Cytokine composition and method of use thereof

Abstract

A novel construct is provided comprising gold nanoparticles bound to two cytokines, wherein the two cytokines include tumor necrosis factor alpha (TNFα) and a cytokine selected from the group consisting of interferon-gamma (IFNγ) and interleukin-12. Furthermore, a method for treating cancer in a target area is provided.

Description

Technical Field

[0001] Embodiments of the present disclosure generally relate to novel nanoparticle and cytokine compositions, as well as constructs, and methods for making and using them.

Background Art

[0002] If a therapeutic agent can be concentratedly delivered to a target site in the body without reducing the titer or effectiveness of the therapeutic agent, improvement of the treatment regimen becomes possible. For example, current cancer treatments include administration of chemotherapeutic agents that act on the whole body and other biologically active factors such as cytokines and immune factors. Side effects due to non-specific delivery include organ damage, loss of sensations such as taste and touch, and hair loss. While these existing treatments provide treatment for specific conditions, they require adjuvant treatment to treat the resulting side effects.

[0003] For formulations containing a therapeutic payload that potentially has toxic side effects when administered systemically, technological advances that improve site-specific delivery and stability of the formulation while reducing the indiscriminate release of the payload would be effective and would thereby improve the overall therapeutic effect of the drug.

[0004] A further drawback of currently available treatments relates to the ability to maintain or increase the titer of the therapeutic agent being administered. Delivering such an agent to the site of a disease such as a tumor is a priority, but it is also important to maintain the activity of such an agent so that it can have its maximum effect.

[0005] What is needed are compositions and methods for delivery systems that deliver drugs acting on desired cells or sites while preserving or improving the therapeutic effect of such drugs. Such systems can be used to deliver all types of drugs to specific cells. There is also a need for delivery systems that facilitate targeted delivery of therapeutic payloads and do not cause undesirable side effects throughout the body. [Overview of the Initiative]

[0006] In one embodiment, the disclosure relates to compositions and methods relating to constructs comprising gold colloid particles and cytokines, optionally combined with one or more therapeutic agents, one or more polyethylene glycol molecules. In one embodiment, the gold colloid particles comprise nanoparticles, and in another embodiment, the nanoparticles consist of gold colloid nanoparticles. In another embodiment, the gold nanoparticles are bound to any two cytokines, including 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 bonded to the gold colloid nanoparticles.

