Transition metal dichalcogenide (TMD)-based antibody mimetic, immunotherapeutic agent comprising same, and preparation method therefor

Abstract

Provided are transition metal dichalcogenide (TMD)-based antibody mimetic comprising: a transition metal dichalcogenide nanosheet; and a tripeptide bound to the nanosheet. The antibody mimetic according to the present invention can strongly bind to PD-L1 with a dissociation constant at the nanomolar level, and can selectively bind to albumin and immunoglobulin (IgG1), which are major plasma proteins, at least 1.5 times more selectively. As a result, imaging of PD-L1-overexpressing cancer cells is possible by treating PD-L1-overexpressing cancer cells with a TMD nanosheet antibody mimic so as to detect the Raman scattering signal, which is an intrinsic optical characteristic of the material. In addition, the PD-L1 / PD-1 binding blocking effect of the TMD nanosheet antibody mimetic, according to the present invention, can be identified at the cell level and the cancer cell transplantation animal model level, and thus the TMD nanosheet antibody mimetic can also function as an immunotherapeutic agent.

Description

Transition metal dichalcogenide (TMD)-based antibody mimetics, immunotherapeutic agents containing the same, and methods for producing the same

[0001] The present invention relates to a transition metal dichalcogenide (TMD)-based antibody mimetic, an immunotherapeutic agent comprising the same, and a method for producing the same. More specifically, the antibody mimetic has a strong binding to PD-L1 with a dissociation constant at the nanomolar level and can selectively bind 1.5 times more than albumin and immunoglobulin G1, which are major plasma proteins, and an immunotherapeutic agent comprising the same, and a method for producing the same.

[0002] In a normal immune response, immune checkpoint molecules regulate T cell activation to prevent excessive inflammation or autoimmunity. Tumor cells exploit this system by overexpressing a protein called PD-L1 (Programmed Death Ligand-1), which binds to the PD-1 receptor on the T cell surface, inhibiting T cell function or inducing T cell exhaustion. Currently, anti-PD-1 or anti-PD-L1 antibody therapies are being used clinically for various solid tumors and have shown remarkable therapeutic effects in some patients. However, only about 20-30% of patients in clinical trials show a substantial therapeutic response, and the low success rate due to resistance or lack of efficacy is a significant limitation. This is the result of a complex interplay of factors including heterogeneous expression of PD-L1, inhibitory factors in the tumor microenvironment, and limited tissue penetration of antibodies. Therefore, new therapeutic strategies that induce immune cell function and modulate the tumor microenvironment are required.

[0003] Therefore, the problem to be solved by the present invention is to provide a novel antibody mimetic and an immunotherapeutic agent containing the same that can detect a target and treat a disease while effectively overcoming the shortcomings of existing antibodies.

[0004] To solve the above problem, the present invention provides the following configuration.

[0005] The present invention provides a transition metal dichalcogenide (TMD)-based antibody mimetic comprising a transition metal dichalcogenide nanosheet and a tripeptide bound to the nanosheet.

[0006] In one embodiment of the present invention, the tripeptide may be a complex of a metal and a tripeptide capable of binding to a transition metal dichalcogenide.

[0007] In one embodiment of the present invention, the metal may be copper.

[0008] In one embodiment of the present invention, the tripeptide may be at least one selected from the group consisting of FNP, FNW, HSW, LSW and YSA.

[0009] In one embodiment of the present invention, the type of target protein to which the antibody mimetic selectively binds and the binding density can be determined depending on the type of tripeptide.

[0010] In one embodiment of the present invention, the target protein may be PD-L1.

[0011] In one embodiment of the present invention, the tripeptide may be FNP or HSW.

[0012] In one embodiment of the present invention, an immunotherapeutic agent comprising the antibody mimetic may be provided.

[0013] In one embodiment of the present invention, the immunotherapeutic agent may be an immuno-oncology agent targeting PD-L1.

[0014] The present invention also provides a multimodal TMD antibody mimic having both a photothermal effect and a PD-L1 immune checkpoint inhibitory effect, comprising a MoSe₂ nanosheet and a tripeptide self-assembled on the surface of the nanosheet.

[0015] In one embodiment of the present invention, the tripeptide may comprise a sequence that selectively binds to the PD-L1 protein.

[0016] In one embodiment of the present invention, the tripeptide can be attached to MoSe₂ nanosheets via Cu-NTA complexation.

[0017] In one embodiment of the present invention, the antibody mimetic can induce a photothermal reaction upon near-infrared (nIR) irradiation.

