Cytokine compositions and uses thereof

Abstract

The present invention provides a novel construct comprising gold nanoparticles bound to two types of cytokines, where the two types of cytokines comprise Tumor Necrosis Factor alpha (TNF) and a cytokine selected from the group consisting of Interferon Gamma (IFN) and Interleukin-12. The invention also provides a method for cancer treatment of a subject in need thereof.

Description

Technical Field

[0001] Embodiments of this disclosure generally relate to novel nanoparticle and cytokine compositions and constructs, as well as methods for manufacturing and using them. Background Technology

[0002] Precisely delivering therapeutic agents to target areas of the body without diminishing their potency or efficacy will help improve treatment options. For example, current cancer treatments involve administering chemotherapy agents and other bioactive factors such as cytokines and immune factors, which affect the entire organism. Side effects of non-specific delivery include organ damage, sensory loss such as taste and sensation, and hair loss. While these existing therapies provide treatment for specific diseases, adjunctive therapies are needed to manage the resulting side effects.

[0003] When formulations containing therapeutic payloads with potential toxic side effects are administered systematically, there is an advantage to improved site-specific delivery and formulation stability, as well as technological improvements that reduce the non-selective release of therapeutic payloads, thereby improving the overall therapeutic effect of the drug.

[0004] Another shortcoming of existing therapies relates to maintaining or enhancing the potency of the applied therapeutic agents. While delivering such agents to the site of disease, such as a tumor, is the primary objective, maintaining their activity to maximize their effectiveness is equally important.

[0005] What is needed are compositions and methods for drug delivery systems capable of acting on desired cells or sites while maintaining or improving the efficacy of such drugs. Such systems could be used to deliver all types of drugs to specific cells. Furthermore, delivery systems that facilitate targeted delivery of therapeutic payloads without causing adverse side effects throughout the organism are also needed. Summary of the Invention

[0006] In one embodiment, this disclosure relates to compositions and methods for constructs comprising colloidal gold particles and cytokines, the constructs optionally being combined with one or more therapeutic agents and one or more polyethylene glycol molecules. In some embodiments, the colloidal gold particles comprise nanoparticles, and in other embodiments, the nanoparticles comprise colloidal gold nanoparticles. In some embodiments, the gold nanoparticles are bound to two types of cytokines, which may be tumor necrosis factor alpha (TNFα) (1) and interferon gamma (IFNγ), TNFα and interleukin-2 (IL-2) (2), or TNFα and interleukin-12 (IL-12) (3). The polyethylene glycol molecules may include polyethylene glycol derivatives covalently bound to the colloidal gold nanoparticles. Attached Figure Description

[0007] A better understanding of this disclosure can be achieved by reading the following detailed description in conjunction with the accompanying drawings. It should be emphasized that, by convention, the various features in the drawings are not necessarily drawn to scale. Instead, for clarity, the dimensions of the various features may be arbitrarily enlarged or reduced. Throughout the specification and drawings, the same reference numerals denote the same features.

[0008] Figure 1 A schematic diagram of cytokine-interferon-γ-tumor necrosis factor-α (CYT-IFNγ-TNFα) nanoparticles is provided.

[0009] Figures 2A to 2B A schematic diagram of a cross-antibody enzyme-linked immunosorbent assay (ELISA) is provided. Figure 2A ) and the results of cross-antibody ELISA performed using this method ( Figure 2B For this assay, nanoparticles containing only TNFα were used as a control group. Both experimental groups of CYT-IFNγ-TNFα nanoparticles and the control group were captured by TNFα monoclonal antibody (mAb), but the control group did not produce a signal in the ELISA due to the lack of IFNγ. The formulation containing IFNγ produced a significant signal, the intensity of which depended on the amount of IFNγ added during binding.

[0010] Figures 3A to 3C Micrographs of the binding and internalization of CYT-IFNγ-TNFα with the follicular thyroid cancer cell line FTC-133(4) are provided. CYT-IFNγ-TNFα was added to FTC-133 cells, and its binding and internalization were observed by bright-field microscopy. Figure 3AThe untreated cells are shown. Figure 3B and 3C The figures show the results after adding nanoparticles for 45 minutes. Figure 3B ) and 8 hours ( Figure 3C The position of the nanoparticles.

[0011] Figures 4A to 4D Micrographs of cytotoxicity induced by cytokine-based nanoparticles in FTC-133 cells are provided. FTC-133 cells were seeded in 6-well tissue culture plates in complete DMEM. 24 to 48 hours after seeding (to allow cell attachment), various nanoparticles at a concentration of 1 µg / mL were added to the cells. Nanoparticle treatments were as follows: untreated ( Figure 4A ); CYT-6091 (TNFα single agent); Figure 4B ); CYT-INFγ (IFNγ monotherapy; Figure 4C ); and CYT-IFNγ-TNFα ( Figure 4D Cells were cultured for another 5 to 7 days, and cytotoxicity was assessed using a microscope.

[0012] Figure 5 illustrates that CYT-IFNγ-TNFα exhibits stronger cytotoxicity against FTC-133 cells compared to solutions of the same doses of natural cytokines IFNγ and TNFα. The data presented are from two independent experiments, with four replicates for each concentration. The data clearly demonstrate that natural IFNγ-TNFα shows low cytotoxicity against FTC-133 cells compared to the same dose of cytokines added as CYT-IFNγ-TNFα. A similar pattern was observed with single-drug nanoparticles: CYT-IFNγ-TNFα exhibited higher potency compared to CYT-6091 (single-drug TNFα nanoparticles) or CYT-IFNγ (single-drug IFNγ nanoparticles).

[0013] Figure 6 The provided charts show that in the genetically engineered cell line HEK-IFNγ, the titer of CYT-IFNγ-TNFα exhibits a similar increase compared to natural IFNγ. HEK cells have been stably transfected with IFNγ receptors and signaling complexes. Titer is based on IFNγ concentration (…). Figure 6 ).

[0014] Figure 7 The provided charts show that the slight increase in cytokine stability is not the underlying mechanism for the increased potency observed in CYT-IFNγ-TNFα formulations (see Figures 4 and 5 for details).

[0015] Figure 8The provided micrographs show that CYT-IFNγ-TNFα induces receptor aggregation. Three hours after the addition of CYT-IFNγ-TNFα, FTC-133 cells were imaged in either the control group (untreated) or those treated with CYT-IFNγ-TNFα. These images illustrate different stages of nanoparticle uptake by FTC-133 cells.

[0016] Figures 9A to 9S provide micrographs illustrating the induction of human leukocyte antigen AC (HLA-AC) in human cancer cell lines treated with CYT-IFNγ-TNFα. 20,000 FTC-133, H-460, or A549 cells were seeded in 6-well tissue culture plates. Forty-eight hours after seeding, the cells were treated with different doses of CYT-IFNγ-TNFα (experimental details are provided in the "Examples" section below, particularly Example 3). Figure 9 shows images of three control wells and six wells treated with CYT-IFNγ-TNFα. Figures 9A to 9C illustrate the induction of HLA-AC in FTC-133 cells by 1 µg of CYT-IFNγ-TNFα. Figures 9D to 9G This demonstrates the uptake and internalization of CYT-IFNγ-TNFα by the human lung cancer cell line H-460. Its uptake pattern is similar to that of FTC-133 cells shown in Figure 3. Figures 9H to 9K The results showed that, similar to FTC-133 cells, CYT-IFNγ-TNFα induced the expression of HLA-AC in the three-dimensional cell clusters that H460 cells typically form. Figures 9L to 9O Images 9P to 9S show the uptake and intracellular processing of CYT-IFNγ-TNFα in A549 lung cancer cells. Consistent with the observations discussed herein, CYT-IFNγ-TNFα induces HLA-AC expression in A549 cells.