[0007] This disclosure is best understood in conjunction with the accompanying drawings. It is emphasized, as is customary, that various features in the drawings are not necessarily to a constant scale. Rather, the dimensions of various features have been arbitrarily enlarged or reduced for clarity. Throughout the specification and drawings, similar reference numbers indicate similar features. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1 shows a schematic diagram of CYT-IFNγ-TNFα nanoparticles. [Figure 2]Figures 2A and 2B show a schematic diagram of the cross-antibody ELISA (Figure 2A) and the results of the cross-antibody ELISA using this method (Figure 2B). For this assay, TNFα-only nanoparticles were used as a control. Both the experimental CYT-IFNγ-TNFα nanoparticles and the control were captured by the TNFα mAb, but the control did not produce a signal in the ELISA because it lacked IFNγ. Preparations containing IFNγ produced a significant signal, the magnitude of which depended on the amount of IFNγ added during binding. [Figure 3] Figures 3A-3C show micrographs illustrating the binding and internalization of CYT-IFNγ-TNFα by the follicular thyroid cancer cell line FTC-133(4). CYT-IFNγ-TNFα was added to FTC-133 cells, and its binding and internalization were observed by bright-field microscopy. Figure 3A shows untreated cells. Figures 3B and 3C show the localization of nanoparticles 45 minutes (Figure 3B) and 8 hours (Figure 3C) after nanoparticle addition. [Figure 4-1] Figures 4A–4D show micrographs illustrating cytokine-based nanoparticle-induced cytotoxicity in FTC-133 cells. FTC-133 cells were plated into 6-well tissue culture clusters in complete DMEM. 24–48 hours after plating (to allow cell adhesion), various nanoparticles were added to the cells at 1 μg / mL. Nanoparticle treatments are shown as follows: no treatment (Figure 4A); CYT-6091 (TNFα monotherapy; Figure 4B); CYT-INFγ (IFNγ monotherapy; Figure 4C); and CYT-IFNγ-TNFα (Figure 4D). Cells were cultured for a further 5–7 days, and cytotoxicity was evaluated microscopically. [Figure 4-2] Same as above. [Figure 5-1]Figure 5 shows a graph demonstrating that CYT-IFNγ-TNFα is more cytotoxic to FTC-133 cells than the same doses of the native cytokines IFNγ and TNFα in solution. The data presented are from two separate experiments using quadruple wells per concentration. The data clearly show that native IFNγ-TNFα was slightly more cytotoxic to FTC-133 cells compared to the same dose of cytokine added as CYT-IFNγ-TNFα. A similar pattern was observed with single-agent nanoparticles: CYT-IFNγ-TNFα showed higher titers than CYT-6091 (single-agent TNFα nanoparticles) or CYT-IFN (single-agent IFNγ nanoparticles). [Figure 5-2] Same as above. [Figure 6] Figure 6 provides a graph showing that CYT-IFNγ-TNFα exhibits a similar increase in titer compared to native IFNγ in the genetically engineered cell line HEK-IFNγ. HEK cells were stably transfected with the IFNγ receptor and signaling complex. Titer is based on IFNγ concentration (Figure 6). [Figure 7] Figure 7 provides a graph showing that a slight increase in cytokine stability is not the underlying mechanism for the increased titer observed in the CYT-IFNγ-TNFα preparation (see Figures 4 and 5 for details). [Figure 8] Figure 8 provides micrographs showing that CYT-IFNγ-TNFα induces receptor cluster formation. Control (untreated) or CYT-IFNγ-TNFα-treated FTC-133 cells were imaged 3 hours after the addition of CYT-IFNγ-TNFα. The images show different stages of nanoparticle uptake by FTC-133 cells. [Figure 9-1]Figures 9A–9S provide micrographs demonstrating the induction of HLA-ACs in human cancer cell lines treated with CYT-IFNγ-TNFα. 20,000 FTC-133, H-460, or A549 cells were plated into 6-well tissue culture clusters. 48 hours after plating, cells were administered varying doses of CYT-IFNγ-TNFα (see the Examples section below, particularly Example 3, for experimental details). Figure 9 provides three photographs of the control wells and six photographs of the CYT-IFNγ-TNFα-treated wells. Figures 9A–9C demonstrate the induction of HLA-ACs in FTC-133 cells by 1 μg of CYT-IFNγ-TNFα. Figures 9D–9G show the uptake and internalization of CYT-IFNγ-TNFα by the human lung cancer cell line H-460. The uptake pattern is similar to that of the FTC-133 cells shown in Figure 3. Figures 9H–9K show that, similar to FTC-133 cells, CYT-IFNγ-TNFα induced HLA-AC expression across the entire three-dimensional cluster typically formed by H460 cells. Figures 9L–9O and 9P–9S show the uptake and intracellular processing of CYT-IFNγ-TNFα by A549 lung cancer cells. Consistent with the observations discussed herein, CYT-IFNγ-TNFα induced HLA-AC expression by A549 cells. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above. [Figure 9-5] Same as above. [Figure 9-6] Same as above. [Figure 9-7] Same as above. [Figure 10-1]Figures 10A–10D provide micrographs showing splenocyte activation induced by treatment of mouse cancer cells with CYT-IFNγ-TNFα. Figures 10A–10B show proliferation of naive splenocytes isolated from naive Balb / c mice induced by Colo26 cancer cells treated with CYT-mIFNγ-TNFα. Note that in co-culture with control (untreated) cells, splenocytes were present on the surface of cancer cells but did not proliferate. Figures 10C–10D show early and late stage proliferation of naive splenocytes isolated from naive C57Bl / 6 mice induced by B16F10 melanoma cancer cells treated with CYT-m-IFNγ-hTNFα. [Figure 10-2] Same as above. [Figure 11-1] Figures 11A–11D provide plots showing that CYT-IFNγ-TNFα increases the blood residence time for both IFNγ and TNFα after intraperitoneal injection (Figures 11A–11B) and intravenous injection (Figures 11C–11D). [Figure 11-2] Same as above. [Figure 12] Figure 12 provides a graph showing the stability of CYT-IFNγ-TNFα in circulation. Blood samples collected at the beginning of the pharmacokinetic study were analyzed using the cross-antibody ELISA described in Example 1. In this assay, the sample was captured using a neutralizing monoclonal antibody against TNFα and detected with a rabbit anti-human IFNγ / alkaline phosphatase-conjugated goat anti-rabbit polyclonal antibody. [Figure 13] Figures 13A and 13B provide images of experimental animals showing visual confirmation of CYT-mIFNγ-hTNFα uptake by Colo-26 (solid) tumors. The presence of gold nanoparticles was recorded by digital photography of mice 4 hours after injection. Colo-26 tumors exhibited a reddish color due to the gold nanoparticles (compare Figures 13A and 13B). Figure 13A shows a control animal (no injection) at T=3.5 hours post-injection. Figure 13B provides an image taken 3.5 hours after intravenous injection of CYT-mIFNγ-hTNFα. [Figure 14]Figure 14 provides a graph showing the accumulation of CYT-IFNγ-TNFα in B16F10 tumors. [Figure 15] Figure 15 shows the saturation binding kinetics of IL-12 to gold colloid nanoparticles. [Figure 16] Figure 16 provides a graph showing the increase in the titer of CYT-IL-12-TNFα relative to the same concentration of native IL-12+TNFα in solution. The assay was performed using HEK-IL-12 cells that secrete an alkaline phosphatase reporter protein, similar to HEK-IFNg cells. [Figure 17] Figure 17 presents confirmation data regarding the presence of human IL-2 and TNFα on the same particles of CYT-IL-2-TNFα. [Figure 18] Figure 18 provides a schematic diagram of the multimodal immunooncology nanoparticle. The novel nanoparticle consists of human IFNγ, human TNFα, and a thiolated paclitaxel prodrug conjugated to the same gold nanoparticles. [Figure 19-1] Figure 19 provides micrographs comparing the uptake of CYT-IFNγ-TNFα versus CYT-IFNγ-TNFα-paclitaxel by FTC-133 cells. Paclitaxel-containing nanoparticles induced a significant change in nuclear morphology, which caused the formation of multilobed nuclei. [Figure 19-2] The same as above. [Figure 20] Figure 20 provides micrographs showing the mechanism by which CYT-IFNγ-TNFα-paclitaxel can generate tumor antigens. When lysed, dead cells can release particles containing putative tumor antigens. By doing so, the new construct can generate tumor antigens to initiate an anti-tumor immune response. [Figure 21]Figure 21 provides composite fluorescence / bright-field images of FTC-133 cells treated with the CYT-IFNγ-TNFα-paclitaxel construct. Intracellular transport of particles was again readily evident by the red color of the particles (see §). Furthermore, when the nanoparticles reached the perinuclear region, a fluorescent marker became apparent, supporting the release of the prodrug (or small molecule payload). These data are consistent with the release of the prodrug substitute from the particles (Δ). The images also show post-internalization release of the alternative drug active ingredient (API) at different stages. For example, API release is well underway in cells highlighted by triangles (Δ). However, release at the initial stage is also shown in cells indicated by asterisks (*). [Modes for carrying out the invention]

[0009] The following detailed descriptions are illustrative and explanatory and are intended to provide further explanation of the disclosures set forth herein. Other advantages and novel features will be readily apparent to those skilled in the art from the following detailed descriptions of the disclosures herein. The texts and references referred to herein, including U.S. Patents No. 7,387,900, 7,790,167, 7,951,614, 7,960,145, RE 42524, 8,435,801, 8,486,666, and 8,785,202, are incorporated herein by reference in their entirety.

[0010] Where used herein, the term “subject” should be interpreted to include, for example, medical or surgical subjects such as humans and other animals requiring therapeutic intervention.

[0011] In this disclosure, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include the plural forms, and references to specific numerical values ​​include at least that specific value. Thus, for example, a reference to “a bead” or “a nanostructure” is a reference to one or more such structures and their equivalents known to those skilled in the art. When a value is expressed as an approximation by the use of the antecedent “about,” it is understood that a specific value constitutes another embodiment. Where used herein, “about X” (where X is a numerical value) preferably refers to ±10% (inclusive) of the stated value. For example, the phrase “about 8” preferably refers to a value between 7.2 and 8.8 (inclusive), and as another example, the phrase “about 8%” preferably (but not always) refers to a value between 7.2% and 8.8% (inclusive). Where present, all ranges include both ends and are combinable. For example, if a range of "1-5" is described, the described range should be interpreted to include ranges such as "1-4", "1-3", "1-2", "1-2 and 4-5", "1-3 and 5", and "2-5". In addition, if a list of options is explicitly presented, such a list may be interpreted to mean that any of the options may be excluded, for example, by a negative limitation in the claims. For example, if a range of "1-5" is described, the described range may be interpreted to include situations in which any of 1, 2, 3, 4, or 5 is negatively excluded, and for example, the description "1-5" may be interpreted as "1 and 3-5, but not 2", or simply "2 is not included". Any component, element, attribute, or step explicitly described herein is intended to be expressly excluded in the claims, whether such component, element, attribute, or step is listed as an option or listed individually.

