Composition for enhancing antitumor efficacy of car-t cell therapeutic agent, comprising microbiome metabolite

Abstract

The present invention relates to: a composition for enhancing the antitumor efficacy of CAR-T cells, the composition comprising indole-3-pyruvic acid, which is a microbiome metabolite; and a pharmaceutical composition for treating cancer, the pharmaceutical composition comprising indole-3-pyruvic acid and CAR-T cells, wherein the indole-3-pyruvic acid has an excellent CAR-T cell proliferation effect, and thus can enhance the antitumor efficacy of CAR-T cells, and can be used together with CAR-T cells to treat cancer.

Description

Composition for enhancing the antitumor efficacy of CAR-T cell therapy containing microbiome metabolites

[0001] The present invention relates to a composition for enhancing the antitumor efficacy of CAR-T cells comprising indole-3-pyruvic acid, a microbiome metabolite, and a pharmaceutical composition for treating cancer comprising indole-3-pyruvic acid and CAR-T cells.

[0002] CAR-T cells are cell therapies created by utilizing genetic recombination technology to enable a patient's T cells to possess potent anticancer capabilities. In particular, anti-CD19 CAR-T cells (CAR-T19) have demonstrated high complete remission rates (40-80%) in patients with relapsed or refractory B-cell acute lymphoblastic leukemia and B-cell lymphoma. However, they have limitations in terms of universality, as they show significantly lower anticancer efficacy in some patients or fail to maintain stable long-term therapeutic effects, leading to relapse; furthermore, their therapeutic efficacy has not yet been verified for other types of cancer, such as solid tumors. Therefore, there is a need for methods to increase the anticancer efficacy of CAR-T therapies across a wider range of patients and cancer types.

[0003] Under the circumstances described above, the inventors investigated a method to increase the anticancer efficacy of CAR-T therapeutics and, as a result of screening various substances, confirmed that indole-3-pyruvic acid, a microbiome metabolite, promotes the cell proliferation of CAR-T.

[0004] Accordingly, the objective of the present invention is to provide a composition for enhancing the antitumor efficacy of CAR-T cells comprising indole-3-pyruvic acid.

[0005] Another object of the present invention is to a pharmaceutical composition for cancer treatment comprising indole-3-pyruritic acid and CAR-T cells.

[0006] To achieve the above objective, one aspect of the present invention provides a composition for T cell proliferation comprising indole-3-pyruvic acid (IPYA) as an active ingredient. Indole-3-pyruvic acid is a microbiome metabolite produced when intestinal microorganisms metabolize tryptophan and is known as an aryl hydrocarbon receptor (AhR) agonist. However, its effects associated with the proliferation of T cells or lymphocytes are not known.

[0007] T cells (T lymphocytes) are cells that play a crucial role in adaptive immunity. They specifically recognize antigens to directly destroy virus-infected cells or tumor cells (cytotoxic T cells), or to coordinate and direct other immune cells (B cells, macrophages, etc.) to eliminate virus-infected cells or tumor cells (helper T cells). Additionally, they produce memory T cells that remember past infections to enable a faster response in the event of reinfection.

[0008] Recently, chimeric antigen receptors (CARs) are being expressed on T cells and used in tumor treatment. The chimeric antigen receptor (CAR) is a synthetic receptor that combines the antigen recognition ability of antibodies with the cytotoxic function of T cells, and it directly recognizes tumor antigens independently of the major histocompatibility complex (MHC) to convert T cells into potent cytotoxic effector cells.

[0009] According to one embodiment of the present invention, the T cells may express a chimeric antigen receptor (CAR). Specifically, the chimeric antigen receptor (CAR) may be one or more selected from the group consisting of anti-CD19 CAR, anti-PSMA (prostate-specific membrane antigen) CAR, anti-Fra (Folate Receptor-alpha) CAR, anti-BCMA (B-cell maturation antigen) CAR, anti-CD33 CAR, anti-FLT3 (Fms-like tyrosine kinase 3) CAR, anti-CD123 CAR, and anti-GPRC5D (G protein-coupled receptor class C group 5 member D) CAR, but is not limited thereto.

[0010] Meanwhile, the cell proliferation composition of the present invention may additionally include, in addition to the active ingredient indole-3-pyruvic acid (IpyA), ingredients useful for cell growth, such as FBS, cytokines, amino acids, and other trace elements.

[0011] In addition, the composition for cell proliferation of the present invention may be provided in the form of a cell culture medium, and may be provided in a form in which the active ingredient indole-3-pyruvic acid (IpyA) is added to MEM (Minimal Essential Medium), DMEM (Dulbecco's Modified Eagle Medium), BME (Basal Medium Eagle), RPMI, MEM-α (Minimal Essential Medium-α), IMDM (Iscove's Modified Dulbecco's Medium), MacCoy's 5A medium, Swim's S-77 medium, CTS OpTmizer, and EBM (Endothelial Basal Medium) media commonly used for cell culture.

[0012] The inventors isolated PBMC from a donor and produced T cells expressing anti-CD19 CAR, anti-BCMA CAR, anti-PSMA CAR, and anti-Fra CAR, and confirmed that indole-3-pyruvic acid (IpyA) significantly promotes the proliferation of each CAR-T cell.