[0017] Figures 10A to 10D The provided micrographs show the activation of spleen cells induced by treatment of mouse cancer cells with CYT-IFNγ-TNFα. Figures 10A to 10B This diagram illustrates the proliferation of naïve spleen cells isolated from naïve Balb / c mice induced by Colo 26 cancer cells treated with CYT-mIFNγ-TNFα. Notably, in co-culture with control (untreated) cells, spleen cells, although located on the surface of cancer cells, did not proliferate. Figures 10C to 10D The early and late proliferation of initial spleen cells isolated from C57Bl / 6 mice induced by B16F10 melanoma cancer cells treated with CYT-m-IFNγ-hTNFα are shown.

[0018] Figures 11A to 11D The provided charts show that CYT-IFNγ-TNFα, when injected intraperitoneally ( Figures 11A to 11B) and intravenous injection ( Figures 11C to 11D After that, it will increase the blood retention time of IFNγ and TNFα.

[0019] Figure 12 The provided charts show the stability of CYT-IFNγ-TNFα in circulation. Blood samples collected in the early stages of the pharmacokinetic studies were analyzed according to the cross-antibody ELISA described in Example 1. In this assay, samples were captured using a neutralizing monoclonal antibody against TNFα and detected using a rabbit anti-human IFNγ / alkaline phosphatase-conjugated goat anti-rabbit polyclonal antibody.

[0020] Figure 13A and 13B Images of experimental animals are provided to visually confirm the uptake of CYT-mIFNγ-hTNFα by Colo-26 (solid) tumors. The presence of gold nanoparticles was recorded by digital photography of mice 4 hours after injection. Colo-26 tumors appear reddish to the gold nanoparticles (comparatively...). Figure 13A and 13B ). Figure 13A The control animals (not injected) are shown at T=3.5 hours after injection. Figure 13B Images were taken 3.5 hours after intravenous injection of CYT-mIFNγ-hTNFα.

[0021] Figure 14 The provided charts show the accumulation of CYT-IFNγ-TNFα in B16F10 tumors.

[0022] Figure 15 The saturation binding kinetics of IL-12 with colloidal gold nanoparticles are shown.

[0023] Figure 16 The provided charts show the potency enhancement of CYT-IL-12-TNFα relative to native IL-12 and TNFα in solution at the same concentration. This assay was performed using HEK-IL-12 cells, which are similar to HEK-IFNγ cells and secrete alkaline phosphatase reporter proteins.

[0024] Figure 17 presents confirming data that human IL-2 and TNFα exist on the same particle of CYT-IL-2-TNFα.

[0025] Figure 18 A schematic diagram of a multimodal immunotumor nanoparticle is provided. This novel nanoparticle consists of human IFNγ, human TNFα, and a thiolated paclitaxel prodrug bound to the same gold nanoparticle.

[0026] Figure 19 provides micrographs comparing the uptake of CYT-IFNγ-TNFα and CYT-IFNγ-TNFα-paclitaxel by FTC-133 cells. The paclitaxel-containing nanoparticles induced significant changes in nuclear morphology, leading to the formation of multi-segmented nuclei.

[0027] Figure 20 Micrographs are provided illustrating the mechanism by which CYT-IFNγ-TNFα-paclitaxel may generate tumor antigens. Following cell lysis, dead cells can release particles containing putative tumor antigens. In this way, the new construct can generate tumor antigens, thereby initiating an anti-tumor immune response.

[0028] Figure 21 Fluorescence / bright-field composite images of FTC-133 cells treated with the CYT-IFNγ-TNFα-paclitaxel construct are provided. Intracellular transport of the particles is again clearly visible as the particles appear red (see §). Furthermore, fluorescent labeling becomes prominent as the nanoparticles reach the perinuclear region, supporting the release of the prodrug (or small molecule payload). These data are consistent with the release of the prodrug substitute from the particles (∆). The image also depicts the post-internalization release of the alternative active pharmaceutical ingredient (API) at different stages. For example, API release is largely complete in cells marked with triangles (∆). However, early release is also shown in cells marked with asterisks (*). Detailed Implementation

[0029] The following detailed description is exemplary and illustrative only, and is intended to further illustrate the disclosure described herein. Other advantages and novel features will become apparent to those skilled in the art from the following detailed description of the disclosure. All texts and references mentioned herein are incorporated herein by reference, including U.S. Patent Nos. 7,387,900, 7,790,167, 7,951,614, 7,960,145, RE42,524, 8,435,801, 8,486,666, and 8,785,202.

[0030] The term “subject” as used in this article should be interpreted to include, for example, subjects undergoing medical or surgical procedures, such as humans and other animals requiring therapeutic intervention.

[0031] In this disclosure, the singular forms “a,” “an,” and “the” include the plural forms, and references to a particular numerical value include at least that particular numerical value unless the context clearly specifies otherwise. Thus, references such as “a bead” or “a nanostructure” refer to one or more such structures and their equivalents known to those skilled in the art, and so on. When numerical values ​​are expressed as approximations, the preposition “about” is used, and it should be understood that the specific numerical value would constitute another embodiment. As used herein, “about X” (where X is a numerical value) preferably inclusively refers to ±10% of the numerical value. For example, “about 8” preferably inclusively refers to 7.2 to 8.8; as another example, “about 8%” preferably (but not always) inclusively refers to 7.2% to 8.8%. All ranges mentioned should be inclusive and composable. For example, when referring to a range of “1 to 5,” the range should be interpreted as including ranges such as “1 to 4,” “1 to 3,” “1 to 2,” “1 to 2 and 4 to 5,” “1 to 3 and 5,” “2 to 5,” etc. Furthermore, when alternatives are explicitly listed, the list can be interpreted as any alternative being excluded, for example, by a negative limitation in the claims. For instance, when referring to the range “1 to 5,” the range can be interpreted as including the exclusion of any one of 1, 2, 3, 4, or 5; thus, “1 to 5” can be interpreted as “1 and 3 to 5, but excluding 2,” or simply as “excluding 2.” Any component, element, feature, or step expressly recited herein can be expressly excluded in the claims, whether such component, element, feature, or step is listed alternatively or recited separately.

[0032] As used herein, the term "cytokine" refers to a broad class of small proteins, such as interferons, interleukins, and growth factors, which are secreted by certain cells of the immune system and affect other cells. Cytokines in this disclosure include interferon-γ (IFNγ), TNFα, interleukin-2 (IL-2), and interleukin-12 (IL-12).

[0033] As used in this article, the term "cytotoxicity" refers to the degree to which a substance can damage cells. Substances or processes that can cause cell damage or death are described as cytotoxic. Treatment of cells with cytotoxic compounds can lead to a variety of cell fates. Cells may undergo apoptosis or necrosis, in which they lose cell membrane integrity and die rapidly due to cell lysis. Cells may also cease active growth and division (leading to decreased cell viability), or they may activate pathway-controlled cell death (i.e., apoptosis).