[0012] As used herein, the term “cytokine” refers to a broad class of small molecules, such as interferons, interleukins, and growth factors, that are secreted by certain cells of the immune system and affect other cells. Cytokines in this disclosure include interferon-gamma (IFNγ), TNFα, interleukin-2 (IL-2), and interleukin-12 (IL-12).

[0013] As used herein, the term “cytotoxicity” refers to the degree to which a substance can damage cells. A substance or process that causes cell damage or cell death is called cytotoxic. By treating cells with cytotoxic compounds, a variety of cell fates can be brought about. Cells may undergo apoptosis or necrosis, which leads to rapid death as a result of cell lysis, as they lose membrane integrity. Cells may also cease active growth and division (resulting in reduced cell viability), or cells may activate pathway-controlled cell death (i.e., apoptosis).

[0014] As used herein, the term “paclitaxel” refers to a type of chemotherapeutic agent used to treat cancer. Paclitaxel is a taxane chemotherapeutic agent. A “paclitaxel prodrug” includes a compound that has little or no pharmacological activity and is converted to a pharmacologically active paclitaxel drug compound in the region of interest in vivo. Such prodrugs may include thiol-derivative paclitaxel prodrugs.

[0015] For the purposes of the following description, it should be understood that the embodiments described below may be subject to alternative variations and embodiments. It should also be understood that the specific articles, compositions, and / or processes described herein are illustrative and should not be considered limiting.

[0016] Tumor necrosis factor alpha (TNFα)(1) is a multifaceted cytokine that affects almost every aspect of human health. Its discovery in the 1970s was of great significance for treating solid tumors because a single injection of this protein caused hemorrhagic necrosis of solid tumors, regardless of whether cancer cells were sensitive to the protein or not. Continued research has shown that TNFα selectively destroys the tumor vascular system(5), reduces tumor interstitial fluid pressure(6), increases the uptake of subsequent chemotherapy agents(7), and mobilizes immune system cells to the tumor site. Taken together, these effects can produce a significant antitumor response.

[0017] Shortly after its discovery, recombinant forms of the cytokine were produced to support clinical trials in cancer patients. Unfortunately, in nearly 200 clinical trials, systemically delivered TNFα was shown to be highly toxic, thus limiting the dose that could be safely administered to cancer patients. The main dose-limiting toxicities of TNFα are hypotension and hepatotoxicity (6). In all of these clinical trials, no sustained antitumor response was observed.

[0018] Furthermore, in early phase 1 clinical trials of TNFα, it was found that many of the potentially dangerous side effects caused by TNFα occur even at low doses. Some of these, including severe fever and chills, can be pharmacokinetically controlled and therefore do not pose a significant barrier to patient treatment or treatment compliance. Other adverse effects, such as tachycardia, can be eligibility-disadvantaging AEs (8). This condition can lead to stroke and heart attack in patients at risk. Notably, many cancer treatments, such as doxorubicin (9), which can be administered in combination with CYT-6091 (see below), are known to be cardiotoxic.

[0019] Based on these data, the use of TNFα in cancer treatment is limited to limb-sparing procedures known as isolated limb perfusion (ILP) (10). In this procedure, patients with melanoma or sarcoma in the limbs have the blood vessels of the affected limb connected to a cardiopulmonary device, the limb is perfused with TNFα, and then chemotherapeutic agents are perfused through the limb. ILP achieves two main objectives: firstly, local delivery of TNF increases the concentration of cytokines at the disease site; secondly, local perfusion of cytokines within the limb reduces systemic exposure to cytokines and thus avoids most toxic side effects. However, due to the remarkable antitumor response (60-75% complete and persistent (10 years)), the inventors have developed the first patented gold nanoparticle CYT-6091.

[0020] CYT-6091(11~12) consists of 27 nm gold nanoparticles covalently bonded to TNFα and PEG-THIOL via the formation of coordination covalent bonds. Novel cytokine constructs comprising gold nanoparticles and TNFα are disclosed herein. In one embodiment, a construct comprising gold nanoparticles bound to two cytokines is provided, the two cytokines comprising tumor necrosis factor α (TNFα) and a cytokine selected from the group consisting of interferon gamma (IFNγ) and interleukin-12 (IL-12) or interleukin-2 (IL-2). One embodiment consists of gold nanoparticles bound to TNFα and IFNγ, in one embodiment the ratio of TNFα to IFNγ is about 20:1 (w / w). Another embodiment consists of gold nanoparticles bound to TNFα and interleukin-2 (IL-2) or TNFα and interleukin-12 (IL-12).

[0021] In one embodiment, the cytokine construct of the present disclosure comprises cytokines that can be bound to the surface of gold nanoparticles using one or more binding chemistrys, including thiols or other covalent, ionic, or hydrophobic interactions.

[0022] In some embodiments, the cytokine construct may further comprise polyethylene glycol, a polyethylene glycol derivative, or a polyethylene glycol-thiol. The cytokine constructs of this disclosure, comprising tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ), may further comprise paclitaxel, paclitaxel analogues, or paclitaxel prodrugs.

[0023] In one embodiment, this specification provides a method for increasing cytokine cytotoxicity, comprising the steps of: preparing a construct by combining gold nanoparticles with two cytokines, wherein the cytokines consist of tumor necrosis factor alpha (TNFα) and IFN-gamma (IFNγ); introducing the construct into a biological sample containing cells; and evaluating the cytotoxicity of the construct to the cells, wherein the cytotoxicity is increased compared to the introduction of (a) natural cytokines and / or (b) individual cytokines bound to gold nanoparticles. Such a method can be performed on biological samples containing cancer cells, more specifically thyroid cancer cells. Cytotoxicity can be measured using a cell viability assay.

[0024] In one embodiment, this specification provides a method for increasing cytokine titer, comprising the steps of: preparing a construct by combining gold nanoparticles with two cytokines, wherein the cytokines are tumor necrosis factor α (TNFα) and interleukin-12 (IL-12); introducing the construct into a biological sample containing cells; and evaluating the titer of the construct to the cells, wherein the titer is increased compared to the introduction of innate cytokines. The titer may be measured using an HEK bioassay for evaluating receptor activation. The HEK bioassay used herein is an HEK cell-based functional assay for examining receptor activity via fluorescence output.

