Patent Application
20220401374
The present disclosure generally relates to a composition matter and a method for cancer treatment comprising albumin (alb) coated tannic acid-Fe nanoparticles (NPs) with a therapeutic compound, and one or more diluents, excipients or carriers. In particular, the present invention provides a treatment by enhancing antitumor immunity using tannic acid-based nanoparticles containing a therapeutic cancer treatment that can induce immunogenic cell death (ICD).
This section introduces aspects that may help facilitate a better understanding of the disclosure. Accordingly, these statements are to be read in this light and are not to be understood as admissions about what is or is not prior art.
Chemotherapy is one of the main cancer treatments along with surgery, radiation and immunotherapy. Chemotherapeutic drugs interfere with the growth of tumor cells by damaging or blocking the synthesis of nucleic acids, preventing cell division, or inhibiting the homeostatic control of regular cellular functions. Some of the chemotherapeutic drugs have gained increasing interest as immunogenic cell death (ICD) inducers. ICD-inducing chemotherapy kills tumor cells such that it induces the production of tumor-associated antigens and damage-associated molecular patterns (DAMPs), thereby helping the host to develop adaptive immunity to the tumor cells1. Therefore, ICD inducers can play an important role in cancer immunotherapy as standalone therapeutics to induce specific anti-tumor immune responses2 as well as companion drugs to enhance the effect of immune checkpoint blockade therapy3.
Several existing chemotherapeutic drugs are identified to be ICD inducers, which include anthracyclines (doxorubicin, idarubicin, epirubicin), mitoxantrones, oxaliplatin, cyclophosphamide, bortezomib, and paclitaxel4,5. In addition, carfilzomib (CFZ), a second-generation proteasome inhibitor, is considered an emerging ICD inducer6, with improved efficacy and safety profiles over the first-generation bortezomib7, 8, 9. CFZ irreversibly inhibits the proteolytic activity of the proteasome9, prevents the degradation of misfolded and other key signaling proteins, causing a significant endoplasmic reticulum (ER) stress10, 11, the main mechanism of ICD induction6. Therefore, it is expected that CFZ delivered to tumors may produce a spatially defined set of tumor-associated antigens and DAMPs, i.e., in situ tumor vaccines and endogenous immune adjuvants.
Proteasome inhibitors are drugs that block the action of proteasomes, cellular complexes that break down proteins. They are used in the treatment of cancer; and three are approved for use in treating multiple myeloma. Proteasome inhibition may prevent degradation of pro-apoptotic factors such as the p53 protein, permitting the activation of programmed cell death in neoplastic cells dependent upon suppression of pro-apoptotic pathways.
However, a critical challenge in using chemotherapeutic agents to promote cancer immunotherapy is that their antiproliferative effects damage not only tumor cells but also immune cells in the tumor microenvironment, impairing their ability to mount immune responses to dying tumor cells12, 13. Given the paradoxical effect of chemotherapy, it is recognized that optimal regimen is required in order to maximize its therapeutic benefit in the context of cancer immunotherapy14. In fact, compared to the standard maximum tolerated dose (MTD) regimen, prolonged administration of low doses of chemotherapeutics, called metronomic chemotherapy, has shown reduced immunotoxicity, thereby improving antitumor efficacy15. Moreover, metronomic dosing of chemotherapeutics can selectively deplete immunosuppressive cell populations, such as myeloid-derived suppressor cells and regulatory T cells, from the tumor microenvironment16, 17, 18. These studies suggest that sustained delivery of ICD inducers to tumors may protect antitumor immune cells and help them to develop effective tumor-specific immune responses16.
Nanoparticles (NPs) have been widely pursued in the delivery of chemotherapeutics. They have been used to help disperse water-insoluble drugs and/or protect metabolically labile drugs from the hostile physiological environments19. Moreover, NPs may be designed to control the drug release rate over a prolonged period, facilitating metronomic delivery of chemotherapeutics20. A recent study also reports that NPs can capture tumor neoantigens and DAMPs from dying tumor cells and facilitate their delivery to dendritic cells to activate the antitumor immunity21. Therefore, NPs may provide multiple benefits to the delivery of ICD inducers: First, NPs can control the release of ICD inducers to prevent damaging immune cells involved in antitumor immunity. Second, with the relatively large size and drug release control, NPs can retain a drug in tumors longer than the free drug counterpart. Third, NPs can capture the tumor-associated antigens and DAMPs produced by dying tumor cells, improving their exposure to antigen presenting cells.