[0034] As used in this article, "paclitaxel" refers to a chemotherapy drug used to treat cancer. Paclitaxel is a taxane-based chemotherapy drug. "Paclitaxel prodrugs" include compounds with little or no pharmacological activity that are converted into pharmacologically active paclitaxel drug compounds in specific regions of the body. Such prodrugs may include thiol-derived paclitaxel prodrugs.

[0035] For ease of description below, it should be understood that alternative variations and implementations may be used in the following embodiments. It should also be understood that the specific articles, compositions, and / or processes described herein are illustrative only and should not be considered limiting.

[0036] Tumor necrosis factor-α (TNFα) (1) is a pleiotropic cytokine that affects almost every aspect of human health. Its discovery in the 1970s had a profound impact on the treatment of solid tumors, as a single injection of this protein could induce hemorrhagic necrosis in solid tumors, regardless of whether the cancer cells were sensitive to the protein. As research progressed, it was found that TNFα could selectively disrupt tumor blood vessels (5), reduce tumor interstitial fluid pressure (6), increase the uptake of subsequent chemotherapeutic drugs (7), and recruit immune system cells to the tumor site. These effects collectively contribute to a significant anti-tumor response.

[0037] Shortly after its discovery, a recombinant form of the cytokine was prepared to support clinical trials in cancer patients. However, in nearly 200 clinical trials, systemically delivered TNFα was found to be highly toxic, thus limiting the safe dose for administration to cancer patients. The main dose-limiting toxicities of TNFα were hypotension and hepatotoxicity (6). In all of these clinical trials, no durable antitumor response was observed.

[0038] Furthermore, early phase I clinical trials of TNFα revealed that even at low doses, TNFα can cause a number of potentially dangerous side effects. Some of these side effects, such as severe fever and chills, are manageable with medication and therefore do not pose a significant obstacle to patient treatment or adherence. Other adverse reactions, such as tachycardia, can lead to disqualification events (8). This can result in stroke and heart attack in high-risk patients. It is worth noting that many cancer therapies, such as doxorubicin (9), which may be used in combination with CYT-6091 (as described below), are known to have cardiotoxicity.

[0039] Given these data, the application of TNFα in cancer treatment was limited to a limb-sparing procedure called Isolated Limb Perfusion (ILP) (10). In this procedure, blood vessels in the affected limb of a patient with melanoma or sarcoma of the extremities are connected to a cardiopulmonary bypass machine, which first infuses TNFα into the affected limb and then administers chemotherapy through that limb. ILP achieves two main goals: first, local delivery of TNF increases cytokine concentration at the site of disease; second, local perfusion of cytokines within the limb reduces systemic exposure to cytokines, thus avoiding most toxic side effects. However, the significant antitumor response (60 to 75% complete remission rate and durability (10 years)) led the inventors to develop the first patented gold nanoparticle, CYT-6091.

[0040] CYT-6091 (11 to 12) consists of 27 nm gold nanoparticles that are covalently linked to TNFα and thiolated polyethylene glycol (PEG-THIOL) via coordination covalent bonds.

[0041] This document discloses novel cytokine constructs comprising gold nanoparticles and TNFα. In one embodiment, the constructs provided herein comprise gold nanoparticles that bind to two types of cytokines, wherein the two types of cytokines include tumor necrosis factor-α (TNFα) and cytokines selected from the group consisting of interferon-γ (IFNγ) and interleukin-12 (IL-12) or interleukin-2 (IL-2). One embodiment comprises gold nanoparticles that bind to TNFα and IFNγ, wherein in some embodiments, the ratio of TNFα to IFNγ is approximately 20:1 (w / w). Another embodiment comprises gold nanoparticles that bind to TNFα and interleukin-2 (IL-2) or TNFα and interleukin-12 (IL-12).

[0042] In some embodiments, the cytokine constructs of this disclosure include cytokines bound to the surface of nanoparticles using one or more chemical binding mechanisms, including thiols or other covalent binding, ionic binding, or hydrophobic interactions.

[0043] In some embodiments, the cytokine construct may also include polyethylene glycol, polyethylene glycol derivatives, or polyethylene glycol thiols.

[0044] The cytokine constructs of this disclosure, including tumor necrosis factor α (TNFα) and interferon γ (IFNγ), may also include paclitaxel, paclitaxel analogs, or paclitaxel prodrugs.

[0045] In one embodiment, this document provides a method for enhancing the cytotoxicity of cytokines, comprising the steps of: conjugating gold nanoparticles with two types of cytokines to construct a construct, wherein the cytokines are composed of tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ); introducing the construct into a biological sample containing cells; and evaluating the cytotoxicity of the construct to the cells, wherein cytotoxicity is enhanced compared to the introduction of (a) a natural cytokine and / or (b) a single cytokine conjugated with gold nanoparticles. This method can be used for biological samples including cancer cells, and more specifically, for thyroid cancer cells. Cytotoxicity can be measured using a cell viability assay.

[0046] In one embodiment, this paper provides a method for enhancing cytokine titers, comprising the following steps: conjugating gold nanoparticles with two types of cytokines to construct a construct, wherein the cytokines consist of tumor necrosis factor-α (TNFα) and interleukin-12 (IL-12); introducing the construct into a cell-containing biological sample; and evaluating the titer of the construct on the cells, wherein the titer is enhanced compared to the introduction of natural cytokines. The titer can be measured using an HEK cell bioassay for assessing receptor activation. The HEK cell bioassay used herein is a HEK cell-based functional assay for studying receptor activity through fluorescence output.

[0047] In one embodiment, this document provides a method for inducing the expression of Major Histocompatibility Complex-1 (MHC-1) (13) in cancer cells, comprising introducing a cytokine construct into the cancer cells, wherein the cytokine construct comprises tumor necrosis factor α (TNFα) and interferon γ (IFNγ) bound to gold nanoparticles. The cancer cells may include lung cancer cells or thyroid cancer cells.

[0048] In one embodiment, this article provides a method for activating naïve lymphocytes in a biological sample containing cancer cells, comprising: introducing a cytokine construct into the biological sample to induce MHC-1 (HLA-AC) expression, and then adding lymphocytes; wherein the cytokine construct is composed of tumor necrosis factor α (TNFα) and interferon γ (IFNγ) bound to gold nanoparticles, wherein the cytokine construct has cytotoxic effects on cancer cells and activates lymphocytes.

[0049] This article provides a method for treating cancer in a subject of need, comprising administering a cytokine construct to the subject, wherein the cytokine construct comprises tumor necrosis factor α (TNFα) and interferon γ (IFNγ) bound to gold nanoparticles. In this embodiment, the construct may further comprise polyethylene glycol, a polyethylene glycol derivative, or polyethylene glycol thiol, and the TNFα to IFNγ ratio is approximately 20:1 (w / w). In this embodiment, the construct may further comprise paclitaxel or a paclitaxel analog or prodrug.

[0050] This document provides compositions and methods relating to structures comprising colloidal gold nanoparticles and cytokines, optionally combined with one or more therapeutic agents and polyethylene glycol molecules. The cytokines are of the types comprising tumor necrosis factor-α (TNFα), interferon-γ (IFNγ), interleukin-12 (IL-12), or interleukin-2 (IL-2). The polyethylene glycol molecules may include polyethylene glycol derivatives covalently bound to the colloidal gold nanoparticles.