[0025] In one embodiment, this specification provides a method for inducing MHC-1(13) expression in cancer cells, comprising introducing a cytokine construct into the cancer cells, the cytokine construct comprising tumor necrosis factor alpha (TNFα) and IFN-gamma (IFNγ) bound to gold nanoparticles. The cancer cells may include lung cancer cells or thyroid cancer cells.

[0026] In one embodiment, this specification provides a method for activating naive lymphocytes in a biological sample containing cancer cells, comprising introducing a cytokine construct into the biological sample to induce MHC-1 (HLA-AC) expression, followed by the addition of lymphocytes, wherein the cytokine construct consists of tumor necrosis factor alpha (TNFα) and IFN gamma (IFNγ) bound to gold nanoparticles, and the cytokine construct has a cytotoxic effect on cancer cells and an activating effect on lymphocytes.

[0027] In one embodiment, the Specified provides a method for treating cancer in a subject requiring cancer treatment, comprising administering a cytokine construct to the subject, wherein the cytokine construct comprises tumor necrosis factor alpha (TNFα) and IFN gamma (IFNγ) conjugated to gold nanoparticles. In such embodiments, the construct may further comprise polyethylene glycol, a polyethylene glycol derivative, or a polyethylene glycol-thiol, with a TNFα to IFNγ ratio of approximately 20:1 (w / w). In such embodiments, the construct may further comprise paclitaxel, or an analogue or prodrug of paclitaxel.

[0028] This specification provides compositions and methods relating to constructs comprising gold colloid nanoparticles and cytokines, which may optionally be combined with one or more therapeutic agents and optionally with polyethylene glycol molecules. The types of cytokines consist of tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ) or interleukin-12 (IL-12) or interleukin-2 (IL-2). The polyethylene glycol molecules may include polyethylene glycol derivatives covalently bonded to the gold colloid nanoparticles.

[0029] One embodiment of the present invention provides a method for treating diseases and disorders, comprising the administration of a cytokine construct. In such an embodiment, a method for treating a solid tumor comprises administering a composition to a living organism having a solid tumor, the composition comprising a cytokine construct consisting of gold colloid particles and two cytokines, and optionally combined with one or more therapeutic agents and one or more polyethylene glycol molecules, wherein the gold colloid particles consist of gold nanoparticles, and the two cytokines bound to these gold nanoparticles consist of TNFα and IFNγ.

[0030] In one embodiment, the cancer is melanoma. In another embodiment, the cancer is a solid tumor. In another embodiment, a method for treating a solid tumor comprises administering a novel cytokine construct composition to a body having a solid tumor, the novel cytokine construct comprising gold nanoparticles bound to two cytokines, the two cytokines comprising tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ).

[0031] The following examples are given to illustrate exemplary embodiments of the present disclosure. However, it should be understood that the present disclosure is not limited to the specific conditions or details described in these examples. Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary ways of constructing and carrying out the present invention.

[0032] Examples background Production of CYT-IFNγ-TNF Two methods were used to produce CYT-IFNγ-TNFα. The first method involved the simultaneous binding of TNFα, IFNγ, and PEG-THIOL to the surface of gold nanoparticles. The second method involved sequential binding, in which IFNγ and PEG-THIOL were simultaneously bound after TNFα.

[0033] For these studies, the pH of the gold colloid solution was adjusted to approximately 8.0 by stepwise addition of a 50 mM sodium borate (NaBo) solution. Similarly, the binding buffer (BB), which was used to dilute TNFα (CytImmune Sciences, Inc.), IFNγ (R&D Systems), and PEG-THIOL in 20 kDa form (SunBio, Inc.), was also adjusted to 8.0 with NaBo. TNFα, IFNγ, and PEG-THIOL were diluted in BB to final concentrations of 0.25, 5.0, and 15 μg / mL, respectively. Equivolutes of both gold nanoparticles and BB (containing various reagents) were mixed by rapidly adding the BB solution to the gold nanoparticle solution under strong vortexing. The solutions were incubated for a minimum of 3 hours.

[0034] Next, the particle-bound reagent and the free reagent were separated by centrifugation, but those skilled in the art can readily use ultrafiltration with suitable equipment such as a UF cartridge or hollow fiber assembly. The concentrated nanoparticles were washed twice with an isotonic solution, and during the final concentration step, the gold nanoparticles were dispensed and frozen at -80°C. Alternatively, lyophilization cycles may be used for long-term storage of nanopharmaceuticals at -20°C to +4°C.

[0035] Measurement of bound and free TNFα and IFNγ Cytokine-specific sandwich ELISAs were used to measure both particle-bound and free fractions of TNFα and IFNγ. Both ELISAs used commercially available cytokine-specific neutralizing monoclonal antibodies (R&D Systems) to capture either TNFα or IFNγ bound to particles or free TNFα or IFNγ in solution. Once captured, cytokine-specific rabbit polyclonal antibodies (CytImmune Sciences, Inc.) were added to the wells, and the complexes were detected with alkaline phosphatase-conjugated goat anti-rabbit antibody (Sigma). The concentration of each cytokine was determined by regression analysis against known standards. Depending on the scale, TNFα and IFNγ concentrations typically ranged from 2 to 40 μg / mL, respectively, with 90–95% of the measured cytokines bound to gold nanoparticles.

[0036] Example 1 Cross-antibody ELISA: Qualitative demonstration that both IFNγ and TNFα are present on the same gold nanoparticles. The ELISA described in Example 1 is useful for quantifying the relative amounts of both IFNγ and TNFα on the particle surface, but it does not demonstrate the presence of both cytokines on the same particle.

[0037] To address this need, we developed a cross-antibody (XAb) ELISA 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 (Figure 2A).

[0038] In one version of the XAb ELISA, the inventors used a mouse monoclonal antibody against TNFα to capture single-agent controls bound to either CYT-IFNγ-TNFα or TNFα alone. After the nanoparticles were captured by the mAb (after incubation at room temperature for 4–24 hours), the plates were washed and a rabbit polyclonal antibody against human IFNγ was used as the detection system. As shown in Figure 2B, the single-agent TNFα nanoparticles produced little to no signal in the XAb ELISA. CYT-IFNγ-TNFα not only produced a significant signal in the XAb ELISA, but the amount of color produced depended on the amount of IFNγ initially bound to the nanoparticles during preparation.

[0039] Binding, uptake, and internalization of CYT-IFNγ-TNFα by the follicular thyroid cancer cell line FTC-133 For these studies, 5,000–10,000 FTC-133 cells were plated into 6-well tissue culture clusters in 2 mL of complete DMEM. The cells were maintained under standard tissue culture conditions (37°C, 95% relative humidity). After 24–48 hours, CYT-IFNγ-TNFα (0.05–2.0 μg of IFNγ) was added to the culture medium, and the uptake of nanoparticles by the cells was imaged under bright-field or phase-contrast microscopy at various time points after nanoparticle addition.