In the present study, we develop a nanocapsule formulation of CFZ, which can retain the drug in tumors for a prolonged period, control the drug release, and serve as a reservoir of tumor antigens and DAMPs. We hypothesize that a prolonged delivery of CFZ puts tumor cells under extended ER stress to induce ICD6, 22, 23, 24. Sustained release of CFZ at low dose may minimize damages to chemosensitive immune cells recruited to tumors. In addition, as a favored substrate of phagocytes25, nanocapsules capturing tumor antigens and DAMPs may enhance their delivery to dendritic cells (DCs) and subsequent activation of the cells. For this purpose, we encapsulate CFZ in a supramolecular assembly of tannic acid (TA) and iron26 and modified the surface with albumin (CFZ-pTA-alb) to control the drug release. We envision that locally (intratumorally) injected CFZ nanocapsules will activate antitumor immune responses, which can translate to systemic protection against tumors. We evaluate the ability of CFZ nanocapsules to control the drug release and capture soluble proteins released from dying tumor cells. We then compare the effects of CFZ nanocapsules and unformulated CFZ on tumor cells and immune cells in vitro and test if they control tumor growth and develop local and systemic antitumor immunity using mouse models of B16F10 and CT26 tumors.
The above and other objects, features, and advantages of the present invention will become more apparent when taken in conjunction with the following description and drawings wherein identical reference numerals have been used, where possible, to designate identical features that are common to the figures, and wherein:
While the concepts of the present disclosure are illustrated and described in detail in the figures and the description herein, results in the figures and their description are to be considered as exemplary and not restrictive in character; it being understood that only the illustrative embodiments are shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.
As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art.
In the present disclosure the term “about” can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range. In the present disclosure the term “substantially” can allow for a degree of variability in a value or range, for example, within 90%, within 95%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more of a stated value or of a stated limit of a range.
In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section heading may occur within or outside of that particular section. Furthermore, all publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.
The term “pharmaceutically acceptable carrier” is art-recognized and refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof. Each carrier must be “acceptable” in the sense of being compatible with the subject composition and its components and not injurious to the patient. Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
As used herein, the term “administering” includes all means of introducing the compounds and compositions described herein to the patient, including, but are not limited to, oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal, and the like. The compounds and compositions described herein may be administered in unit dosage forms and/or formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles.
Illustrative formats for oral administration include tablets, capsules, elixirs, syrups, and the like. Illustrative routes for parenteral administration include intravenous, intraarterial, intraperitoneal, epidural, intraurethral, intrasternal, intramuscular and subcutaneous, as well as any other art recognized route of parenteral administration.
Illustrative means of parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques, as well as any other means of parenteral administration recognized in the art. Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably at a pH in the range from about 3 to about 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. The preparation of parenteral formulations under sterile conditions, for example, by lyophilization, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art. Parenteral administration of a compound is illustratively performed in the form of saline solutions or with the compound incorporated into liposomes. In cases where the compound in itself is not sufficiently soluble to be dissolved, a solubilizer such as ethanol can be applied.
The dosage of each compound of the claimed combinations depends on several factors, including: the administration method, the condition to be treated, the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the person to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular patient may affect the dosage used.
It is to be understood that in the methods described herein, the individual components of a co-administration, or combination can be administered by any suitable means, contemporaneously, simultaneously, sequentially, separately or in a single pharmaceutical formulation. Where the co-administered compounds or compositions are administered in separate dosage forms, the number of dosages administered per day for each compound may be the same or different. The compounds or compositions may be administered via the same or different routes of administration. The compounds or compositions may be administered according to simultaneous or alternating regimens, at the same or different times during the course of the therapy, concurrently in divided or single forms.
The term “therapeutically effective amount” as used herein, refers to that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated. In one aspect, the therapeutically effective amount is that which may treat or alleviate the disease or symptoms of the disease at a reasonable benefit/risk ratio applicable to any medical treatment. However, it is to be understood that the total daily usage of the compounds and compositions described herein may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically-effective dose level for any particular patient will depend upon a variety of factors, including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, gender and diet of the patient: the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidentally with the specific compound employed; and like factors well known to the researcher, veterinarian, medical doctor or other clinician of ordinary skill.