[0051] In one embodiment of the present invention, a method for treating diseases and symptoms is provided, comprising administering a cytokine construct. In this embodiment, the method for treating a solid tumor includes administering a composition to an organism having the solid tumor, wherein the composition includes a cytokine construct composed of colloidal gold particles and two types of cytokines, and optionally combined with one or more therapeutic agents and one or more polyethylene glycol molecules; wherein the colloidal gold particles are composed of gold nanoparticles, and wherein the two types of cytokines bound to the gold nanoparticles are TNFα and IFNγ.

[0052] In some embodiments, the cancer is melanoma. In some embodiments, the cancer is a solid tumor. In some embodiments, a method of treating a solid tumor includes administering a novel cytokine construct composition to an organism suffering from a solid tumor, wherein the novel cytokine construct comprises gold nanoparticles bound to two types of cytokines, including tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ).

[0053] The following examples are intended to illustrate exemplary embodiments of this disclosure. However, it should be understood that this disclosure is not limited to the specific conditions or details described in these examples. The following examples are intended to help to provide a more complete understanding of the invention. The following examples illustrate exemplary manufacture and implementation methods of the invention. Example background Production of CYT-IFNγ-TNFα

[0054] There are two methods for preparing CYT-IFNγ-TNFα. The first method involves simultaneously binding TNFα, IFNγ, and PEG-THIOL to the surface of gold nanoparticles. The second method involves first binding TNFα to the surface of gold nanoparticles, and then simultaneously binding IFNγ and PEG-THIOL to the surface of the gold nanoparticles.

[0055] In these studies, the pH of the colloidal gold solution was adjusted to approximately 8.0 by stepwise addition of 50 mM sodium borate (NaBo) solution. Similarly, the binding buffer (BB) used to dilute TNFα (CytImmune Sciences, Inc.), IFNγ (R&D Systems), and 20 kDa PEG-THIOL (SunBio, Inc.) was also adjusted to 8.0 using NaBo. TNFα, IFNγ, and PEG-THIOL were diluted in BB to final concentrations of 0.25, 5.0, and 15 µg / mL, respectively. An equal volume of gold nanoparticle solution and BB solution (containing various reagents) were mixed, and the BB solution was rapidly added to the gold nanoparticle solution under vigorous vortexing. The solution was incubated for at least 3 hours.

[0056] Subsequently, the reagents bound to the particles and those free from the particles are separated by centrifugation, although ultrafiltration can be easily performed by those skilled in the art using suitable devices such as ultrafiltration cartridges or hollow fiber modules. The concentrated nanoparticles are washed twice with an isotonic solution. After the final concentration step, the gold nanoparticles are aliquoted and frozen at -80°C. Alternatively, a freeze-drying cycle for long-term storage of nanomedicines at -20°C to +4°C can also be used. Determination of bound and free TNFα and IFNγ

[0057] Cytokine-specific sandwich ELISA was used to determine the particle-bound and free fractions of TNFα and IFNγ. Both ELISAs used commercially available cytokine-specific neutralizing monoclonal antibodies (R&D Systems) to capture TNFα or IFNγ in solution, whether bound to or free of the particles. Once captured, a cytokine-specific rabbit polyclonal antibody (CytImmune Sciences, Inc.) was added to the wells, and the complex was detected using alkaline phosphatase-conjugated goat anti-rabbit antibody (Sigma). The concentrations of each cytokine were determined by regression analysis against known standards. Depending on the assay scale, the concentrations of TNFα and IFNγ typically ranged from 2 to 40 µg / mL, with 90 to 95% of the cytokines bound to the gold nanoparticles. Example 1

[0058] Cross-antibody ELISA: Qualitatively demonstrated that both IFNγ and TNFα exist on the same gold nanoparticle.

[0059] While the ELISA described in Example 1 can be used to quantify the relative amounts of IFNγ and TNFα on the particle surface, they do not prove the presence of both cytokines.

[0060] To meet this need, a cross-antibody (XAb) ELISA (Figure 2A) was developed, in which nanoparticles are captured by a monoclonal antibody specific to the first cytokine and detected by a polyclonal (rabbit) antibody against the second cytokine.

[0061] In one XAb ELISA, a mouse monoclonal antibody against TNFα is used to capture CYT-IFNγ-TNFα or a single-reagent control that binds only to TNFα. Once the nanoparticles are captured by the mAb (incubated at room temperature for 4 to 24 hours), the plate is washed and a rabbit polyclonal antibody against human IFNγ is used as the detection system. Figure 2B As shown, single-drug TNFα nanoparticles produce almost no signal in XAb ELISA. CYT-IFNγ-TNFα not only produced a significant signal in XAb ELISA, but the color intensity also depended on the amount of IFNγ initially bound to the nanoparticles during preparation.

[0062] Follicular thyroid cancer cell line FTC-133's binding, uptake, and internalization of CYT-IFNγ-TNFα

[0063] In these studies, 5,000 to 10,000 FTC-133 cells were seeded in 6-well tissue culture plates, with 2 mL of complete DMEM added to each well. The cells were cultured under standard tissue culture conditions (37°C, 95% relative humidity). After 24 to 48 hours, CYT-IFNγ-TNFα (IFNγ concentration of 0.05 to 2.0 µg) was added to the culture medium, and bright-field or phase-contrast microscopy was used to image the uptake of nanoparticles by the cells at different time points after the addition of the nanoparticles.

[0064] As shown in Figure 3, when FTC-133 cells take up CYT-IFNγ-TNFα, the cells appear reddish due to the colloidal gold nanoparticles. Within 15 to 45 minutes after the addition of the nanoparticles, the particles are evenly distributed on the cell surface. Over the next 90 minutes, the staining pattern is localized to specific regions of the cell (see the example of receptor aggregation below). Finally, after 8 to 12 hours, black aggregates of the particles are observed in the perinuclear region.

[0065] Cytotoxicity assessment

[0066] Given the uptake of nanoparticles and the known cytotoxicity of both TNFα and IFNγ in certain cancer cell lines, the experiment was repeated, but cell growth was measured after an additional 5 to 7 days of incubation. In addition, other nanoparticles such as CYT-6091 (see below) and single-drug interferon γ nanoparticles were also added to the experiment.

[0067] Figure 4 shows micrographs of the various cultures described above. The data in Figure 4 indicate that both CYT-6091 and CYT-IFNγ are cytotoxic to FTC-133 cells because the confluence of pores is lower compared to the untreated control group. Figure 4A However, CYT-IFNγ-TNFα exhibited the strongest cytotoxicity, as no surviving cells were observed in the culture. Example 2

[0068] CYT-IFNγ-TNFα enhances the potency of IFNγ and TNFα.

[0069] This study compared the potency of CYT-IFNγ-TNFα with solutions of the same doses of IFNγ and TNFα. As described above, small batches of nanoparticles were prepared, and the concentrations of cytokines bound to the particles were determined by quantitative ELISA. Subsequently, IFNγ and TNFα in progressively increasing concentrations were added to FTC-133 cells cultured in 96-well tissue culture plates in the form of CYT-IFNγ-TNFα. In another set of wells, the same doses of native IFNγ and TNFα were added at the same concentration as CYT-IFNγ-TNFα. The native cytokines were added as a single solution.