[0040] As shown in Figure 3, uptake of CYT-IFNγ-TNFα by FTC-133 cells was observed when the cells exhibited a reddish color due to the gold colloid nanoparticles. Within 15–45 minutes of nanoparticle addition, the particles were uniformly distributed on the cell surface. Over the next 90 minutes, the staining pattern localized to different regions of the cells (see the example below discussing receptor cluster formation). Finally, after 8–12 hours, the particles were visualized as black aggregates in the perinuclear region of the cells.

[0041] Evaluation of cytotoxicity Considering the uptake of nanoparticles and the fact that both TNFα and IFNγ are known to induce cytotoxicity in certain cancer cell lines, the experiment was repeated, but cell proliferation was determined after a further incubation period of 5–7 days. In addition, additional nanoparticles such as CYT-6091 (see below) and mono-interferon-gamma nanoparticles were also included.

[0042] Figure 4 shows micrographs of the various cultures described above. The data in Figure 4 demonstrate that both CYT-6091 and CYT-IFNγ were cytotoxic to FTC-133 cells because the wells were not confluent compared to the untreated control (Figure 4A). However, CYT-IFNγ-TNFα showed the highest degree of cytotoxicity, as no viable cells were observed in the culture.

[0043] Example 2 CYT-IFNγ-TNFα increases the titers of IFNγ and TNFα. In this study, the inventors compared the titer of CYT-IFNγ-TNFα with that of the same doses of IFNγ and TNFα in solution. Small batches of nanoparticles were produced as described above, and the concentrations of cytokines bound to the particles were determined by quantitative ELISA. Subsequently, IFNγ and TNFα, added as CYT-IFNγ-TNFα, were added to FTC-133 cells growing in 96-well tissue culture clusters, increasing in concentration. In another group of wells, the same doses of native IFNγ and TNFα were added at the same concentrations as CYT-IFNγ-TNFα. Native cytokines were added as single solutions.

[0044] The cells were incubated at 37°C and 95% relative humidity for a further 2-3 days. Subsequently, the incubation medium was removed, and the cells were gently washed three times in serum-free DMEM. After the final wash, 100 μL of complete DMEM was added to the cells, followed by 10 μL of Alamar Blue®. Fluorescence in the untreated / control well was 10 4The plates were incubated at 37°C until the relative fluorescence unit value was reached.

[0045] As shown in Figure 5, CYT-IFNγ-TNFα increased the dose-to-dose cytotoxicity of IFNγ / TNFα in FTC-133 cells. These data are consistent with IFNγ / TNFα interacting more efficiently with their respective receptors, likely by inducing receptor clustering (see below).

[0046] CYT-IFNγ-TNFα increases the titer of IFNγ / TNFα in genetically modified cell lines. HEK-IFNγ (InvivoGen, California, USA) is an 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 amount of alkaline phosphatase produced is directly proportional to the amount of IFNγ present in the sample. The amount of reporter gene released can be assayed by adding a certain volume of tissue culture supernatant to a PNPP (p-nitrophenyl phosphate; Sigma Aldrich, Missouri, USA) substrate.

[0047] To assess whether CYT-IFNγ-TNFα induced a similar increase in titer, as demonstrated in cytotoxicity studies, these cells were used in similar studies, as described in Figures 5A and 6. Briefly, 20,000 HEK-IFNγ cells were plated as outlined by the manufacturer. The following day, IFNγ and TNFα were added as single solutions or as CYT-IFNγ-TNFα, increasing in concentration. The cells were incubated for a further 48 hours. Subsequently, 10 μL of tissue culture supernatant was collected and added to 200 μL of PNPP substrate. The reaction was monitored by measuring OD at 405 nm, and the process was terminated when the optical density for the highest dose reached an OD of 2.0–3.0 OD units.

[0048] The native IFNγ / TNFα combination induced a dose-dependent increase in the relative amount of alkaline phosphatase secreted by HEK-INFg cells. Unlike FTC-133, which showed only slight activity with the native cytokine combination, the HEK-IFNγ data suggest that the cells are being manipulated to secrete the reporter protein in a dose-dependent manner. However, a similar titer increase (EC) was observed in cells treated with CYT-IFNγ-TNFα. 50 A 15-fold decrease was observed, so the data shown in Figure 6 is consistent with that reported in Figure 5.

[0049] Cytokine stability provided by gold nanoparticles The increased titer observed in CYT-IFNγ-TNFα may be partly due to a mechanism by which gold nanoparticles enhance cytokine stability. To test this in a simple matrix, equal concentrations of both native IFNγ and TNF, or CYT-IFNγ-TNFα, were added to FTC-133 cells, which were then cultured at 37°C for 2 days. On day 2, aliquots of the samples from each set were collected, frozen at -80°C, and analyzed by ELISA.

[0050] The data shown in Figure 7 indicates that a slight decrease of approximately 20% in the recovered cytokine concentration was measured in the natural cytokine sample. While significant, this decrease is unlikely to explain the difference in titer between the two preparations. However, it should be noted that the increased cytokine stability obtained by binding cytokines to the particle surface is desirable.

[0051] Evidence of receptor cluster formation While we do not wish to be bound by the following theory, it is hypothesized that the titer increase reported in Figure 5 may be mediated by receptor cluster formation. Given that both IFNγ and TNFα are biologically active on the particle surface (quantitative ELISA utilizes cytokine-specific neutralizing mAbs to capture CYT-IFNγ-TNFα), it was hypothesized that when presented on the particle surface, IFNγ and possibly TNFα would induce avidity events for their respective receptors. Based on these observations, the first evidence supporting receptor cluster formation was obtained using a gold nanoparticle platform.

[0052] To test this hypothesis, the imaging test outlined in Example 1 was repeated using cells imaged between 90 minutes and 8 hours after the addition of CYT-IFNγ-TNFα. The cells were imaged using both bright-field and phase-contrast microscopy to confirm the transport of nanoparticles.

[0053] The data shown in Figure 8 are likely consistent with the receptor clustering and internalization of particles within endosomes. Within the endosomes, the particle-bound components (TNFα, IFNγ, and PEG-THIOL) were either degraded or released from the particle surface, and these so-called "naked particles," lacking passivators, aggregated as they moved toward the nucleus.