[0018] In one embodiment of the present invention, an immunotherapeutic agent for treating cancer can be provided that includes the antibody mimetic as an active ingredient.

[0019] In one embodiment of the present invention, the immunotherapeutic agent can improve the tumor microenvironment by combining the PD-L1 immune checkpoint inhibition function and photothermal response.

[0020] In one embodiment of the present invention, the immunotherapeutic agent can be applied to solid cancers including CT-26 colon cancer.

[0021] The antibody mimetic according to the present invention has a strong binding to PD-L1 with a dissociation constant at the nanomolar level, and can selectively bind 1.5 times more than albumin and immunoglobulin (IgG1), which are major plasma proteins. As a result, by treating the TMD nanosheet antibody mimetic to cancer cells overexpressing PD-L1 and detecting the Raman scattering signal, which is an optical characteristic unique to the material, the cancer cells overexpressing PD-L1 can be imaged. In addition, since the PD-L1 / PD-1 binding blocking effect of the TMD nanosheet antibody mimetic according to the present invention can be confirmed at the cellular level and the cancer cell transplantation animal model level, it can also function as an immunotherapeutic agent.

[0022] Figure 1 is a TEM image of WS2 and five types of WS2-TPs structures in which tripeptides are introduced into WS2 nanosheets.

[0023] Figure 2 shows a) C1s XPS data of the WS2-TPs structure. b) Results confirming the fractional coverage of the tripeptide introduced into the structure.

[0024] Figure 3 is a graph showing the degree of binding of the WS2-TPs construct to PD-L1 protein and major plasma proteins (albumin, IgG1) normalized to the degree of binding of the construct to albumin.

[0025] Figure 4 shows the dissociation constant for the PD-L1 protein of the Phe-Asn-Pro(FNP) or WS2-TPs structure.

[0026] Figure 5 is a schematic diagram and results of identifying the PD-L1 protein binding site of the WS2-FNP structure.

[0027] Figure 6 shows the results of measuring the Raman scattering signal of WS2-FNP treated with cancer cells with low or overexpression of PD-L1.

[0028] Figure 7 shows the results of comparing the amount of cytokine (IL-2) secretion through cell-level PD-L1 and PD-1 binding inhibition.

[0029] Figure 8 shows a comparison of the anticancer effects of CT26 animal models injected with WS2-FNP or immunotherapy (Avelumab, Ab). a) Diagram of anticancer treatment, b) tumor images extracted from experimental animals sacrificed 19 days after cancer cell transplantation, c) tumor weights, and de) tumor growth graphs for each group. fi) Cytokine release and immune cell capture effects of tumors or spleens extracted from CT26 animal models injected with WS2-FNP or immunotherapy (Avelumab, Ab) sacrificed on the end day of the experiment (D+19). f) Interferon gamma (IFN-γ) secretion per gram of tumor tissue, g) Killer T cell ratio, h) Mature macrophage ratio, and i) Differentiation of regulatory T cells among cells extracted from the spleen are analyzed.

[0030] FIG. 9 is a schematic diagram illustrating the synthesis principle of a MoSe₂ nanosheet-based antibody mimic (MoSe₂-FNP) according to one embodiment of the present invention.

[0031] FIG. 10 is a schematic diagram illustrating the structure of a MoSe₂-FNP complex according to one embodiment of the present invention.

[0032] Figure 11 is a transmission electron microscope (TEM) image to confirm the structure of the MoSe₂-FNP complex into which the peptide has been introduced.

[0033] Figure 12 is a diagram showing an FT-IR spectrum analyzing whether the MoSe₂-FNP complex is functionalized.

[0034] Figure 13 is a graph showing the results of comparing the binding affinities of the MoSe₂-FNP complex to PD-L1 and albumin.

[0035] Figure 14 is a Langmuir adsorption isotherm graph measuring the dissociation constant (Kd) of the MoSe₂-FNP complex for PD-L1.

[0036] Figure 15 is a graph showing the temperature rise change according to the near-infrared (nIR) irradiation time for PBS, MoSe₂, and WS₂, respectively.

[0037] Figure 16 is a graph showing the temperature change over time during nIR irradiation for MoSe₂-FNP complexes of various concentrations.

[0038] Figure 17 is (a) a schematic diagram showing the photothermal immunotherapeutic mechanism of action of the complex of the present invention, and (b) a graph showing the change in survival rate for CT-26 tumor cells.

[0039] Figure 18 is a graph comparing the tumor volume changes among the three groups: PBS, WS₂-FNP, and MoSe₂-FNP + nIR.