[0070] Cells were cultured at 37°C and 95% relative humidity for 2 to 3 days. Then, the culture medium was removed, and the cells were gently washed three times with serum-free DMEM. After the final wash, 100 µL of complete DMEM was added back to the cells, followed by 10 µL of AlamarBlue™. The plates were incubated at 37°C until the fluorescence value of the untreated / control group wells reached 10. 4 Relative fluorescence unit.

[0071] As shown in Figure 5, CYT-IFNγ-TNFα enhanced the dose-to-dose cytotoxicity of IFNγ / TNFα in FTC-133 cells. These data are consistent with the more efficient interaction between IFNγ / TNFα and its respective receptors, which may be achieved through the induction of receptor aggregation (see below).

[0072] CYT-IFNγ-TNFα enhances the potency of IFNγ / TNFα in genetically engineered cell lines.

[0073] HEK-IFNγ (InvivoGen, California, USA) is a genetically engineered HEK cell line stably transfected with the IFNγ receptor / signaling mechanism. In this cell line, the binding of IFNγ to its receptor induces the expression and secretion of an alkaline phosphatase reporter gene. The yield of alkaline phosphatase is directly proportional to the amount of IFNγ in the sample. The amount of reporter gene released can be measured by adding a fixed volume of PNPP (p-Nitrophenyl Phosphat; Sigma-Aldrich, Missouri, USA) substrate to tissue culture supernatant.

[0074] To assess whether CYT-IFNγ-TNFα induces a similar titer enhancement as shown in cytotoxicity studies, according to... Figure 5A and Figure 6 The method described herein applies these cells to similar experiments. In short, following the manufacturer's instructions, 20,000 HEK-IFNγ cells are seeded into a culture plate. The following day, IFNγ and TNFα are added in gradually increasing concentrations, either as a single solution or in the form of CYT-IFNγ-TNFα. Cells are cultured for another 48 hours. Afterward, 10 µL of tissue culture supernatant is collected and added to 200 µL of PNPP substrate. The reaction is monitored by measuring the optical density (OD) at 405 nm, and the reaction is terminated when the optical density in the highest dose group reaches 2.0 to 3.0 OD units.

[0075] The combination of natural IFNγ / TNFα elicited a dose-dependent increase in the relative levels of alkaline phosphatase secreted by HEK-IFNγ cells. Unlike FTC-133, where the natural cytokine combination exhibited low activity, the data for HEK-IFNγ were as expected, as these cells are genetically engineered to secrete reporter proteins in a dose-dependent manner. However, Figure 6 The data in [the figure] are consistent with those in Figure 5, as a similar increase in titer (EC) was also observed in cells receiving CYT-IFNγ-TNFα. 50 (Reduced by 15 times).

[0076] Gold nanoparticles confer stability to cytokines

[0077] The enhanced potency observed in CYT-IFNγ-TNFα may be partly attributed to the mechanism by which gold nanoparticles enhance cytokine stability. To validate this in a simple matrix, equal concentrations of native IFNγ and TNF or CYT-IFNγ-TNF were added to FTC-133 cells, followed by incubation at 37°C for 2 days. On the second day, aliquots of samples from each group were collected, cryopreserved at -80°C, and analyzed by ELISA.

[0078] Figure 7 The data shown indicate that the concentration of recovered cytokines in the natural cytokine sample was measured to be slightly lower by approximately 20%. While this decrease is significant, it is unlikely to explain the difference in potency between the two formulations. However, it is worth noting that improving cytokine stability by binding cytokines to the particle surface is desirable.

[0079] Evidence of receptor aggregation

[0080] While reluctant to be bound by the theories described later, it is hypothesized that the potency enhancement shown in Figure 5 may be mediated by receptor aggregation. Given that both IFNγ and TNFα are biologically active on the particle surface (quantitative ELISA uses specific neutralizing mAbs targeting each cytokine to capture CYT-IFNγ-TNFα), it is hypothesized that when IFNγ and TNFα are present on the particle surface, IFNγ, and possibly TNFα, also induces enhanced affinity for their respective receptors. Based on these observations, preliminary evidence supporting receptor aggregation was generated using a gold nanoparticle platform.

[0081] To verify this hypothesis, the imaging study outlined in Example 1 was repeated, and cells were imaged from 90 minutes to 8 hours after the addition of CYT-IFNγ-TNFα. Bright-field microscopy and phase-contrast microscopy were used to image the cells to confirm the migration of the nanoparticles.

[0082] Figure 8 The presented data are consistent with receptor aggregation and the inferred internalization of particles within endosomes. Within endosomes, the particle-bound components (TNFα, IFNγ, and PEG-THIOL) are degraded or released from the particle surface, and in the absence of any passivating agents, these so-called "naked particles" aggregate as they migrate toward the cell nucleus.

[0083] Unlike the nearly transparent images of control FTC-133 cells (top left), many cells appeared pink, indicating the presence of nanoparticles on the cell surface. Similarly, all FTC-133 cells treated with CYT-IFNγ-TNFα showed perinuclear localization of the particles, appearing as black deposits around the nucleus. Furthermore, in some cells marked by arrows in the bright-field image (bottom left) and phase-contrast image (bottom right), the nanoparticle uptake pattern was a dotted distribution. While no mechanism of action is provided, these preliminary data support the idea that CYT-IFNγ-TNFα induces aggregation of its target receptor. Example 3

[0084] CYT-IFNγ-TNFα induces MHC-1

[0085] Many tumors evade detection and clearance by the immune system due to underexpression or absence of the major histocompatibility antigen (HLA-AC) (13–14). In humans, this complex is named HLA-AC, and its mouse counterpart is called MHC-1. IFNγ is known to upregulate MHC-1 expression (12). To test this hypothesis, baseline (untreated) expression of the HLA-AC and CYT-IFNγ-TNFα-induced expression in human and mouse cancer cell lines were assessed.

[0086] For these studies, a variety of cell lines were used, including FTC-133 (human thyroid cancer), H460 and A549 (human lung cancer), Colo26 (mouse colon cancer isolated from Balb C mice), and B16F10 (melanoma cell line isolated from C57Bl / 6 mice). Approximately 20,000 cells from each cell line were seeded into 6-well tissue culture plates and cultured for 48 hours under standard culture conditions. Subsequently, the cells were co-cultured with human nanoparticles CYT-IFNγ-TNFα or its murine equivalent CYT-mIFNγ-hTNFα.

[0087] Since human IFNγ differs from human TNFα and does not exhibit cross-reactivity in mice, mouse-derived nanoparticles are required. This variant was prepared and studied using the method outlined in the aforementioned examples.

[0088] In these studies, 0.0625 to 1 µg of CYT-IFNγ-TNFα (human nanoparticles) or CYT-mIFNγ-TNFα (mouse nanoparticles) were incubated with FTC-133, H460, and A549 cells, or Colo 26 or B16F10 cancer cells, respectively. After 48 hours, the cells were washed twice with incomplete DMEM, and 1 µg of a mouse monoclonal antibody (Sigma Aldrich) recognizing the HLA-AC and mouse MHC-1 complex was added to the culture. The antibody was diluted with complete DMEM and incubated with the cells for 1 hour. Subsequently, the cells were washed twice more with incomplete DMEM, and a FITC-conjugated goat anti-mouse polyclonal antibody (Sigma Aldrich) was added to the culture. The antibody was incubated for another hour. Finally, the cells were washed once with incomplete DMEM, and the presence of the HLA-AC or MHC-1 complex was recorded using fluorescence microscopy.