[0054] Unlike the nearly transparent image of control FTC-133 cells (upper left panel), many cells show a pink color that highlights the presence of nanoparticles on the cell surface. Similarly, all FTC-133 cells treated with CYT-IFNγ-TNFα showed perinuclear localization of particles, which is evidenced by the black precipitate around the nucleus. Furthermore, in some cells highlighted by arrows in the bright-field (lower left panel) and phase-contrast (lower right panel) images, the pattern of nanoparticle uptake was essentially punctate. Although studies on the mechanism of action have not been shown, these initial data support the idea that CYT-IFNγ-TNFα induced clustering of its target receptors.

[0055] Example 3 MHC-I induction by CYT-IFNγ-TNFα Many tumors evade detection and elimination by the immune system by expressing little to no major histocompatibility complex (13-14). In humans, this complex is called HLA AC, and its mouse equivalent is called MHC-1. IFNγ is known to upregulate MHC-1 expression (12). To test this hypothesis, we evaluated both baseline (untreated) and CYT-IFNγ-TNFα-inducible expression of the histocompatibility complex in both human and mouse cancer cell lines.

[0056] For these studies, we used numerous cell lines, 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 in C57Bl / 6 mice). Approximately 20,000 cells from each cell line were plated into 6-well tissue culture plates and incubated for 48 hours under standard culture conditions. Subsequently, the cells were incubated with either human nanoparticle CYT-IFNγ-TNFα or the mouse equivalent CYT-mIFNγ-hTNFα.

[0057] Unlike human TNFα, human IFNγ is not cross-reactive in mice, so the generation of mouse nanoparticles was necessary. These mutants were produced and investigated using the method outlined in the examples above.

[0058] For these studies, 0.0625–1 μg of CYT-IFNγ-TNFα (human nanoparticles) or CYT-mIFNγ-TNFα (mouse nanoparticles) were incubated with FTC-133, H460, and A549 cells or Colo26 or B16F10 cancer cells, respectively. After 48 hours, the cells were washed twice with incomplete DMEM, and 1 μg of mouse monoclonal antibody (Sigma Aldridge), which recognizes both HLA-AC and mouse MHC-1 complexes, 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 again with incomplete DMEM, and FITC-conjugated goat anti-mouse polyclonal antibody (Sigma Aldridge) was added to the culture. The antibody was incubated for a further 1 hour. The cells were washed once more with incomplete DMEM, and the presence of HLA-AC or MHC-1 complexes was recorded by fluorescence microscopy.

[0059] Figure 9 shows confirmation data that CYT-IFNγ-TNFα induced HLA expression in the FTC-133, HL-460, and A549 human cancer cell lines (see Figures 9A-9C, 9D-9K, and 9L-9O, respectively). Induction of HLA-AC in both H460 and A549 (Figures 9P-S) occurred over a wide dose range. Consistent with the hypothesis presented by Angell et al., (12), control cells showed no or only slight expression of the HLA-AC complex, while cultures treated with nanoparticles showed significant staining for the antigen.

[0060] Furthermore, both human lung cancer cell lines exhibited similar patterns of CYT-IFNγ-TNFα uptake, as evidenced by the pink / black staining of cells highlighting nanoparticle binding and internalization, as well as subsequent HLA complex expression (Figures 9D-9G for H-460 cancer cell line and 9L-9O for A549 cancer cell line).

[0061] Generation of anti-cancer immune response by CYT-mIFNγ-TNFα In the following study, the inventors determined whether MHC-1 induction by CYT-IFNγ-TNFα leads to a novel anti-cancer immune response. For this study, either Colo26 or B16F10 cancer cells were plated and treated with a mouse variant of CYT-IFNγ-TNFα (CYT-mIFNγ-hTNFα) as described above. The control was untreated cancer cells. After confirming MHC-1 induction, approximately 10 cells derived from naive Balb / c cells were used. 6 A number of splenocytes were added to the Colo26 culture. A similar number of splenocytes, isolated from C57Bl / 6 mice, were added to either a control or a nanoparticle-treated culture, and the induction of an anti-cancer immune response by splenocyte proliferation was confirmed.

[0062] The data presented in Figure 10 support the finding that CYT-mIFNγ-hTNFα induced proliferation of splenocytes isolated from naive Balb / c mice. Several observations were made in these cultures. Consistent with the data shown in Figures 4-5, CYT-mIFNγ-TNFα induced significant inhibition of Colo26 cell proliferation compared to the untreated control (Figure 10B) (Figure 10A). Furthermore, the nanoparticles activated and directly proliferated naive splenocytes on the surface of the remaining cancer cells. These data are consistent with the nanoparticle-induced proliferation of naive B cells reported by Paciottii et al. (15). Finally, consistent with poor MHC expression, splenocytes added to the untreated control did not proliferate and remained quiescent, as shown in Figure 10B, despite being directly present on the surface of proliferating cancer cells.

[0063] Similar data were obtained in CYT-mIFNγ-hTNFα-treated B16F10 cancer cell cultures. As shown in Figure 10C, lymphocyte proliferation was evident within 24 hours of adding naive splenocytes isolated from naive C57Bl / 6 to CYT-mIFNγ-hTNFα-treated B16F10 melanoma cells. These data further support the idea that nanoparticles induce antigen presentation by cancer cells, ultimately leading to lymphocyte proliferation, in these studies.

[0064] Example 4 Pharmacology of CYT-IFNγ-TNFα CYT-6091 (11-12) and CYT-21625 (16) are tumor-targeted nanoparticles that target the delivery of TNFα as a monotherapy (CYT-6091) or as a TNFα + paclitaxel prodrug (CYT-21625) to solid tumors. Both nanoparticles are designed on 27 nm particles of PEGylated gold colloid. Pharmacokinetic and tumor uptake studies have shown that CYT-6091 and CYT-21625 exhibit a higher terminal phase half-life (T) due to PEGylation compared to their natural preparations. 1 / 2 ) and the pharmacokinetic exposure, as measured by area under the curve (AUC), were revealed to increase (11 and 16).

[0065] The following experiments evaluated the ability of the particle-bound PEG-THIOL moiety to similarly increase IFNγ pharmacokinetic exposure and tumor uptake. Therefore, after nanoparticle formulation and analytical investigation, both TNFα and IFNγ in single solutions, or the same doses of cytokines formulated as CYT-IFNγ-TNFα, were injected into naive Balb / c mice or Colo-26 tumor-loaded Balb / c mice. Given that IFNγ in its currently approved form is administered subcutaneously, the PK profile was determined by injecting the drug either intravenously or intraperitoneally.