[0013] Specifically, when CAR-T cells were co-cultured with cancer cells under conditions where indole-3-pyrrous acid (IpyA) was treated at 100 uM, the proliferation of CAR-T cells was significantly increased. Therefore, indole-3-pyrrous acid (IpyA) may be included at concentrations of 10 to 200 uM, 15 to 180 uM, 20 to 150 uM, 25 to 150 uM, 30 to 120 uM, 50 to 120 uM, and 50 to 100 uM based on 1 L of cell culture composition, and preferably at a concentration of 80 to 100 uM.

[0014] The proliferation of CAR-T cells by indole-3-pyrbic acid (IpyA) continued until depletion (Figs. 17 and 18), and given these effects, a composition containing indole-3-pyrbic acid (IpyA) can be usefully used for T cell proliferation.

[0015] In addition, another aspect of the present invention provides a composition for cell proliferation comprising indole-3-pyruvic acid (IpyA) as an active ingredient.

[0016] Another aspect of the present invention provides CAR-T cells treated with the cell proliferation composition.

[0017] The present invention also provides a method for proliferating T cells, comprising the step of co-culturing T cells and cancer cells in the presence of a composition for T cell proliferation containing the indole-3-pyruvic acid (IpyA) as an active ingredient.

[0018]

[0019] Meanwhile, since CAR-T is a cell therapy, it is important to proliferate rapidly in the early stages to effectively control cancer cells, and it is also important to survive continuously for a long period to eliminate remaining cancer cells for the final enhancement of anticancer efficacy. Therefore, the enhancement of CAR-T proliferation by indole-3-pyruvic acid can enhance the anticancer efficacy of CAR-T.

[0020] Accordingly, another aspect of the present invention provides a pharmaceutical composition for cancer treatment comprising indole-3-pyruvic acid and CAR-T cells as active ingredients.

[0021] In the present invention, "prevention" refers to any act of suppressing the occurrence of cancer or delaying the progression of cancer by administering a pharmaceutical composition according to the present invention.

[0022] In the present invention, "treatment" refers to any act in which symptoms of cancer are improved or beneficially altered by the administration of a pharmaceutical composition according to the present invention.

[0023] In the present invention, the cancer may be one or more selected from lymphoma, multiple myeloma, prostate cancer, breast cancer, colorectal cancer, rectal cancer, brain tumor, lung cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, stomach cancer, bladder cancer, ovarian cancer, bile duct cancer, gallbladder cancer, uterine cancer, cervical cancer, head and neck cancer, pancreatic cancer, and squamous cell carcinoma, but is not limited thereto. In the experimental examples of the present invention, lymphoma, multiple myeloma, prostate cancer, and breast cancer cells were used.

[0024] The above CAR-T cells are the same as those described in the above T cell proliferation composition. In the above pharmaceutical composition, the amount of CAR-T cells included in the composition may vary depending on the type of cancer to be treated and the type of CAR.

[0025] Specifically, CAR-T cells, also known as effector cells, recognize antigens on the surface of tumor cells, form immune synapses, and release perforin and granzyme to induce apoptosis in the tumor cells. Since this process is time-consuming and the number of tumor cells a CAR-T cell can process at one time is limited, the ratio of tumor cells (T) to effector cells (T), or the E:T ratio, is important.

[0026] If the number of CAR-T cells is low, the number of tumor cells that must be managed is high, so the consumption of CAR-T cells accelerates and they transition to an exhausted state, failing to maintain sufficient tumor-removing ability.

[0027] According to one embodiment of the present invention, the E:T ratio may be 0.2 to 1.5, 0.4 to 1.2, or 0.4 to 1.0, but is not limited thereto.

[0028] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. In this case, the pharmaceutically acceptable carrier is one that is commonly used in formulations and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, in addition to the above components, it may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc.

[0029] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., by topical application, intravenous, subcutaneous, intraperitoneal injection, or topical application) depending on the intended method, but parenteral administration is preferred.

[0030] When the active ingredient of the present invention is formulated into a preparation such as a tablet, capsule, chewing tablet, powder, liquid, or suspension for oral administration, it may include a binder such as gum arabic, corn starch, microcrystalline cellulose, or gelatin; an excipient such as dicalcium phosphate or lactose; a disintegrant such as alginic acid, corn starch, or potato starch; a lubricant such as magnesium stearate; a sweetener such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavor.

[0031] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, a "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0032] The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, or administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art.

[0033] The present invention also provides a method for treating cancer comprising the step of administering the above-mentioned pharmaceutical composition for cancer treatment to an individual requiring treatment.

[0034] Using a composition containing indole-3-pyrrous acid according to the present invention can increase the antitumor efficacy by proliferating CAR-T cells, and using indole-3-pyrrous acid and CAR-T cells together can also increase the antitumor efficacy.

[0035] Figure 1 shows the results of confirming the degree of cell activity after treating CAR-T cells with various microbiome metabolites.

[0036] Figure 2 shows the results of confirming CAR expression (A), cell doubling time (B), and cell volume (C) after generating anti-CD19 CAR-T cells from donor PBMCs: UTD = untreated group; and iRFP702-CAR19 = lentivirus-treated group containing the anti-CD19 CAR gene.

[0037] Figure 3 shows the results of confirming CAR expression (A), cell doubling time (B), and cell volume (C) after generating anti-BCMA CAR(scFv)-T cells from donor PBMCs: UTD = untreated group; and iRFP702-BCMA CAR = lentivirus-treated group containing the anti-BCMA CAR gene.