[0089] Figure 9 shows validation data for HLA expression induced by CYT-IFNγ-TNFα in human cancer cell lines FTC-133, HL-460, and A549 (see Figures 9A to 9C, 9D to 9K, and 9A to 9C, respectively). Figures 9L to 9O In H460 and A549 cells, HLA AC induction ( Figure 9P The effect of HLA-AC on the control group occurred over a wide dose range. Consistent with the hypothesis proposed by Angell et al. (12), control group cells expressed little or no HLA-AC complex, while cell cultures treated with nanoparticles showed significant antigen staining.

[0090] Furthermore, the binding and internalization of nanoparticles, as highlighted by pink / black staining of cells, and subsequent expression of HLA complexes (see [link to H-460 cancer cell line] for details). Figures 9D to 9G For the A549 cancer cell line, see [link to relevant documentation]. Figures 9L to 9O As confirmed by [the study], both human lung cancer cell lines exhibit similar CYT-IFNγ-TNFα uptake patterns.

[0091] Anti-cancer immune response generated by CYT-mIFNγ-TNFα

[0092] In a later study, it was determined whether MHC-1 induction via CYT-IFNγ-TNFα induced a new anti-cancer immune response. In this study, Colo 26 or B16F10 cancer cells were seeded into culture plates and treated with a murine variant of CYT-IFNγ-TNFα (CYT-mIFNγ-hTNFα), as described above. The control group consisted of untreated cancer cells. After confirming MHC-1 induction, approximately 10 6Spleen cells from naïve Balb / c mice were added to Colo 26 cell cultures. The same number of spleen cells from C57Bl / 6 mice were added to either the control group or nanoparticle-treated cultures, and the induction of anticancer immune responses was confirmed by spleen cell proliferation.

[0093] The data presented in Figure 10 support the fact that CYT-mIFNγ-hTNFα induces the proliferation of spleen cells obtained from naïve Balb / c mice. Several patterns were observed in these cultures. Consistent with the data shown in Figures 4 and 5, compared to the untreated control group (… Figure 10B Compared to CYT-mIFNγ-TNFα, CYT-mIFNγ-TNFα significantly inhibited the proliferation of Colo26 cells. Figure 10A Furthermore, the nanoparticles activated naïve spleen cells, causing them to proliferate directly on the surface of the remaining cancer cells. These data are consistent with the findings reported by Paciotti et al. (15) that nanoparticles induce the proliferation of naïve B cells. Finally, consistent with the low MHC expression scenario, spleen cells added to the untreated control group did not proliferate, and as... Figure 10B As shown, although these cells are located directly on the surface of proliferating cancer cells, they remain quiescent.

[0094] Similar data were obtained in B16F10 cancer cell cultures treated with CYT-mIFNγ-hTNFα. Figure 10C As shown, within 24 hours of adding spleen cells isolated from naïve C57Bl / 6 mice to B16F10 melanoma cells treated with CYT-mIFNγ-hTNFα, lymphocytes significantly proliferated. These data further support the conclusion in this study that nanoparticles induce cancer cell antigen presentation, ultimately leading to lymphocyte proliferation. Example 4

[0095] Pharmacology of CYT-IFNγ-TNFα

[0096] CYT-6091 (11 to 12) and CYT-21625 (16) are tumor-targeting nanoparticles that deliver TNFα as a single drug (CYT-6091) or TNFα plus a paclitaxel prodrug (CYT-21625) to solid tumors. Both nanoparticles are engineered based on 27 nm polyethylene glycol-modified colloidal gold particles. Pharmacokinetic and tumor uptake studies showed that, compared with the natural formulations (11 and 16), CYT-6091 and CYT-21625 exhibited enhanced pharmacokinetic exposures due to polyethylene glycol modification, as measured by terminal half-life (T½) and area under the curve (AUC).

[0097] In the experiments described later, the ability of the PEG-THIOL portion of the bound particles to enhance the pharmacokinetic exposure and tumor uptake of IFNγ in a similar manner was evaluated. Therefore, after formulation and analysis of the nanoparticles, TNFα and IFNγ were injected in a single solution, or in a formulation of CYT-IFNγ-TNFα with the same dose of cytokines, into Balb / c mice either naïve or carrying Colo-26 tumors. Given that currently approved IFNγ formulations are for subcutaneous injection, pharmacokinetic characteristics were determined by intravenous or intraperitoneal injection.

[0098] Animals were sacrificed at different time points after injection, and whole blood was collected and mixed with 18 mg / mL EDTA solution (10% v / v). The levels of TNFα and IFNγ in the samples were determined by cytokine-specific ELISA. To demonstrate that CYT-IFNγ-TNFα remained stable in circulation, blood samples were collected after injection and analyzed using the XAb assay described in Example 1.

[0099] The data presented in Figure 11 show that CYT-IFNγ-TNFα increased the pharmacokinetic exposure of both cytokines compared to treatment with natural cytokines. These data are consistent with the previously reported pharmacokinetic characteristics of CYT-6091 and CYT-21625.

[0100] CYT-IFNγ-TNFα does not undergo burst release after being injected into the bloodstream.

[0101] One of the common obstacles in nanoparticle development is burst release, where nanoparticle components are released almost immediately after injection. For TNFα and IFNγ, this can lead to toxicities such as hypotension, and in the case of TNFα, it can also cause hepatotoxicity. Therefore, to test the stability of CYT-IFNγ-TNFα in circulation, circulating samples collected early in pharmacokinetic studies were analyzed using the cross-antibody (XAb) ELISA described in Example 1. In short, if the TNFα or IFNγ component undergoes burst release in circulation, it is expected that the blood sample will not produce a signal in the XAb ELISA because the particles are not captured or detected.

[0102] Figure 12 The presented data support the early stability of CYT-IFNγ-TNFα, as samples collected early in the pharmacokinetic studies produced significant signals in XAb ELISA, while samples collected from animals receiving the natural formulation did not produce significant signals in ELISA. Example 5

[0103] Accumulation of CYT-IFNγ-TNFα in solid tumors

[0104] In the subsequent study, the accumulation of human or murine forms of CYT-IFNγ-TNFα in two murine tumor models was tracked. In the first study, the presence of nanoparticles was tracked using imaging control in either the control group or the nanoparticle-treated animals. In Figure 13, CYT-mIFNγ-hTNFα was intravenously injected into Balb / c mice carrying Colo-26 tumors. The presence of nanoparticles was easily recorded 3 to 4 hours post-injection because the Colo-26 tumors exhibited a reddish hue, similar to that of gold nanoparticles (see Figure 13). Figure 13A and 13B ).

[0105] Figure 13A Images of Balb / c mice carrying Colo-26 tumors, taken from control groups (e.g., untreated). Figure 13B Images of Balb / c mice carrying Colo-26 tumors 3.5 hours after intravenous injection of CYT-mIFNγ-TNFα are shown. The accumulation of the CYT-mIFNγ-TNFα nanomedicine in the tumors is evident because the tumors appear reddish, similar to gold nanoparticles.