[0066] At various time points after injection, animals were sacrificed, whole blood was collected, and 18 mg / mL of EDTA solution was added (10% v / v). The samples were measured for the content of both TNFα and IFNγ by cytokine-specific ELISA. To demonstrate that CYT-IFNγ-TNFα remains stable in circulation, blood samples were collected after injection and analyzed using the XAb assay described in Example 1.

[0067] The data shown in Figure 11 demonstrate that CYT-IFNγ-TNFα increases pharmacokinetic exposure to both cytokines compared to innate cytokine treatment. These data are consistent with the pharmacokinetic profiles reported for both CYT-6091 and CYT-21625.

[0068] CYT-IFNγ-TNFα does not cause burst release when injected into the circulation. A common obstacle in nanoparticle development is burst release, where nanoparticle components are released almost immediately upon injection. For both TNFα and IFNγ, this can lead to toxicity such as hypotension, and in the case of TNFα, hepatotoxicity. Therefore, to test the stability of CYT-IFNγ-TNFα in circulation, samples collected early in pharmacokinetic studies were analyzed using the cross-antibody (XAb) ELISA described in Example 1. In short, if either the TNFα or IFNγ component undergoes burst release in circulation, the particles will not be captured or detected, and therefore, it is expected that the blood sample will not produce a signal in the XAb ELISA.

[0069] The data presented in Figure 12 support the initial stability of CYT-IFNγ-TNFα, as samples collected early in the pharmacokinetic study generated a significant signal in the XAb ELISA. Samples taken from animals administered the natural formulation did not generate a significant signal in the ELISA.

[0070] Example 5 Accumulation of CYT-IFNγ-TNFα in solid tumors The following studies tracked the accumulation of human or mouse morphology CYT-IFNγ-TNFα in two mouse tumor models. In the first study, the presence of nanoparticles was tracked by imaging control or nanoparticle-treated animals. In Figure 13, CYT-mIFNγ-hTNFα was intravenously injected into Colo-26 tumor-loaded Balb / c mice. Three to four hours after injection, the Colo-26 tumors exhibited a reddish color due to the presence of gold nanoparticles, thus easily demonstrating the presence of nanoparticles (see Figures 13A and 13B).

[0071] Figure 13A shows images taken from control (e.g., untreated) Colo-26 tumor-loaded Balb / c mice. Figure 13B shows images of Colo-26 tumor-loaded Balb / c mice taken 3.5 hours after intravenous injection of CYT-mIFNγ-TNFα. Accumulation of the CYT-mIFNγ-TNFα nanopharmaceutical in the tumors was readily apparent, as the tumors exhibited a reddish color due to the presence of gold nanoparticles.

[0072] Accumulation of CYT-IFNγ-TNFα in B16F10 tumors For this study, B16F10 tumors were established on the abdominal surface of C57Bl / 6, as described in Paciotti et al. (14). Once the tumors were established, mice (n=5 / group) were intraperitoneally injected with CYT-IFNγ-TNFα, or the same cytokine dose administered in solution. The animals were sacrificed after 4 hours, the tumors were collected, and frozen at -80°C. The samples were then homogenized using a glass homogenizer. The homogenates were analyzed for intratumoral IFNγ content by ELISA.

[0073] Consistent with data reported for CYT-6091(10) and CYT-21625(14), the data shown in Figure 14 support the accumulation of IFNγ in solid tumors by the nanoparticles.

[0074] Example 6 Saturated binding of interleukin-2 or interleukin-12: Formation of CYT-interleukin-12 (CYT-IL-12) and CYT-interleukin-2 (CYT-IL-2) The data presented in the previous examples support the idea that binding highly potent immune molecules such as INFγ and TNFα to the surface of PEGylated gold nanoparticles increases cytokine titers, improves their pharmacokinetic profiles, and is advantageous for accumulation in solid tumors.

[0075] Using the method described in Example 1, we pursued the development of additional subsequent nanoparticles with the potential to induce and drive potent anti-cancer vascular and immune responses. After production, the nanoparticles were investigated using the analytical methods described above.

[0076] Saturation binding curves were created for interleukin-2 (IL-2) or interleukin-12 (IL-12) by adding cytokines in increasing amounts relative to a fixed volume of gold. After 1 hour incubation, the particles were centrifuged at 14,000 rpm, and the supernatant was collected and set aside. The particles were reconstituted to 0.1 times their original volume, and both the particle-bound fraction and the free (supernatant) fraction were assayed by in-house IL-2 or IL-12 ELISA.

[0077] The data shown in Figure 15 indicates that IL-12 exhibited saturation binding to gold particles. At relatively low masses, most of the cytokines were associated with the particles (black circles). As the particle surface became saturated with cytokines, more cytokines were detected as free (white circles).

[0078] Example 7 The formation of the construct CYT-IL-12-TNFα significantly increases the titer of IL-12.

[0079] The two-agent IL-12-TNFα, CYT-IL-12-TNFα, was prepared using the method described in Example 1. The titer of CYT-IL-12-TNFα was tested using the HEK-12 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 either 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 from each replication well was added to 200 μL of PNPP substrate. Similar to the data reported for the CYT-IFNγ-TNFα nanopharmaceutical, CYT-IL-12-TNFα induced a significant increase in IL-12 titer, which was nearly 70-fold higher (Figure 16).

[0080] Example 8 Generation of IL-2-TNF dual-component nanoparticles In the following series of studies, 5 μg of interleukin-2 (IL-2), 0.25 μg of TNF, and 15 μg of PEG-THIOL were introduced into a single solution of gold nanoparticles. A monotherapy control containing only one of the cytokines was also generated. The bound cytokine fraction and the free cytokine fraction were isolated by centrifugation. To demonstrate the presence of both cytokines on the same particle, the monotherapy and dual-agent nanoparticles were serially diluted and tested in both forms of cross-antibody ELISA. For these assays, the dual-agent or monotherapy nanoparticles were captured using either a TNFα or IL-2 monoclonal antibody. The monoclonal antibody capture material was detected using a rabbit polyclonal antibody (Cytimmune Sciences, Inc., Maryland, USA) and alkaline phosphatase-conjugated goat anti-rabbit antibody to detect complementary cytokines.

[0081] The data shown in Figure 17 confirm the presence of both cytokines on the same particle. In both assays, the single-agent control produced minimal color, while the dual-agent particles produced a clearer signal than the signal produced by the single-agent control. Furthermore, the magnitude of the signal produced in each XAb ELISA was consistent with the amount of cytokine initially bound. For example, in the TNFα capture XAb assay, IL-2 detection showed a significant signal across the dilution curve. Conversely, while a dilution curve was observed in the IL-2 XAb assay, the TNFα signal showed a greater decrease in signal for each dilution in the dilution curve.