[0040] Figure 19 is a schematic diagram showing the treatment mechanism of the present invention that combines photothermal response and immune checkpoint inhibition.

[0041] Figure 20 is a graph showing the tumor growth curves of each experimental group (PBS, nIR, MoSe₂-FNP, MoSe₂-FNP + nIR).

[0042] Figure 21 is a graph comparing the weight of tumors extracted from each experimental group.

[0043] Figure 22 is a graph showing the change in body weight of mice in each experimental group.

[0044] Figure 23 is a graph comparing the level of interferon gamma (IFN-γ) expression in tumor tissue in each experimental group.

[0045] Figure 24 shows the results of flow cytometry analysis of the proportion of CD3+ T cells in tumor tissue.

[0046] Figure 25 shows the results of flow cytometry analysis showing the proportion of Treg cells in tumor tissue.

[0047] Figure 26 is a flow cytometry analysis result showing the proportion of dendritic cells in tumor tissue.

[0048] Figure 27 is a graph showing the proportion of CD3+CD8+ cytotoxic T cells confirmed in the spleen.

[0049] Figure 28 is a graph showing the ratio of CD3+CD4+ helper T cells confirmed in the spleen.

[0050] Figure 29 is a graph showing the proportion of Treg cells confirmed in the spleen.

[0051] Hereinafter, a preferred embodiment of the present invention will be described in detail with reference to the attached drawings.

[0052] Before describing the present invention in detail, it should be noted that the terms or words used in this specification should not be interpreted as being unconditionally limited to their usual or dictionary meanings, and the inventor of the present invention may appropriately define and use the concepts of various terms in order to describe his or her invention in the best possible manner.

[0053] Furthermore, it should be noted that these terms and words should be interpreted with meanings and concepts that are consistent with the technical idea of ​​the present invention.

[0054] That is, the terms used in this specification are only used to describe preferred embodiments of the present invention, and are not intended to specifically limit the contents of the present invention.

[0055] It should be noted that these terms are defined taking into account the various possibilities of the present invention.

[0056] Additionally, in this specification, a singular expression may include a plural expression unless the context clearly indicates a different meaning.

[0057] Also, it should be noted that even if similarly expressed in plural, it can contain singular meaning.

[0058] Throughout this specification, whenever a component is described as "including" another component, it may mean that the component may further include any other component, rather than excluding any other component, unless specifically stated otherwise.

[0059] In order to solve the above-described problem, the present invention provides an antibody mimetic based on a transition metal dichalcogenides (TMD) nanosheet that selectively and strongly binds to a Programmed cell death ligand-1 (PD-L1) protein.

[0060] The antibody mimic based on TMD nanosheet according to the present invention has a structure in which a tripeptide is assembled on the surface of a TMD nanosheet, and the tripeptide forms a complex with a metal capable of binding to the chalcogenide of the TMD nanosheet, and in particular, the binding selectivity and binding affinity for PD-L1 can be controlled and determined depending on the type and density of the tripeptide.

[0061] The present invention is described below through preferred examples, but the scope of the present invention is not limited by the following examples and experimental examples.

[0062] Example 1

[0063] Synthesis of TMD nanosheet-based antibody mimetics (TMD-TPs)

[0064] After adjusting the pH of the solution (0.5 mM) in which TMD nanosheets were dispersed to 5.8 using NaOH solution, 500 μL of Cu-NTA (nitrilotriacetic acid)-tripeptide (5 mM) was added to 5 mL of the TMD nanosheet solution (0.5 mM).

[0065] That is, in one embodiment of the present invention, a tripeptide complex (TP) in which Cu capable of binding to S of TMD and the following five types of tripeptides are combined was used, and for the synthesis of WS2-TP, the reaction mixture was stirred at 25°C for 24 hours.

[0066] Afterwards, the reaction solution was centrifuged at 20,000 xg to collect TMD-TP, and the obtained TMD-TP was washed twice with H2O through centrifugation. In this example, WS2 was used as the TMD nanosheet, and Phe-Asn-Pro (FNP), Phe-Asn-Trp (FNW), His-Ser-Trp (HSW), Leu-Ser-Trp (LSW), and Tyr-Ser-Ala (YSA) were used as the tripeptides, and nanosheets without added tripeptides were used as the control.