[0106] Accumulation of CYT-IFNγ-TNFα in B16F10 tumors

[0107] In this study, as described by Paciotti et al. (14), B16F10 tumors were established on the ventral surface of C57Bl / 6 mice. After tumor establishment, mice (n=5 / group) received intraperitoneal injections of CYT-IFNγ-TNFα or the same dose of cytokine solution. Animals were sacrificed 4 hours later, and the tumors were removed and frozen at -80°C. The samples were then homogenized using a glass homogenizer. The intratumoral IFNγ content in the homogenate was analyzed by ELISA.

[0108] Consistent with the data reported by CYT-6091(10) and CYT-21625(14), Figure 14 The data supports the idea that nanoparticles can accumulate IFNγ in solid tumors. Example 6

[0109] Saturation binding of interleukin-2 or interleukin-12: production of cytokines interleukin-12 (CYT-IL-12) and interleukin-2 (CYT-IL-2).

[0110] The data presented in the previous example support the following phenomenon: binding highly potent immune molecules such as INFγ and TNFα to the surface of PEGylated gold nanoparticles can enhance the potency of cytokines and improve their pharmacokinetic characteristics, thereby facilitating their accumulation in solid tumors.

[0111] Using the method described in Example 1, we further sought to develop other follow-up nanoparticles with the potential to induce and drive potent anticancer vascular and immune responses. Once fabricated, the nanoparticles were studied using the aforementioned analytical methods.

[0112] Saturation binding curve experiments for interleukin-2 (IL-2) or interleukin-12 (IL-12) were performed by adding incremental amounts of cytokines to a fixed volume of gold. After incubation for 1 hour, the particles were centrifuged at 14,000 rpm, and the supernatant was collected and set aside. The particles were restored to 0.1 times their original volume, and the bound and free (supernatant) fractions of the particles were determined using an internally developed IL-2 or IL-12 ELISA.

[0113] Figure 15 The data shown indicate that IL-12 exhibits saturation binding to gold particles. At relatively low masses, most cytokines bind to the particles (solid dots). As the particle surface becomes saturated with cytokines, the detected amount of free cytokines increases (hollow dots). Example 7

[0114] The formation of the CYT-IL-12-TNFα construct significantly enhances the potency of IL-12.

[0115] A dual-effect IL-12-TNFα complex, CYT-IL-12-TNFα, was prepared using the method described in Example 1. The titer of CYT-IL-12-TNFα was detected using an HEK-12 cell bioassay. Similar to HEK-IFNγ cells, HEK-IL-12 cells respond to IL-12 by secreting the same alkaline phosphatase reporter protein. Therefore, for this study, equal doses of native IL-12 or CYT-IL-12 were incubated with HEK-IL-12 cells according to the manufacturer's instructions. After 48 hours, 10 μL of sample was taken from each replicate well and added to 200 μL of PNPP substrate. Similar to the reporting data for the CYT-IFNγ-TNFα nanomedicine, CYT-IL-12-TNFα induced a significant increase in IL-12 titer, nearly 70-fold (…). Figure 16 ). Example 8

[0116] Generation of IL-2-TNF dual agent nanoparticles

[0117] In subsequent studies, 5 μg of interleukin-2 (IL-2), 0.25 μg of TNF, and 15 μg of PEG-THIOL were added to a single solution of gold nanoparticles. A single-drug control group containing only one cytokine was also prepared. Bound and free cytokines were separated by centrifugation. To verify that both cytokines were present on the same particle, single-drug and dual-drug nanoparticles were serially diluted and detected in two forms of cross-antibody ELISA. In these assays, either dual-drug or single-drug nanoparticles were captured using TNFα or IL-2 monoclonal antibodies. The substances captured by the monoclonal antibodies were detected using a rabbit polyclonal antibody (Cytimmune Sciences, Inc., Maryland, USA), and complementary cytokines were detected using an alkaline phosphatase-conjugated goat anti-rabbit antibody.

[0118] The data presented in Figure 17 confirm that both cytokines are present on the same particle. In both assays, the colorimetric reaction produced by the single-drug control group was weak, while the dual-drug particle produced a significantly stronger signal than the single-drug control group. Furthermore, the signal intensity produced in each XAb ELISA was consistent with the amount of cytokine initially bound. For example, in the XAb assay for capturing TNFα, the detection of IL-2 showed a significant signal across the entire dilution curve range. Conversely, although dilution curves were also observed in the IL-2 XAb assay, the signal of TNFα showed a more significant decrease with each dilution. Example 9

[0119] Development of Multimodal IO Nanoparticles

[0120] Long-standing evidence suggests that many chemotherapy drugs activate the immune system as part of their anti-tumor efficacy (17–18). Conceptually, chemotherapy may generate cancer antigens needed to initiate and drive cancer immunotherapy. This view is also supported by the fact that many current clinical trials focused on tumor immunotherapy have added chemotherapy groups (19–20). Other therapies, including precision medicine, have the potential to restore or block certain biological pathways, which may enhance or drive anti-cancer efficacy, and are used in combination with cytokines; therefore, a growing number of small molecule immuno-oncology drugs are under development.

[0121] To verify the ability to generate such drugs, a multimodal immunotumor nanoparticle composed of TNFα and IFNγ was developed and a small molecule prodrug was added to it. Figure 18 A schematic diagram of this novel nanostructure is shown. Specifically, a previously reported thiol-derived paclitaxel prodrug form is incorporated into existing CYT-IFNγ-TNFα parent nanoparticles.

[0122] Using the method described in Example 1, 2.5 µg of a thiolized paclitaxel analog was added to an existing formulation. The free drug was separated from the particle-bound components by centrifugation, and the cytokine concentrations of the nanoparticles were analyzed.

[0123] To preliminarily understand the effect of adding paclitaxel prodrug CYT-IFNγ-TNFα, FTC-133 cells were incubated with similar doses of IFNγ and TNFα, which were added in the form of CYT-IFNγ-TNFα or CYT-IFNγ-TNFα-paclitaxel nanoparticles. Significant morphological changes were observed within two days of incubation. For example, with CYT-IFNγ-TNFα, particles were observed located around the nucleus, as described above. In the case of CYT-IFNγ-TNFα-paclitaxel, the nucleus became segmented, and particles were located around each segment.

[0124] The micrographs presented in Figure 19 demonstrate that CYT-IFNγ-TNFα represents a unique mechanism for the intracellular targeted delivery of small molecule immuno-oncology drugs using TNFα and IFNγ. To further validate this concept, a derived nanoparticle was generated in which a thiolized paclitaxel prodrug was replaced by a fluorescently labeled 2kDa PEG-THIOL. Adding this construct to FTC-133 cells allowed for the tracking of particle entry into the cells and the release of the prodrug substitute (e.g., the fluorescently labeled 2kDa PEG-THIOL).

[0125] The nanoparticles discussed in this article propose a possible therapeutic strategy for solid tumors. Figure 20 The image shows FTC-133 cells treated with CYT-IFNγ-TNFα-paclitaxel. CYT-IFNγ-TNFα-paclitaxel treatment leads to cell lysis and the production of cell debris (from...). Figure 20 (Circles in the image indicate this). These cellular debris may contain tumor antigens that can initiate an immune response. These findings suggest that using CYT-IFNγ-TNFα-paclitaxel to initiate an immune response, followed by the use of other immunotumor nanoparticles, may be a therapeutic strategy to further drive an antitumor immune response.