[0038] Figure 4 shows the results of confirming CAR expression (A), cell doubling time (B), and cell volume (C) after generating anti-BCMA CAR(VHH)-T cells from donor PBMCs: UTD = untreated group; and iRFP702-Cilta cel = lentivirus-treated group containing the anti-BCMA CAR(VHH) gene.

[0039] Figure 5 shows the results of confirming CAR expression (A), cell doubling time (B), and cell volume (C) after generating anti-PSMA CAR-T cells from donor PBMCs: UTD = untreated group; and iRFP702-PSMA CAR = lentivirus-treated group containing the anti-PCMA CAR gene.

[0040] Figure 6 shows the results of confirming CAR expression (A), cell doubling time (B), and cell volume (C) after generating anti-Fra CAR-T cells from donor PBMCs: UTD = untreated group; and iRFP702-Fra CAR = lentivirus-treated group containing the anti-Fra CAR gene.

[0041] Figure 7 shows the results of confirming the degree of proliferation of anti-CD19 CAR-T cells after co-culturing human leukemia cell line NALM6 cells (tumor cells = target, T) with anti-CD19 CAR-T cells (effector cells = E) at various ratios under treatment with indole-3-pyruvic acid (IPyA).

[0042] Figure 8 shows the results of confirming the number of anti-CD19 CAR-T cells after co-culturing human leukemia cell line NALM6 cells (T) with anti-CD19 CAR-T cells (E) at various ratios under IpyA treatment.

[0043] Figure 9 shows the results of confirming the degree of proliferation of anti-CD19 CAR-T cells after co-culturing human leukemia cell line NALM6 cells with anti-CD19 CAR-T cells derived from various donors under IpyA treatment.

[0044] Figure 10 shows the results of confirming the number of anti-CD19 CAR-T cells after co-culturing NALM6 cells with anti-CD19 CAR-T cells derived from various donors under IpyA treatment.

[0045] Figure 11 shows the results of confirming the degree of proliferation of anti-CD19 CAR-T cells after co-culturing NALM6 cells with anti-CD19 CAR-T cells under treatment with various concentrations of IpyA.

[0046] Figure 12 shows the results of confirming the number of anti-CD19 CAR-T cells after co-culturing NALM6 cells with anti-CD19 CAR-T cells under various concentrations of IpyA treatment.

[0047] Figure 13 shows the results of confirming changes in cytotoxicity of anti-CD19 CAR-T cells after co-culturing NALM6 cells with anti-CD19 CAR-T cells at various ratios (E:T ratio) under IpyA treatment.

[0048] Figure 14 shows the results of confirming changes in cytokine secretion after co-culturing NALM6 cells with anti-CD19 CAR-T cells derived from various donors under IpyA treatment.

[0049] Figure 15 shows the results of confirming changes in cytokine secretion after co-culturing OPM2 cells with anti-BCMA CAR-T cells under IpyA treatment.

[0050] Figure 16 shows the results of confirming changes in cytokine secretion after co-culturing PC3 cells with anti-PSMA CAR-T cells under IpyA treatment.

[0051] Figure 17 shows the results of confirming the degree of proliferation of anti-CD19 CAR-T cells by co-culturing NALM6 cells with anti-CD19 CAR-T cells derived from various donors for a long period under IpyA treatment.

[0052] Figure 18 shows the results of confirming the number of anti-CD19 CAR-T cells while co-culturing NALM6 cells with anti-CD19 CAR-T cells derived from various donors for a long period under IpyA treatment.

[0053] Figure 19 shows the results of confirming the degree of proliferation of anti-CD19 CAR-T cells after co-culturing human lymphoma cell lines MINO, Daudi, SUDHL5, and SUDHL4 with anti-CD19 CAR-T cells under IpyA treatment.

[0054] Figure 20 shows the results of confirming the number of anti-CD19 CAR-T cells after co-culturing MINO, Daudi, SUDHL5, and SUDHL4 cells with anti-CD19 CAR-T cells under IpyA treatment.

[0055] Figure 21 shows the results of confirming the degree of proliferation of CAR-T cells after co-culturing solid tumor cell lines PC3 and OVCAR3 with anti-PSMA or anti-FRa CAR-T cells for 120 hours under IpyA treatment.

[0056] Figure 22 shows the results of confirming the number of CAR-T cells after co-culturing PC3 and OVCAR3 cells with anti-PSMA or anti-FRa CAR-T cells for 120 hours under IpyA treatment.

[0057] Figure 23 shows the results of confirming the degree of proliferation of CAR-T cells after co-culturing PC3 cells with anti-PSMA CAR-T cells for 72 hours under IpyA treatment.

[0058] Figure 22 shows the results of confirming the number of CAR-T cells after co-culturing PC3 cells with anti-PSMA CAR-T cells for 72 hours under IpyA treatment.

[0059] Figure 25 shows the results of confirming the degree of proliferation of anti-BCMA CAR-T cells after co-culturing multiple myeloma cell lines OPM2, MM1S, and RPMI8226 with anti-BCMA CAR-T cells under IpyA treatment.

[0060] Figure 26 shows the results of confirming the number of anti-BCMA CAR-T cells after co-culturing OPM2, MM1S, and RPMI8226 cells with anti-BCMA CAR-T cells under IpyA treatment.