[0067]

[0068] In vitro cell imaging

[0069] A sterile 24-well plate containing a cover glass was prepared. Cancer cells (10,000 cells mL-1) were seeded in the wells, cultured for 48 h, and washed twice with 1x PBS. Then, 400 μL of a 0.2 mM WS2-FNP solution (PBS, pH 7.4) was added to the wells. The mixture was gently shaken at 50 rpm for 6 h in a shaking incubator, the cover glass was removed, and the unbound TMD-TP was washed twice with PBS. The cover glass was inverted and fixed to a custom-made well-shaped PDMS mold containing PBS, and the adsorbed cells were sealed by making complete contact with the 1x PBS solution. Raman imaging was obtained at 200 to 3000 cm-1 with an excitation laser of 532 nm and a power of 9 W.

[0070]

[0071] extracorporeal immunotherapy

[0072] To evaluate in vitro immunosuppression, interleukin-2 (IL-2) release from activated Jurkat cells was assessed. A549 cells were cultured in 96-well plates (10,000 cells / well) for 24 h and treated with interferon γ (IFN-γ, 10 ng ml -1 ) were pretreated for 36 hours to induce PD-L1 overexpression. Jurkat cells were cultured in T flasks (10,000 cells mL -1 ) and cultured in phytohemagglutinin (PHA, 1 ug ml -1 ) / phorbol myristate acetate (PMA, 50ng mL -1) for 36 h. Then, IFN-γ-treated A549 cells were treated with Avelumab (80 nM in 0.3x PBS 7.4) or WS2-FNP (80 nM in 0.3x PBS 7.4) for 6 h and then co-cultured with Jurkat T cells at a 1:4 (tumor cells:Jurkat) ratio. After 48 h of incubation, cell culture supernatants were collected and centrifuged at 10,000 rpm for 5 min in a centrifugal filter. The filtrate was analyzed using an Interleukin-2 (IL-2) ELISA kit according to the manufacturer's protocol.

[0073]

[0074] In vivo cancer immunotherapy

[0075] All animals were maintained in a pathogen-free environment. All animal experimental procedures were reviewed and approved by the Hanyang University Institutional Animal Care and Use Committee (Approval No. 2023-0135A). All animal experiments were conducted in accordance with the guidelines of the Korea Food and Drug Administration. As previously described, a synthetic tumor model was designed using 5-week-old male BALC / C mice (Koatech, Gyeonggi-do, Korea). Prior to tumor cell and material inoculation, mice were anesthetized using inhaled isoflurane (0.5–2%).

[0076] CT-26 cells (3×10 6 100 μL portion of the cells) was subcutaneously inoculated into the right flank of BALB / C mice. CT-26 tumor-bearing mice (approximately 60 mm 3, Four days after tumor inoculation), mice were randomly assigned to four groups (n = 5), and the substance (1 mg / kg) was injected intratumorally five times at two-day intervals. Tumor volumes and body weights of mice receiving various treatments were monitored every other day.

[0077] Next, the tumor volume of the tumor-bearing mice was monitored using a caliper. Tumor volume (V) was calculated as V = (W 2× L) / 2. (W: width, L: length). Because of the standard animal protocol of this study, the tumor was measured as 2000 mm 3 When reached, mice were presumed dead. After sacrifice, mouse organs were harvested for ex-vivo analysis.

[0078]

[0079] Experimental Example 1

[0080] Figure 1 is a TEM image of WS2 and five types of WS2-TPs structures in which tripeptides are introduced into WS2 nanosheets.

[0081] Referring to FIG. 1, in one embodiment of the present invention in which a bulk TMD is dispersed in nanosheets and then a material containing a tripeptide (Cu-NTA-tripeptides) is mixed to introduce a tripeptide structure onto the surface of the nanosheets, it can be confirmed that the TMD-TPs structure is in the form of a nanosheet having an average diameter of about 43 nm.

[0082] Figure 2 shows a) C1s XPS data of the WS2-TPs structure. b) Results confirming the fractional coverage of the tripeptide introduced into the structure.

[0083] Referring to FIG. 2a, it can be confirmed that the ratio of C=C and O=CO bond energies corresponding to the tripeptide increases in the TMD-TP structure manufactured according to the present invention.

[0084] In Fig. 2b, the amount of tripeptide structure (Cu-NTA-tripeptide) introduced into the nanosheet was quantified based on the amount of tungsten and Cu, and the coverage of the tripeptide introduced to the nanosheet surface was calculated by converting this.

[0085] Referring to Fig. 2b, it can be seen that the amount of tripeptide binding to TMD is different depending on the type of tripeptide, and in the case of WS2-FNP, it can be seen that it has a coverage of nearly 40%.

[0086] Figure 3 is a graph showing the degree of binding of the WS2-TPs construct to PD-L1 protein and major plasma proteins (albumin, IgG1) normalized to the degree of binding of the construct to albumin.