[0126] Figure 21 Fluorescence / bright-field composite images of FTC-133 cells treated with this construct are shown. The red color (§) of the particles indicates intracellular transport. Furthermore, the fluorescent markers become prominent when the nanoparticles reach the perinuclear region, supporting the release (Δ) of the prodrug analog from the particles. References 1.Alexander, H.R., & Feldman, A. 2000. Tumor Necrosis Factor: BasicPrinciples and clinical applications in systemic and regional cancertreatment. In Principles and Practice of the Biologic Therapy of Cancer.Eds. S. Rosenberg. Lippincott Williams and Wilkins. 2.Lode, H.N., Xiang, R., Duncan, S.R., Theofilopoulos, A.N., Gillies,S.D., & Reisfeld, R.A. 1999. Tumor-targeted IL-2 amplifies T cell-mediatedimmune response induced by gene therapy with single-chain IL-12. Proc. Natl.Acad. Sci. USA. 96:8591. 3.Nagarajan, S., & Selvaraj, P. 2002. Glycolipid-anchored IL-12expressed on tumor cell surface induces antitumor immune response. CancerRes. 62, 2869. 4.FTC-133cell line: For a description of the cell line please seehttps: / / www.sigmaaldrich.com / US / en / product / sigma / 94060901?gclid=Cj0KCQjwn_OlBhDhARIsAG2y6zNT-b7BKWDWPBO6l6OjVUjNF0Vus40EnabHKjdser6KPxowvAzmFegaAiSFEALw_wcB&gclsrc=aw.ds (last accessed July 24, 2023) 5.Farma, J.M., Puhlmann, M., Soriano, P.A., Cox, D., Paciotti G.F.,Tamarkin, L., Alexander, H.R. (2007). Direct evidence for rapid and selectiveinduction of tumor neovascular permeability by tumor necrosis factor and anovel derivative, colloidal gold bound tumor necrosis factor. Internationaljournal of cancer. 120(11):2474-80. 6.Koonce, N.A., Quick, C.M., Hardee, M.E., Jamshidi-Parsian, A.,Dent, J.A., Paciotti, G.F., Nedosekin, D., Dings-Ruud,P.M., Griffin, R.J.(2015). Combination of Gold Nanoparticle-Conjugated Tumor Necrosis Factor-aand Radiation Therapy Results in a Synergistic Antitumor Response in MurineCarcinoma Models. Int J Radiation OncolBiol Phys. 93(3):588. 7.Tamarkin., L., Yuan, Z., Maggi, EC, Adem, A., Schorzman, AN,Zamboni, WC, Oarr, D. and Libutti, SK 2017. Cancer Nanomedicines:Opportunities and Challenges. TechConnect Briefs 2017: 126Libutti, SK,Paciotti, GF, Byrnes, AA, Alexander, HR, 1008 Gannon, WE, Walker, M., Seidel, GD, Yuldasheva, N.; a novel PEGylated colloidal gold-rhTNF nanomedicine. Clin. 1011 Cancer Res. 16 , 6139−6 8.Pfreundschuh et al., Phase I Study of Intratumoral Application ofRecombinant Human Tumor Necrosis Factor. European Journal of Cancer ClinicalOncology, Vol 25, No 2, pp. 379–388, 9.Ichikawa, Y., Ghanefar, M., Bayeva, M., Wu, R., Khechaduri, R., Prasad, SVN, Mutharasan, RK, Naik, TJ, and Ardehali1, H. 2014.Cardiotoxicity of doxorubicin is mediated through mitochondrial iron accumulation. 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Claims

1. A construct comprising gold nanoparticles that bind to two types of cytokines, wherein, The two types of cytokines include tumor necrosis factor-α (TNFα) and cytokines selected from the group consisting of interferon-γ (IFNγ) and interleukin-12 (IL-12).

2. The construct according to claim 1, wherein, The two cytokines mentioned include TNFα and IFNγ.

3. The construct according to claim 2, wherein, The ratio of TNFα to IFNγ was approximately 20:1 (w / w).

4. The construct according to claim 1, wherein, The two cytokines mentioned include TNFα and IL-12.

5. The construct according to any one of claims 1 to 4, wherein, The cytokines bind to the surface of the nanoparticles using one or more chemical binding mechanisms, including thiols or other covalent binding, ionic binding, or hydrophobic interactions.

6. The construct according to any one of claims 1 to 5 further comprises polyethylene glycol, a polyethylene glycol derivative, or polyethylene glycol thiol.

7. The construct according to claim 2 or 3 further comprises paclitaxel, a paclitaxel analogue, or a paclitaxel prodrug.

8. The construct according to claim 7, wherein the cytokines are primarily bound to the nanoparticles via thiol chemical binding.

9. A method for enhancing the cytotoxicity of cytokines, comprising the following steps: Gold nanoparticles were combined with two types of cytokines to construct an construct, wherein the cytokines consisted of tumor necrosis factor α (TNFα) and interferon γ (IFNγ); the construct was introduced into a biological sample containing cells; and the cytotoxicity of the construct to the cells was evaluated, wherein the cytotoxicity was improved compared to the introduction of (a) a natural cytokine and / or (b) a single cytokine combined with gold nanoparticles.

10. The method according to claim 9, wherein, The cells in question are cancer cells.

11. The method according to claim 10, wherein, The cancer cells in question are thyroid cancer cells.

12. The method according to claim 9, wherein, The cytotoxicity is measured using a cell viability assay.

13. A method for increasing cytokine potency, comprising the following steps: Gold nanoparticles were combined with two types of cytokines to construct a construct, wherein the cytokines consisted of tumor necrosis factor-α (TNFα) and interleukin-12 (IL-12); the construct was introduced into a biological sample containing cells; and the potency of the construct for the cells was evaluated, wherein the potency was improved compared to the introduction of natural cytokines.

14. The method according to claim 13, wherein, Titer was measured using the HEK bioassay to assess receptor activation.

15. A method for inducing MHC-1 expression in cancer cells, comprising introducing a cytokine construct into cancer cells, wherein the cytokine construct comprises tumor necrosis factor α (TNFα) and interferon γ (IFNγ) bound to gold nanoparticles.

16. The method of claim 15, wherein the cancer cells are lung cancer cells or thyroid cancer cells.

17. A method for activating lymphocytes in a biological sample containing cancer cells, comprising: A cytokine construct is introduced into the biological sample and then added to the lymphocytes. The cytokine construct consists of (TNFα) and interferon γ (IFNγ) bound to gold nanoparticles. The cytokine construct has cytotoxic effects on the cancer cells and activates the lymphocytes.

18. A method for administering cancer treatment to a subject in need, comprising administering a cytokine construct to the subject. in, The cytokine construct includes (TNFα) and interferon γ (IFNγ) bound to gold nanoparticles.

19. The method according to claim 18, wherein, The construct also includes polyethylene glycol, polyethylene glycol derivatives, or polyethylene glycol thiols.

20. The method according to claim 18 or 19, wherein, The ratio of TNFα to IFNγ was approximately 20:1 (w / w).

21. The method according to any one of claims 18 to 20, wherein the cancer is melanoma.

22. The method according to any one of claims 18 to 20, wherein the cancer is a solid tumor.

23. The method according to any one of claims 18 to 22, wherein the cytokine construct further comprises paclitaxel, or a paclitaxel analog or prodrug.

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