[0061] Figure 27 shows the results of confirming the degree of proliferation and the number of anti-BCMA CAR-T cells after long-term co-culture of OPM2 cells with anti-BCMA CAR-T cells under IpyA treatment.

[0062] Figure 28 shows the results of comparing tumor sizes after administering anti-CD19 CAR-T cells or anti-CD19 CAR-T cells + IpyA to a tumor-induced animal model.

[0063] Figure 29 shows the results of confirming the degree of proliferation of anti-CD19 CAR-T cells after co-culturing NALM6 cells with anti-CD19 CAR-T cells under various indole analog treatments.

[0064] Figure 30 shows the results of confirming the number of anti-CD19 CAR-T cells after co-culturing NALM6 cells with anti-CD19 CAR-T cells under various indole analog treatments.

[0065] Figure 31 shows the results of confirming the degree of proliferation of anti-CD19 CAR-T cells after co-culturing NALM6 cells with anti-CD19 CAR-T cells under treatment with FICZ, an AHR (aryl hydrocarbon receptor) agonist.

[0066] Figure 32 shows the results of confirming the degree of proliferation of anti-CD19 CAR-T cells after co-culturing NALM6 cells with anti-CD19 CAR-T cells under treatment with the AHR antagonist CH-223191.

[0067] One or more specific examples are described in more detail below through embodiments. However, these embodiments are intended to illustrate one or more specific examples and the scope of the present invention is not limited to these embodiments.

[0068]

[0069] Example 1: Microbiome Metabolite Screening

[0070] Substances capable of increasing the activity of CAR-T cells were screened as follows.

[0071] Specifically, CAR-T cells were treated with substances known as microbiome metabolites, and the degree of cell activation was evaluated by confirming the extent of cell proliferation (absolute number and fold change) using flow cytometry. The absolute number refers to the absolute number of living T cells (cells / μL) actually present at a specific time point within a cultured T cell sample, measured by a flow cytometry instrument, and is a key indicator for quantitatively evaluating the degree of proliferation. This indicator is not a simple percentage (%) but a value directly calculated as the number of cells per sample volume using bead-based absolute counting; it represents the number of proliferated living cells after excluding background noise or dead cells. Fold change is expressed as the ratio of the absolute number to the control group. The control group was treated with DMSO of the same composition as the microbiome metabolites instead of the microbiome metabolites.

[0072] As a result of the evaluation, it was found that ascorbic acid, ascorbic acid (sodium salt), gallic acid, and indole-3-pyruvic acid had excellent activation effects, as shown in Figure 1.

[0073] Ascorbic acid and ascorbic acid (sodium salt) are known as Vitamin C and are essential nutrients with antioxidant properties. Since they are not metabolites directly produced by gut microorganisms, they cannot be considered microbiome-derived substances. Additionally, they are substances whose results of combination therapy with CAR-T have been reported in academic papers.

[0074] Gallic acid is a polyphenol metabolite produced by some intestinal microorganisms as they break down tannins. Therefore, it can be considered a microbiome metabolite, but there are research results regarding its use in combination therapy with CAR-T.

[0075] Indole-3-pyruvic acid is a microbiome metabolite produced when gut microbes metabolize tryptophan. Since the results of combination therapy with CAR-T are unknown and it exhibits excellent CAR-T cell activation effects, it was selected as a final candidate substance to evaluate the potential for CAR-T antitumor activity and enhancement. In the following, indole-3-pyruvic acid is referred to as "IpyA".

[0076]

[0077] Example 2: Production of various CAR-T cells

[0078] 2-1. PBMC / T cell isolation

[0079] PBMCs were isolated from the blood of healthy donors using Ficoll. Subsequently, T cells were finally isolated from the PBMCs using the EasySep™ Human T cell Enrichment Kit (STEMCELL).

[0080]

[0081] 2-2. Production of lentiviruses encoding anti-CD19 CAR-T, anti-BCMA CAR-T, anti-PSMA CAR-T, and anti-FRa CAR-T genes

[0082] The lentivirus-producing cell line HEK 293T was cultured at 5% CO₂ and 37°C using GIBCO® RPMI Media-1640 supplemented with 10% FBS, 1% penicillin streptomycin, 1% Glutamax, and 1% HEPES.

[0083] When HEK 293T cells grew to about 60-70%, 116 µl of Lipofectamine 2000 was used to transfect the cells with a plasmid for virus production (plasmids used: 15 µg of expression plasmid with inserted genes for anti-CD19 CAR (SEQ Nos. 1 and 2), anti-BCMA CAR (SEQ Nos. 7 to 10), anti-PSMA CAR (SEQ Nos. 3 and 4), or anti-FRa CAR (SEQ Nos. 5 and 6), 18 µg of Gag / pol, 18 µg of REV (Rev protein), and 7 µg of VSV-G (G protein of Vesicular Stomatitis Virus). The inventors used the above combination of plasmids to achieve high-efficiency CAR gene delivery while minimizing the risk of recombinant virus generation by isolating the viral structure, enzymes, envelope, and nucleation functions. After 24 and 48 hours, the culture media containing the virus were harvested, respectively, and the virus was concentrated by centrifuging at 15,000 g overnight. The concentrated virus was stored at -80℃.