[0087] Referring to FIG. 3, it can be seen that the WS2-FNP and WS2-HSW structures selectively bind to PD-L1 in comparison with the relative binding affinity with proteins present in excess in plasma, and in particular, it can be seen that the degree of binding and binding selectivity for the target protein can be adjusted depending on the type and density of the tripeptide.

[0088] Figure 4 shows the dissociation constant for the PD-L1 protein of the Phe-Asn-Pro(FNP) or WS2-TPs structure.

[0089] Referring to Fig. 4, it can be seen that the WS2-TPs structure has a low dissociation constant (=strong binding affinity) of nM level to the target protein PD-L1.

[0090] Figure 5 is a schematic diagram and results of identifying the PD-L1 protein binding site of the WS2-FNP structure.

[0091] Referring to FIG. 5, PD-L1 is a cancer cell ligand that binds to the immune cell surface protein PD-1. When PD-1 was first bound to PD-L1 and then the WS2-FNP structure was added dropwise, the degree of binding of WS2-FNP decreased as the concentration of PD-1 increased. This means that the binding site for PD-L1 of the TMD-TP according to the present invention is similar to PD-1.

[0092] Figure 6 shows the results of measuring the Raman scattering signal of WS2-FNP treated with cancer cells with low or overexpression of PD-L1.

[0093] In Fig. 6, cytokines that enable cancer cells (A549 cells) to overexpress PD-L1 were added to prepare overexpressed and underexpressed cells, and after treating with WS2-FNP material to remove unbound material, the Raman scattering signal of WS2-FNP was measured and imaged.

[0094] Referring to the results in Figure 6, the strong Raman scattering signal observed in cells overexpressing PD-L1 confirms at the cellular level that WS2-FNP can bind to PD-L1. Furthermore, when MoSe2 is used, the photothermal effect of the material can be utilized to kill bound cancer cells.

[0095] Figure 7 shows the results of comparing the amount of cytokine (IL-2) secretion through cell-level PD-L1 and PD-1 binding inhibition.

[0096] Referring to FIG. 7, the commercialized antibody treatment (Durvalumab) and the WS2-FNP according to the present invention can bind to PD-L1 and block the PD-L1 / PD-1 pathway, which means that the immune cells are in an activated state (cytokine release).

[0097] Figures 8a to 8d are comparisons of the anticancer effects of CT26 animal models injected with WS2-FNP or immunotherapy (Avelumab, Ab), a) a diagram of anticancer treatment, b) tumor images extracted from experimental animals sacrificed 19 days after cancer cell transplantation, c) tumor weights according to the results, and de) tumor growth graphs for each group. fi) Cytokine release amounts and immune cell capture effects of tumors or spleens extracted from CT26 animal models injected with WS2-FNP or immunotherapy (Avelumab, Ab) sacrificed on the experimental end day (D+19). f) Interferon gamma (IFN-γ) secretion amount per gram of tumor tissue, g) Killer T cell ratio, h) mature macrophage ratio, and i) differentiation amount of regulatory T cells among cells extracted from the spleen.

[0098] Referring to Figure 8, after cancer cells were inoculated into experimental mice, the cancer was allowed to grow, and the immunotherapy effect was confirmed by injecting an antibody treatment (Ab, avelumab) and WS2-FNP. The WS2-FNP according to the present invention and the commercialized antibody treatment as a comparative example exhibited similar anticancer effects, and analysis of immune cells and cytokine release in the organs of sacrificed mice confirmed that an immunotherapy effect was induced.

[0099] As described above, the antibody mimetic according to the present invention has a structure based on TMD nanosheets, strongly binds to PD-L1 with a dissociation constant at the nanomolar level, and can selectively bind 1.5 times more than albumin and immunoglobulin G1, which are major plasma proteins. Therefore, it can be confirmed that PD-L1-overexpressing cancer cells can be imaged by treating the TMD nanosheet antibody mimetic to the cancer cells overexpressing PD-L1 and detecting the Raman scattering signal, which is an optical property unique to the material. In addition, since the PD-L1 / PD-1 binding blocking effect of the TMD nanosheet antibody mimetic according to the present invention can be confirmed at the cellular level and the cancer cell transplantation animal model level, it can be utilized as an immune anticancer agent for PD-L1.