[0082] Example 9 Development of multimodal IO nanoparticles There is long-standing evidence suggesting that many chemotherapy regimens activate the immune system as part of their antitumor effect (17-18). Conceptually, chemotherapy may generate cancer antigens needed to initiate and drive cancer immunotherapy. This is supported by the fact that many current clinical trials focused on immuno-oncology have added chemotherapy treatment groups (19-20). Other therapies, including precision medicines, have the potential to restore or block biological pathways that can enhance or drive anti-cancer effects in combination with cytokines, and for these purposes, the list of small molecule immuno-oncological drugs under development is growing.

[0083] To demonstrate the ability to generate such drugs, we developed multimodal immuno-oncology nanoparticles composed of both TNFα and IFNγ, to which we attached small molecule prodrugs. A schematic diagram of the new nanoconstruct is shown in Figure 18. Specifically, we conjugated the previously described thiol-derivative prodrug form of paclitaxel to existing CYT-IFNγ-TNFα parent nanoparticles.

[0084] Using the method outlined in Example 1, 2.5 μg of thiolated paclitaxel analog was added to an existing formulation. The free drug was separated from the particle-bound components by centrifugation, and the nanoparticles were analyzed for cytokine concentration.

[0085] To initially understand the effects of adding paclitaxel prodrugs to CYT-IFNγ-TNFα, FTC-133 cells were incubated with similar doses of IFNγ and TNFα added as either 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, the previously described perinuclear localization of particles was observed. In the case of CYT-IFNγ-TNFα-paclitaxel, the nucleus was segmented, and particles localized around each segment.

[0086] The micrograph shown in Figure 19 demonstrates that CYT-IFNγ-TNFα represents a unique mechanism for targeting the intracellular delivery of small molecule immuno-oncological drugs accompanied by TNFα and IFNγ. To further demonstrate this concept, a thiolated paclitaxel prodrug was used to generate derivative nanoparticles substituted with fluorescently labeled 2kDa PEG-THIOL. By adding this construct to FTC-133 cells, it was possible to track both the entry of the particles into the cells and the release of the prodrug substitute (e.g., fluorescently labeled 2kDa PEG-THIOL).

[0087] The nanoparticles discussed in this document suggest possible therapeutic strategies for solid tumors. Figure 20 shows FTC-133 cells treated with CYT-IFNγ-TNFα-paclitaxel. Treatment with CYT-IFNγ-TNFα-paclitaxel results in cell lysis and the generation of cell fragments (highlighted by circles in Figure 20). These cell fragments may contain tumor antigens that can act to initiate an immune response. These findings suggest that initiating an immune response using CYT-IFNγ-TNFα-paclitaxel, followed by the use of other immuno-oncology nanoparticles, could be a possible therapeutic strategy to further drive an anti-tumor immune response.

[0088] Figure 21 shows a combined fluorescence / bright-field image of FTC-133 cells treated with this construct. Intracellular transport of particles was indicated by the red color of the particles (§). Also evident is the increased clarity of the fluorescent marker as the nanoparticles reach the perinuclear region, supporting the release of a prodrug substitute from the particles (△).

[0089] References

[0090] [Table 1-1]

[0091] [Table 1-2]

[0092] [Table 1-3]

[0093] [Table 1-4]

Claims

1. A composition comprising gold nanoparticles bound to a first cytokine and a second cytokine, wherein the first cytokine comprises tumor necrosis factor alpha (TNFα) and the second cytokine comprises interferon gamma (IFNγ).

2. The composition according to claim 1, wherein the first cytokine is TNFα and the second cytokine is IFNγ.

3. The composition according to claim 1, wherein the ratio of the first cytokine to the second cytokine is 18:1 to 22:1 by weight.

4. The composition according to claim 1, wherein the gold nanoparticles are covalently bound to the first cytokine and the second cytokine.

5. The composition according to claim 1, wherein the gold nanoparticles are covalently bonded to the first cytokine via a first thiol group and to the second cytokine via a second thiol group.

6. The composition according to claim 1, further comprising polyethylene glycol, a polyethylene glycol derivative, or polyethylene glycol-thiol.

7. The composition according to claim 1, further comprising paclitaxel, a paclitaxel analog, or a paclitaxel prodrug.

8. The composition according to claim 1, further comprising polyethylene glycol-thiol, wherein the size of the polyethylene glycol-thiol is 2 kDa to 20 kDa.

9. The composition according to claim 1, wherein the size of the gold nanoparticles is about 27 nm.

10. The composition according to claim 1, wherein the first cytokine and / or the second cytokine are bound to the gold nanoparticles by ionic bonding or hydrophobic interaction.

11. A composition comprising gold nanoparticles bound to a first cytokine and a second cytokine, wherein the first cytokine comprises tumor necrosis factor alpha (TNFα) and the second cytokine comprises interleukin-2 (IL-2).

12. The composition according to claim 11, wherein the first cytokine comprises natural TNFα and the second cytokine comprises natural IL-2.

13. The composition according to claim 11, wherein the ratio of the first cytokine to the second cytokine is 18:1 to 22:1 by weight.

14. The composition according to claim 11, wherein the gold nanoparticles are covalently bound to the first cytokine and the second cytokine.

15. The composition according to claim 11, wherein the gold nanoparticles are covalently bonded to the first cytokine via a first thiol group and to the second cytokine via a second thiol group.

16. The composition according to claim 11, further comprising polyethylene glycol, a polyethylene glycol derivative, or a polyethylene glycol-thiol.

17. The composition according to claim 11, further comprising polyethylene glycol-thiol, wherein the size of the polyethylene glycol-thiol is 2 kDa to 20 kDa.

18. The composition according to claim 16, wherein the ratio of the second cytokine to the polyethylene glycol, the polyethylene glycol derivative, or the polyethylene glycol-thiol is about 1:3 by weight.

19. The composition according to claim 11, further comprising paclitaxel, a paclitaxel analog, or a paclitaxel prodrug.

20. The composition according to claim 19, wherein the ratio of the second cytokine to the paclitaxel, the paclitaxel analog, or the paclitaxel prodrug is about 2:1 by weight.

21. The composition according to claim 11, wherein the size of the gold nanoparticles is about 27 nm.

22. The composition according to claim 11, wherein the first cytokine and / or the second cytokine are bound to the gold nanoparticles by ionic bonding or hydrophobic interaction.

23. The composition according to claim 11, further comprising a fluorescent label.

24. The composition according to claim 11, further comprising a fluorescently labeled polyethylene glycol-thiol.

25. The composition according to claim 24, wherein the size of the fluorescently labeled polyethylene glycol-thiol is about 2 kDa.

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