[0084]

[0085] 2-3. Production of T cells expressing anti-CD19 CAR, anti-CD19 CAR, anti-BCMA CAR, anti-PSMA CAR, and anti-FRa CAR

[0086] Anti-CD19 CAR-T cells

[0087] Primary T cells from a healthy donor were activated using anti-CD3 / anti-CD28 beads (Dynabeads™ Human T-Expander CD3 / CD28 Catalog #11141D, Gibco™) at a 1:1 ratio with cells (beads:cells = 1:1). Twenty-four hours after T cell activation, a lentivirus containing the anti-CD19 CAR gene was administered, and the anti-CD3 / anti-CD28 beads were removed five days later. Next, the primary T cells activated with PE-conjugated anti-CD19 CAR antibodies were stained, and CAR expression was confirmed using a flow cytometer.

[0088]

[0089] Anti-BCMA CAR-T cells

[0090] Primary T cells from healthy donors were activated using anti-CD3 / anti-CD28 beads at a 1:1 ratio (beads:cells = 1:1). Twenty-four hours after T cell activation, a lentivirus containing the anti-BCMA CAR gene was administered, and the anti-CD3 / anti-CD28 beads were removed five days later. Next, the scFv-structured BMCA CAR (ABECMA) was treated with an anti-mouse H+L antibody conjugated to Alexa Fluor 647 (Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 647 / Catalog # A-21235, Invitrogen), the VHH-structured BMCA CAR (CARVYKTI) is a PE-conjugated anti-humanized VHH antibody (MonoRab TM Primary T cells activated with Rabbit Anti-Humanized VHH Antibody [PE], mAb / Catalog # A02171-200 FRa CAR, GenScript) were stained, and CAR expression was confirmed using a flow cytometer.

[0091]

[0092] Anti-PSMA CAR-T cells

[0093] Primary T cells from a healthy donor were activated using anti-CD3 / anti-CD28 beads in a 1:1 ratio (beads:cells = 1:1). Twenty-four hours after T cell activation, a lentivirus containing the anti-PSMA CAR gene was administered, and the anti-CD3 / anti-CD28 beads were removed five days later. Next, the activated primary T cells were stained with an anti-mouse H+L antibody conjugated with Alexa Flour 647, and CAR expression was confirmed using a flow cytometer.

[0094]

[0095] Anti-Fra CAR-T cells

[0096] Primary T cells from a healthy donor were activated using anti-CD3 / anti-CD28 beads in a 1:1 ratio (beads:cells = 1:1). 24 hours after T cell activation, a lentivirus containing the anti-FRa CAR gene was administered, and the anti-CD3 / anti-CD28 beads were removed 5 days later. Next, the primary T cells were treated first with a FRA-his tag recombinant protein, then stained with a PE-conjugated anti-his tag antibody (PE anti-His Tag Antibody / Catalog #362603, Biolegend), and CAR expression was confirmed using a flow cytometer.

[0097]

[0098] After that, 0.8×10 every 48 hours 6 cells / ml, 0.5×10 6 Activated primary T cells were subcultured to match the cells / cm² ratio, and when the anti-CD19 CAR-T cells reached a size of 350-400 fl (fluorescence), the cells were frozen and stored in a nitrogen tank (Fig. 2).

[0099] Upon confirming CAR expression, it was found that the proportion of CAR19-positive cells was significantly higher when the anti-CD19 CAR gene expression plasmid was introduced compared to the control group (Fig. 2A). The proliferation cycle of activated primary T cells was found to be somewhat slower than that of the control group (Fig. 2B).

[0100] It was confirmed that the expression of each CAR was increased by gene introduction in anti-BCMA CAR-T cells, anti-PSMA CAR-T cells, and anti-Fra CAR-T cells, and CAR introduction did not significantly affect the cell proliferation cycle (Figs. 3 to 6).

[0101]

[0102] Experimental Example 1: Antitumor activity of combined microbiome metabolite (IpyA) and CAR-T_in vitro

[0103] 1-1. Cell line

[0104] NALM6 cells were used to evaluate the antitumor activity when the anti-CD19 CAR-T produced in Example 2 was combined with a microbiome metabolite. NALM6 cells are a CD19-positive human leukemia cell line derived from a patient with B-cell precursor acute lymphoblastic leukemia (B-ALL). In the experiment, NALM6 CBG GFP cell lines transduced with CBG (Click beetle green luciferase) and GFP (Green fluorescent protein) were used, and will be referred to as "NALM6 cells" below.

[0105] All cell lines are GIBCO ® Incubated in RPMI Media 1640 supplemented with 10% FBS, 1% penicillin streptomycin, 1% glutamate, and 1% HEPES at 5% CO₂ and 37°C.

[0106]

[0107] 1-2. Evaluation of the effect of IpyA on the proliferation of anti-CD19 CAR-T cells (Proliferation assay)

[0108] Confirmation of the ratio of effector cells to tumor cells (effector cell:tumor cell = E:T)

[0109] CAR-T therapy is an immunotherapy that attacks cancer by attaching a 'CAR (Chimeric Antigen Receptor)' that recognizes cancer cells to a patient's T cells, proliferating them, and then re-injecting them. It shows a high response rate, particularly in blood cancers. Cells that actually perform anticancer functions are also called effector cells. Since CAR-T is a cell therapy that involves living, moving cells seeking out and killing tumors rather than drugs, the ratio of effector cells to tumor cells is a crucial factor in the anticancer effect. Accordingly, the inventors confirmed an effective E:T ratio.