[0100] Another embodiment of the present invention provides a so-called "multimodal immunotherapy" that can kill cancer cells by simultaneously inducing a photothermal reaction through near-infrared (nIR) absorption along with the PD-L1 immune checkpoint inhibition effect. This structure has a PD-L1 selectivity that is approximately 1.5 times higher than that of albumin, a major plasma protein, and exhibits excellent binding affinity with a nanomolar-level dissociation constant. Furthermore, by demonstrating excellent cancer treatment efficacy at both the cellular and animal model levels through photothermal reaction as well as immune checkpoint inhibition upon nIR irradiation, it can technically overcome the limitations of existing antibody-based immune checkpoint inhibitors.

[0101]

[0102] Example 2

[0103] Synthesis of TMD nanosheet-based antibody mimetics (MoSe2-FNP) 11 mg of NTA-COOH was dissolved in 3 mL of water (H2O), and 7.5 mg of copper sulfate hydrate (CuSO4·xH₂O) was added. The pH of the reaction solution was adjusted to 5.8 by adding 60 μL of 1 M NaOH aqueous solution, and the solution was stirred at 25°C for 1 h. Next, 4.6 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 4.4 mg of NHS (N-hydroxysulphur succinimide) were added to the Cu 2+ It was added to the NTA-COOH solution (Cu-NTA-COOH) that formed a complex with the ion. This mixed solution was stirred again at 25°C for 30 minutes, and then 500 μL of a 10 mM tripeptide aqueous solution was added and reacted at 25°C for 12 hours to prepare a Cu-NTA-tripeptide complex solution.

[0104] Thereafter, the pH of the TMD nanosheet (MoSe2 as an example) solution having a concentration of 0.5 mM was adjusted to 5.8 using an aqueous NaOH solution, and 500 μL of the above-mentioned Cu-NTA-tripeptide complex solution having a concentration of 5 mM was added to 5 mL of the above-mentioned TMD nanosheet solution having a concentration of 0.5 mM. The mixed reaction solution was stirred at room temperature (25°C) for 24 hours to proceed with the reaction to form a TMD-TP complex.

[0105] After the reaction was completed, the reaction solution was centrifuged at 20,000 xg to recover the generated TMD-TP, and the recovered TMD-TP was washed twice with deionized water and then purified through centrifugation under the same conditions.

[0106] FIG. 9 is a drawing explaining the synthesis principle of a MoSe2 nanosheet-based antibody mimetic (MoSe2-FNP) according to one embodiment of the present invention, and FIG. 10 is a schematic diagram explaining the structure of a MoSe2 nanosheet-based antibody mimetic (MoSe2-FNP) according to one embodiment of the present invention.

[0107] Referring to Figures 9 and 10, it can be confirmed that a multivalent and cumulative recognition phase is formed by self-assembly of a plurality of tripeptides (TPs).

[0108] This composite structure according to the present invention is composed of MoSe2, which functions as a robust photothermal platform, and TP assemblies responsible for biotarget recognition, and it can be confirmed that a large number of TPs are aligned (Fig. 10). That is, the composite manufactured according to the present invention is one in which tripeptides (TPs) are uniformly distributed in a self-assembled form on the surface of MoSe2 nanosheets, and this structure forms a nanocomposite that can be stably maintained in the body, which contributes to reducing the possibility of side effects due to degradation or nonspecific binding in the body, which will be described in more detail below.

[0109]

[0110] Experimental Example 2

[0111] All experimental animals were raised in a pathogen-free environment, and all animal experiments were conducted with the approval of the Hanyang University Institutional Animal Care and Use Committee (Approval No. 2023-0135A). Furthermore, all experiments were conducted in compliance with the Ministry of Food and Drug Safety's guidelines for laboratory animal care.

[0112] The allograft tumor model was established in 5-week-old male BALB / C mice (Koatech, Gyeonggi-do). Anesthesia was induced by inhalation of isoflurane (0.5–2%) before injection of tumor cells and composition, and CT-26 colon cancer cells were injected at a density of 3 × 10 6 Tumors were induced by subcutaneously injecting 100 μL of suspension containing the dog into the right flank of mice.

[0113] Four days after tumor induction, when the average tumor volume reached approximately 60 mm³, the experimental animals (n = 5) were randomly divided into the following groups.

[0114] PBS treatment group; laser (nIR) irradiation group; MoSe2-FNP single administration group (1 mg kg -1 ); MoSe2-FNP + laser combined group (1 mg kg -1 + nIR).

[0115] Afterwards, the composition was administered by direct injection into the tumor site, and if necessary, a near-infrared laser with a wavelength of 808 nm (1 W cm -2 ) was used to investigate for 1 minute, 1 minute stop, and 1.5 minutes additional investigation, and the tumor volume and body weight changes were measured at two-day intervals. At this time, the tumor volume (V) was calculated using the following formula based on the horizontal (W) and vertical (L) lengths measured using a caliper.

