effective against pulmonary mycosis such as pulmonary aspergillosis.

(Mechanism of action)
The mechanism of action of the therapeutic agent of this embodiment is not particularly limited, but it is thought to be due to direct damaging activity due to contact between γδT cells and hyphae of filamentous fungi.

[Use of human γδT cells to produce a therapeutic drug for fungal infections]
Another embodiment of the invention is the use of human γδ T cells to produce a medicament for the treatment of fungal infections.
Human γδT cells are as described above.

[Human γδT cells for use in the treatment of fungal infections]
Yet another embodiment of the invention is human γδ T cells for use in treating fungal infections.
Human γδT cells are as described above.

[Method for treating filamentous fungal infections]
Yet another embodiment of the invention is a method of treating a fungal infection comprising administering a therapeutically effective amount of human γδ T cells to a patient in need of treatment.
Human γδT cells are as described above.
The therapeutically effective amount of human γδ T cells is not particularly limited, but is preferably 10 ³ to 10 ¹² cells per infusion.

The present invention will be explained in more detail below using Examples, but the present invention is not limited to the Examples described below.

<Materials>
(1) Highly purified Vγ9Vδ2-positive γδT cells.
Peripheral blood Vγ9Vδ2-positive γδT cells from healthy individuals or patients infected with filamentous fungi were induced to proliferate/activated ex vivo in the presence of PTA and IL-2 by the method described above (Tanaka et al. (2017)).
(2) Aspergillus fumigatus Af293 (wild strain) provided by the filamentous fungus American Type Culture Collection (ATCC).

<Experimental method-XTT assay->
(1) Af293 suspension (1×10 ⁵ /mL) was inoculated into a 24-well plate (300 μL/well) and cultured at 37° C. and 5% CO ₂ for 24 hours (resting conidia → hyphae).
(2) The wells were washed with PBS (phosphate buffered saline), and Vγ9Vδ2-positive γδT cells (1×10 ⁷ /well, 1×10 ⁶ /well) were added to each well in which Af293 was cultured. Co-cultured for 24 hours at 5% _CO2 .
(3) After washing the wells with PBS, ice-cold water was added to lyse the effector cells for 30 minutes.
(4) The wells were washed twice with PBS, an appropriate proportion of XTT solution was added, and the wells were left standing at 37° C. in the dark for 24 hours.
(5) OD was measured at 450 nm.

<Results>
FIG. 1 shows the purity and number of γδT cells in the peripheral blood of healthy subjects before culture and 10 days after culture.
Figure 2 shows the results of co-culture of Af293 and γδT cells.
A bactericidal effect on Af293 of Vγ9Vδ2 T cells was observed.

The advantage of the present invention is that cell medicine is developed as a new pharmaceutical modality for treating filamentous fungal infections. Specifically, human peripheral blood mononuclear cells are purified, stimulated with the nitrogen-containing bisphosphonic acid prodrug PTA, and IL-2 is added to expand and culture Vγ9Vδ2-positive γδT cells for use. This disease occurs in patients with immunodeficiency, and the concept of activating the immune system is very logical and is thought to have a therapeutic effect that cannot be expected with antifungal drugs. Not affected.

Claims (10)

1. A therapeutic drug for filamentous fungal infections that contains human γδT cells as an active ingredient.
2. The therapeutic agent for filamentous fungal infections according to claim 1, which is administered to patients with decreased immunity.
3. The drug for treating filamentous fungal infections according to claim 2, wherein the patient with decreased immunity is a patient with cellular immunodeficiency.
4. The drug for treating filamentous fungal infections according to claim 3, wherein the cell-mediated immunodeficiency patient is an adult T-cell leukemia/lymphoma patient.
5. The therapeutic agent for a filamentous fungal infection according to any one of claims 1 to 4, wherein the human γδT cells are cultured human γδT cells.
6. Use of human γδT cells to produce a therapeutic agent for filamentous fungal infections.
7. The use according to claim 6, wherein the human γδT cells are cultured human γδT cells.
8. Human γδ T cells for use in the treatment of fungal infections.
9. Cultured human γδT cells for use in the treatment of fungal infections.
10. A method for treating a filamentous fungal infection, comprising administering a therapeutically effective amount of human γδT cells to a patient in need of treatment.

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