[0110] NALM6 CBG GFP cells 0.05X10 6 The cells were seeded into 96-well plates at various concentrations. The number of anti-CD19 CAR-T cells was set so that the E:T ratio (T=1) was 0.125, 0.25, 0.5, or 1. Subsequently, the cells were treated with 100 µM IpyA and co-cultured with NALM6 cells. After 5 days, the medium was carefully pipetted and transferred to isolate the T cells in suspension, which were then stained with dye-conjugated antibodies for flow cytometry analysis. Anti-CD3 antibodies were used to label the T cells, and counting beads were utilized to measure the cell count.

[0111] As a result of confirming the proliferation of CAR-T cells, all CAR-T cells treated with IpyA showed increased cell proliferation ability compared to the control group (DMSO treatment) (Fig. 8). In particular, the proliferation efficiency was found to be the best when the E:T ratio was 0.5:1 (Fig. 8), and subsequently, an E:T ratio of 0.5:1 was used in experiments using anti-CD19 CAR-T.

[0112]

[0113] Confirmation of the effects of IpyA on various donor-derived anti-CD19 CAR-T cells

[0114] NALM6 CBG GFP cells 0.05X10 6 The cells were seeded into 96-well plates at a specific concentration. The cell counts of various donor-derived anti-CD19 CAR-Ts were set so that the E:T ratio was 0.5:1. Subsequently, the cells were treated with 100 µM of IpyA and co-cultured with NALM6 cells. After 5 days, the medium was carefully pipetted and transferred to isolate the T cells in suspension, which were then stained with dye-conjugated antibodies for flow cytometry analysis. Anti-CD3 antibodies were used to label the T cells, and counting beads were utilized to measure the cell count.

[0115] As a result of confirming CAR-T cell proliferation, all CAR-T cells treated with IpyA showed a higher cell proliferation ability compared to the control group (DMSO treatment) (Fig. 9). This increase in CAR-T cell proliferation caused by IpyA treatment was also confirmed by cell number measurement results (Fig. 10).

[0116]

[0117] 1-3. Evaluation of the effect of IpyA on the proliferation of anti-CD19 CAR-T cells (Proliferation assay)

[0118] NALM6 CBG GFP cells 0.05X10 6 Seeds were placed in 96-well plates at various concentrations. The same experiment as in Experimental Example 1-2 was conducted, but NALM6 CBG GFP cells were co-cultured with IpyA at varying concentrations of 0, 10, 25, 50, and 100 uM.

[0119] As a result of confirming CAR-T cell proliferation, it was found that there was a difference in CAR-T cell proliferation when the concentration of IpyA was 25 uM or higher (Fig. 11). This increase in CAR-T cell proliferation caused by IpyA treatment was also confirmed by cell number measurement results (Fig. 12).

[0120]

[0121] 1-4. Evaluation of Changes in Cytotoxicity of CAR-T Cells Caused by IpyA

[0122] NALM6 CBG GFP cells 0.05X10 6 The cells were seeded into 96-well plates at the specified concentrations. The number of anti-CD19 CAR-T cells was set so that the E:T ratio was 0.062, 0.125, 0.25, or 0.5:1. Subsequently, 100 uM of IpyA was added, and the cells were co-cultured with NALM6 cells. After 48 hours, to evaluate the cytotoxicity of the CAR-T, luciferase-expressing Nalm6-CBG-GFP cell lines were treated with D-luciferin and incubated for 10 minutes. Subsequently, luminescence intensity was measured using a luminescence reader, and cytotoxicity was calculated according to the following formula.

[0123] [Equation 1]

[0124] Cytotoxicity (% of killing) = (Emission intensity of control group - Emission intensity of experimental group) / (Emission intensity of control group - Background value) x 100

[0125] (The background value is defined as the luminescence intensity of a well containing only the medium without cells.)

[0126] As a result of the evaluation, it was found that the E:T ratio did not affect the cytotoxicity of CAR-T (Fig. 13).

[0127]

[0128] 1-5. Evaluation of Changes in Cytokine Expression in CAR-T Cells Induced by IpyA

[0129] Anti-CD19 CAR-T cells

[0130] NALM6 CBG GFP cells 0.05X10 6 The cells were seeded into 96-well plates at a certain concentration. The number of anti-CD19 CAR-T cells was set so that the E:T ratio was 0.25:1. Subsequently, 100 uM of IpyA was added, and the cells were co-cultured with NALM6 cells. After 24 hours, the medium was collected, and the secreted cytokines were analyzed using the ELISA technique.

[0131] Analysis results showed that the secretion of interferon gamma (IFN-γ) and interleukin-2 (IL-2) increased significantly with IpyA treatment (Fig. 14).

[0132]

[0133] Anti-BCMA CAR-T cells

[0134] OPM2 CBG GFP cells 0.05 x 10 6 The cells were seeded into 96-well plates at a certain concentration. The number of anti-BCMA CAR-T cells was set so that the E:T ratio was 0.25:1. Subsequently, 100 uM of IpyA was added, and the cells were co-cultured with OPM2 cells. After 24 hours, the medium was collected, and the secreted cytokines were analyzed using the ELISA technique.

[0135] Analysis results showed that the secretion of interferon gamma (IFN-γ) and interleukin-2 (IL-2) increased significantly with IpyA treatment (Fig. 15).