[0116] V = (W 2 × L) / 2

[0117] When the tumor volume reached 2000 mm³, the subject was considered to have reached the end of the experiment according to the experimental protocol. The mice were then euthanized, and major organs were removed for histological and ex vivo analysis.

[0118] The experimental results are explained in more detail using the drawings below.

[0119] Figure 11 is an enlarged image of a transmission electron microscope (TEM) to confirm the morphology of the MoSe2-FNP complex of the present invention.

[0120] Referring to Figure 11, it can be confirmed that the nanosheet form of MoSe2 is maintained even after the peptide is introduced.

[0121] Figure 12 shows an FT-IR (Fourier transform infrared spectroscopy) spectrum to confirm the functionalization of the MoSe2-FNP complex.

[0122] Referring to Fig. 12, the functionalized MoSe2-FNP complex shows characteristic peaks corresponding to the amine group, carboxyl group, and Cu-NTA bond of the peptide in the FT-IR spectrum, which is a result proving stable chemical bonding on the surface of the complex.

[0123] Figure 13 is a graph comparing the binding affinity of the MoSe2-FNP complex to PD-L1 and albumin.

[0124] Referring to FIG. 13, the complex according to the present invention has a selective binding ability to PD-L1, an immune checkpoint protein, and exhibits a significantly higher binding strength compared to albumin, a comparative group, thereby proving its superiority as a target-specific delivery platform.

[0125] Figure 14 is a graph showing the Langmuir adsorption isotherm for calculating the dissociation constant (Kd) of MoSe2-FNP for PD-L1.

[0126] Referring to Fig. 14, the results of the Langmuir isotherm adsorption curve analysis showed that the MoSe2-FNP complex had a low dissociation constant (Kd) for PD-L1, confirming that the binding affinity was high. This shows that the present invention is a basis for performing a high-precision immunomodulatory function.

[0127] Figure 15 is a graph showing the temperature rise change according to near-infrared (nIR) irradiation time for PBS, MoSe2, and WS2.

[0128] Referring to Fig. 15, under the same conditions, only MoSe2 showed a distinct temperature increase depending on the nIR irradiation time, indicating that MoSe2 is a material with an effective photothermal effect.

[0129] Figure 16 shows the results of measuring the temperature change of the solution over time after irradiating it with a near-infrared (nIR, 808 nm) laser to confirm the temperature increase response of the MoSe2-FNP complex.

[0130] Referring to Figure 16, it can be confirmed that as the concentration of the complex increases, the temperature increase upon nIR irradiation significantly increases, and at a concentration of 800 μM, the temperature rises to approximately 22°C or more. This suggests that MoSe2-FNP has excellent photothermal conversion efficiency and is capable of effective photothermal cancer cell killing under nIR conditions.

[0131] Figure 17 (a) is a schematic diagram showing the photothermal immunotherapeutic mechanism of action of the complex of the present invention, and (b) is a graph showing the change in cell viability according to the induction of a photothermal response in CT-26 colon cancer cells.

[0132] Referring to Fig. 17, (a) the conceptual schematic diagram visually represents the complex mechanism of simultaneously inducing a tripeptide assembly-based immune checkpoint inhibition function and a photothermal response in the immunotherapeutic agent according to the present invention, and (b) in an experiment targeting actual CT-26 tumor cells, it was confirmed that cell viability significantly decreased according to near-infrared irradiation when the MoSe2-FNP complex was treated. This means that the present invention can realize a complex anticancer effect that combines a photothermal response and immunosuppression.

[0133] Figure 18 is a graph comparing the change in tumor volume over time for three groups: PBS, WS2-FNP, and MoSe2-FNP + nIR.

[0134] Referring to Figure 18, the group combining MoSe2-FNP and nIR exhibited the most pronounced tumor suppression effect, demonstrating higher therapeutic efficacy than the WS2 group. This is interpreted as a synergistic effect resulting from the combined superior photothermal effect and immune checkpoint inhibition ability of the MoSe2-FNP-based immunotherapy.

[0135] Figure 19 is a schematic diagram schematically illustrating the mechanism of colon cancer immunotherapy combining photothermal response and immune checkpoint inhibition by a MoSe2-FNP complex that induces photothermal response, and Figure 20 shows tumor growth curves in tumor model mice treated with PBS, nIR alone, MoSe2-FNP alone, and MoSe2-FNP + nIR combined groups.