[0136]

[0137] Anti-PSMA CAR-T cells

[0138] PC3 PSMA CBG GFP cells 0.01X10 6The cells were seeded into 96-well plates at a certain concentration. The number of anti-PSMA CAR-T cells was set so that the E:T ratio was 0.5:1. Subsequently, 100 uM of IpyA was added, and the cells were co-cultured with PC3 cells. After 24 hours, the medium was collected, and the secreted cytokines were analyzed using the ELISA technique.

[0139] Analysis results showed that the secretion of interferon gamma (IFN-γ) and interleukin-2 (IL-2) increased significantly with IpyA treatment (Fig. 16).

[0140]

[0141] 1-6. Evaluation of Long-term Improvement in Antitumor Activity of CAR-T Cells Following Repeated IpyA Treatment

[0142] NALM6 CBG GFP cells 0.05X10 6 The cells were seeded at a specific concentration. The number of anti-CD19 CAR-T cells was set so that the E:T ratio was 0.25:1. Subsequently, 10 µM of IpyA was added, and the cells were co-cultured with NALM6 cells. Every 4 days of co-culture, the expression of anti-CD19 CAR-T and NALM6 was monitored using a flow cytometer. If the NALM6 cells were completely controlled, NALM6 cells and 10 µM of IpyA were added again, in an amount corresponding to that of anti-CD19 CAR-T. After monitoring every 4 days in the same manner, the experiment was repeated by adding NALM6 cells until the CAR-T cells reached a state of exhaustion. Upon completion of the experiment, the cells were harvested, and the degree of CAR-T cell proliferation and cell count were evaluated.

[0143] Evaluation results confirmed that continuous treatment with IpyA significantly increased the proliferative capacity of CAR-T (Figs. 17 and 18). These results suggest that the antitumor activity of CAR-T can be increased by continuous treatment with IpyA.

[0144]

[0145] 1-7. Confirmation of the effect of IpyA on CAR-T in lymphoma cell lines

[0146] Various lymphoma cell lines 0.05X10 6 The cells were seeded into 96-well plates at the specified concentrations. The E:T ratio was set to 0.5:1 (MINO), 1:1 (Daudi, SUDHL5), or 4:1 (SUDHL4). Subsequently, each lymphoma cell line was co-cultured with anti-CD19 CAR-T cells after treatment with 100 uM of IpyA. After 5 days, the medium was carefully pipetted and transferred to isolate the T cells in suspension, which were then stained with dye-conjugated antibodies for flow cytometry analysis. Anti-CD3 antibodies were used to label the T cells, and counting beads were utilized to measure the cell count.

[0147] The MINO cell line is a Mantle Cell Lymphoma (MCL) cell line, a rare and aggressive subtype of B-cell Non-Hodgkin lymphoma, and the Daudi cell line is a human B-cell lineage suspension cell line derived from Burkitt's lymphoma in an African-American boy in 1967. The SUDHL5 cell line is a human B-cell lymphoma cell line derived from the lymph nodes of a patient with diffuse large B-cell lymphoma (DLBCL), and the SUDHL4 cell line is a cell line derived from lymphoblast-like cells isolated from peritoneal effusion of a patient with diffuse large B-cell lymphoma.

[0148] As a result of the experiment, IpyA treatment promoted the proliferation of anti-CD19 CAR-T cells in all cell lines tested (Fig. 19), and this result was also confirmed by the cell count results (Fig. 20).

[0149]

[0150] 1-8. Confirmation of the effect of IpyA on CAR-T in solid tumor cell lines

[0151] Solid tumor cell lines PC3, PSMA, CBG, GFP, and OVCAR3 cells were each 0.025 x 10 6 , 0.015X10 6 Seeded at a certain concentration and cultured for 48 hours. Subsequently, the same experiment as in Experimental Examples 1-7 was conducted, with an E:T ratio of 1.5:1 for PC3 cells and 1:1 for OVCAR3 cells. Anti-PSMA CAR-T and anti-FRa CAR-T cells were used as CAR-T cells.

[0152] As a result of the experiment, IpyA treatment promoted the proliferation of CAR-T cells in all cell lines tested (Fig. 21), and this result was also confirmed by the cell count results (Fig. 22).

[0153]

[0154] Additionally, the effect of reducing the co-culture period was also confirmed. PC3 PSMA CBG GFP cells were 0.04 x 10⁻⁶ 6 The cells were seeded at a certain concentration and cultured. The E:T ratio was set to 1.5:1 and co-cultured with anti-PSMA CAR-T cells for 72 hours. Subsequently, the degree of CAR-T cell proliferation was evaluated using the same method as in Examples 1-7.

[0155] As a result of the evaluation, it was found that the proliferation of CAR-T cells was promoted by IpyA treatment even when the co-culture period was reduced (Figs. 23 and 24).

[0156]

[0157] 1-9. Confirmation of the effect of IpyA on CAR-T in multiple myeloma cell lines

[0158] Multiple myeloma cell lines OPM2, MM1S, and RPMI8226 were 0.05X10 6The cells were seeded and cultured at a certain concentration. Subsequently, the same experiment as in Experimental Examples 1-7 was conducted, and an E:T ratio of 0.5:1 was used. As CAR-T cells, cells expressing scFV-based CAR or VHH (Variable heavy chain domains of heavy chain antibody)-based CAR against BCMA were used.

[0159] As a result of the experiment, IpyA treatment promoted the proliferation of CAR-T cells regardless of the type of CAR expressed (Fig. 25), and this result was also confirmed by the cell count measurement results (Fig. 26).

[0160]

[0161] In addition, long-term antitumor efficacy was confirmed using the OPM2 CBG GFP cell line.

[0162] 0.5 x 10⁶ OPM2 CBG GFP cells 6 The cells were seeded at a specific concentration, and the E:T ratio was set to 0.5:1 or 1:1. Subsequently, OPM2 CBG GFP cells were treated with 100 µM of IpyA and co-cultured with scFV-based anti-BCMA CAR-T cells. Every three days of co-culture, the expression of anti-BCMA CAR-T and OPM2 was monitored using a flow cytometer. If the OPM2 cells were completely controlled, OPM2 cells and 10 µM of IpyA were added again, in an amount corresponding to that of the anti-BCMA CAR-T cells. After monitoring every three days in the same manner, the experiment was repeated by adding OPM2 cells until the anti-BCMA CAR-T cells reached a state of exhaustion. Upon completion of the experiment, the cells were harvested, and the degree of proliferation and cell count of the anti-BCMA CAR-T cells were evaluated.

[0163] As a result of the evaluation, it was found that continuous treatment with IpyA significantly increased the proliferative capacity of anti-BCMA CAR-T (Fig. 27). These results suggest that the antitumor activity of anti-BCMA CAR-T can be increased by continuous treatment with IpyA.

[0164]

[0165] Experimental Example 2: Antitumor activity of IpyA and anti-CD19 CAR-T combination therapy_In vivo experiment

[0166] Inject NALM6 CBG GFP cell line into mice (0.05X10 6 Tumor formation was induced by administering 150 µl. When the tumor reached a certain size, anti-CD19 CAR-T cells were administered at a rate of 0.05 x 10⁶ 6 It was injected at a concentration of 150 µl, and IpyA was administered orally at a dose of 15 mg / kg five times a week. Bioluminescence imaging (BLI) was performed every 7 days to check the tumor size.

[0167] Experimental results showed that tumor growth was inhibited when CD19 CAR-T cells and IpyA were used together compared to a vehicle injected with only anti-CD19 CAR-T cells (Fig. 28).

[0168]

[0169] Comparative Example: Confirmation of the effects of various indole metabolites and AHR agonists or antagonists

[0170] We confirmed whether the effects of IpyA were observed in other microbiome metabolites. In addition, since IpyA is known as an AHR (aryl hydrocarbon receptor) agonist, we confirmed the effects of other AHR agonists or antagonists.

[0171] NALM6 CBG GFP cell line 0.05X10 6The cells were seeded at a certain concentration and treated with various indole metabolites (100 µM), AHR agonists, and antagonists. Subsequently, they were cultured with anti-CD19 CAR-T cells at an E:T ratio of 1:1 for 120 hours. After culture, the medium was carefully pipetted and transferred to separate the T cells in suspension, flow cytometry was performed, and the cell count was counted.

[0172] Analysis results showed that, with the exception of IpyA, other indole metabolites had little effect on improving CAR-T cell proliferation (Figs. 29 and 30).

[0173] In addition, it was found that the AHR agonist FICZ and the AHR antagonist CH-223191 also had little effect on improving the proliferation of CAR-T cells (Figs. 31 and 32).

Claims

A composition for T cell proliferation containing indole-3-pyruvic acid as an active ingredient. A composition for T cell proliferation according to claim 1, wherein the T cells are CAR-T cells expressing a chimeric antigen receptor (CAR). A composition for T cell proliferation according to claim 2, wherein the chimeric antigen receptor (CAR) is one or more selected from the group consisting of anti-CD19 CAR, anti-PSMA (prostate-specific membrane antigen) CAR, anti-Fra (Folate Receptor-alpha) CAR, anti-BCMA (B-cell maturation antigen) CAR, anti-CD33 CAR, anti-FLT3 (Fms-like tyrosine kinase 3) CAR, anti-CD123 CAR, and anti-GPRC5D (G protein-coupled receptor class C group 5 member D) CAR. A pharmaceutical composition for cancer treatment comprising indole-3-pyruritic acid and CAR-T cells as active ingredients. A pharmaceutical composition for treating cancer according to claim 4, wherein the CAR-T cell is one or more selected from the group consisting of anti-CD19 CAR-T, anti-PSMA CAR-T, anti-FRa CAR-T, anti-BCMA CAR-T, anti-CD33 CAR, anti-FLT3(Fms-like tyrosine kinase 3) CAR, anti-CD123 CAR, and anti-GPRC5D(G protein-coupled receptor class C group 5 member D) CAR. A pharmaceutical composition for treating cancer according to claim 4, wherein the cancer is one or more selected from lymphoma, multiple myeloma, prostate cancer, breast cancer, colorectal cancer, rectal cancer, brain tumor, lung cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, stomach cancer, bladder cancer, ovarian cancer, bile duct cancer, gallbladder cancer, uterine cancer, cervical cancer, head and neck cancer, pancreatic cancer, and squamous cell carcinoma. A method for treating cancer comprising the step of administering the pharmaceutical composition for treating cancer of claim 4 to an individual requiring treatment.

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