[0136] Additionally, Figure 21 is a graph comparing the weight of tumors excised after treatment, in which the lowest tumor weight was observed in the combined group.

[0137] Figure 22 is a graph comparing the weight changes of mice in each experimental group, confirming that there was no difference in systemic toxicity.

[0138] In other words, the composition of the present invention was repeatedly injected into a mouse model, and no significant weight changes were observed. This supports the structure's low systemic toxicity and guaranteed in vivo safety. This experimentally demonstrates that the nanocomposite-based structure offers superior in vivo adaptability and biocompatibility compared to existing antibody-based immune checkpoint inhibitors.

[0139] Figure 23 is a graph comparing the expression levels of interferon gamma (IFN-γ) in tumor tissues in each treatment group, and the highest immune activity was observed in the combined group. Figure 24 shows the proportion of CD3+ T cells in tumor tissues, and Figure 25 shows the proportion of T reg Cell ratio, Figure 26 is the flow cytometry analysis result analyzing the ratio of dendritic cells.

[0140] Figure 27 shows the proportion of CD3+CD8+ cytotoxic T cells confirmed in the spleen, Figure 28 shows the proportion of CD3+CD4+ helper T cells, and Figure 29 shows the proportion of T reg It shows the ratio of cells.

[0141] These results indicate that the treatment method of the present invention does not simply inhibit PD-L1, but also affects the activity of T cells and the overall immune system, and induces an anti-tumor response.

[0142] In particular, the increase in IFN-γ expression and the increase in the proportion of CD8+ T cells strongly suggest that a cytotoxic immune response against tumor cells has been induced, and thus, it can be strongly confirmed that the complex according to the present invention is very effective against solid tumors that overexpress PD-L1.

[0143] The present invention is recognized as having industrial applicability as a new immunotherapeutic agent.

Claims

1. Transition metal dichalcogenide (TMD)-based antibody mimetics, Transition metal dichalcogenide nanosheets; and A transition metal dichalcogenide (TMD)-based antibody mimetic comprising a tripeptide bound to the above nanosheet.

2. In paragraph 1, A transition metal dichalcogenide (TMD)-based antibody mimetic, characterized in that the tripeptide is a complex of a metal and a tripeptide capable of binding to the transition metal dichalcogenide.

3. In paragraph 1, A transition metal dichalcogenide (TMD)-based antibody mimetic, characterized in that the metal is copper.

4. In paragraph 3, A transition metal dichalcogenide (TMD)-based antibody mimetic, characterized in that the tripeptide is at least one selected from the group consisting of FNP, FNW, HSW, LSW, and YSA.

5. In paragraph 4, A transition metal dichalcogenide (TMD)-based antibody mimetic, characterized in that the type of target protein to which the transition metal dichalcogenide (TMD)-based antibody mimetic selectively binds and the binding density are determined depending on the type of the tripeptide.

6. In paragraph 5, A transition metal dichalcogenide (TMD)-based antibody mimetic characterized in that the target protein is PD-L1.

7. In paragraph 6, A transition metal dichalcogenide (TMD)-based antibody mimetic characterized in that the tripeptide is FNP or HSW.

8. An immunotherapeutic agent comprising a transition metal dichalcogenide (TMD)-based antibody mimetic according to any one of claims 1 to 7.

9. In paragraph 8, The above immunotherapy agent is an anticancer agent characterized in that it is an immune anticancer agent targeting PD-L1.

10. As a multimodal TMD antibody mimic that simultaneously has photothermal and PD-L1 immune checkpoint inhibitory effects, MoSe2 nanosheets; and A multimodal TMD antibody mimic characterized by comprising a tripeptide (TP) self-assembled on the surface of the MoSe2 nanosheet.

11. In paragraph 10, An antibody mimetic characterized in that the tripeptide comprises a sequence that selectively binds to the PD-L1 protein.

12. In paragraph 10, The above tripeptide is an antibody mimic characterized in that it is attached to MoSe2 nanosheets through Cu-NTA (copper-nitrilotriacetic acid) complexation.

13. In paragraph 10, The above antibody mimic is characterized in that it induces a photothermal reaction when irradiated with near-infrared (nIR).

14. An immunotherapeutic agent for cancer treatment, characterized in that it contains an antibody mimic according to Article 10 as an active ingredient.

15. In paragraph 14, The above immunotherapeutic agent is characterized by improving the tumor microenvironment by combining the PD-L1 immune checkpoint inhibition function and photothermal response.

16. In paragraph 15, The above immunotherapeutic agent is characterized in that it is applied to solid cancers including CT-26 colon cancer.

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