Depending upon the route of administration, a wide range of permissible dosages are contemplated herein, including doses falling in the range from about 1 μg/kg to about 1 g/kg. The dosages may be single or divided, and may administered according to a wide variety of protocols, including q.d. (once a day), b.i.d. (twice a day), t.i.d. (three times a day), or even every other day, once a week, once a month, once a quarter, and the like. In each of these cases it is understood that the therapeutically effective amounts described herein correspond to the instance of administration, or alternatively to the total daily, weekly, month, or quarterly dose, as determined by the dosing protocol.
In addition to the illustrative dosages and dosing protocols described herein, it is to be understood that an effective amount of any one or a mixture of the compounds described herein can be determined by the attending diagnostician or physician by the use of known techniques and/or by observing results obtained under analogous circumstances. In determining the effective amount or dose, a number of factors are considered by the attending diagnostician or physician, including, but not limited to the species of mammal, including human, its size, age, and general health, the specific disease or disorder involved, the degree of or involvement or the severity of the disease or disorder, the response of the individual patient, the particular compound administered, the mode of administration, the bioavailability characteristics of the preparation administered, the dose regimen selected, the use of concomitant medication, and other relevant circumstances.
The term “patient” includes human and non-human animals such as companion animals (dogs and cats and the like) and livestock animals. Livestock animals are animals raised for food production. The patient to be treated is preferably a mammal, in particular a human being.
The present disclosure generally relates to a composition matter and a method for cancer treatment comprising albumin (alb) coated tannic acid-Fe nanoparticles (NPs) with a therapeutic compound, and one or more diluents, excipients or carriers. In particular, the present invention provides a treatment by enhancing antitumor immunity using tannic acid-based nanoparticles containing a therapeutic cancer treatment that can induce immunogenic cell death (ICD). The method disclosed herein provides a potential solution to the immunotoxicity accompanying the ICD cancer immunotherapy by intratumoral administration of a nanocapsule formulation of carfilzomib (CFZ), an ICD-inducing proteasome inhibitor, using interfacial supramolecular assembly of tannic acid (TA) and iron, supplemented with albumin coating for better metabolic stability.
In some illustrative embodiments, this present disclosure relates to a composition as a cancer treatment comprising albumin (alb) coated tannic acid-Fe (pTA) nanoparticles (NPs) incorporating a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers.
In some illustrative embodiments, this present disclosure relates to a composition as a cancer treatment comprising albumin (alb) coated tannic acid-Fe (pTA) nanoparticles (NPs) incorporating a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said therapeutic compound is an anthracycline or a proteasome inhibitor.
In some illustrative embodiments, this present disclosure relates to a composition as a cancer treatment comprising albumin (alb) coated tannic acid-Fe (pTA) nanoparticles (NPs) incorporating a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said therapeutic compound comprises doxorubicin, idarubicin, epirubicin, mitoxantrones, oxaliplatin, cyclophosphamide, bortezomib, paclitaxel, or carfilzomib (CFZ).
In some illustrative embodiments, this present disclosure relates to a composition as a cancer treatment comprising albumin (alb) coated tannic acid-Fe (pTA) nanoparticles (NPs) incorporating a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said therapeutic compound is carfilzomib (CFZ).
In some illustrative embodiments, this present disclosure relates to a composition as a cancer treatment comprising albumin (alb) coated tannic acid-Fe (pTA) nanoparticles (NPs) incorporating a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said composition is administered locally at the site of a tumor (intratumorally).
In some illustrative embodiments, this present disclosure relates to a composition as a cancer treatment comprising albumin (alb) coated tannic acid-Fe (pTA) nanoparticles (NPs) incorporating a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said composition as a cancer treatment is administered systemically.
In some illustrative embodiments, this present disclosure relates to a composition as a cancer treatment comprising albumin (alb) coated tannic acid-Fe (pTA) nanoparticles (NPs) comprising a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers, wherein said albumin coated tannic acid-Fe nanoparticles with said therapeutic compound are manufactured according to the process of:
In some other illustrative embodiments, this present disclosure relates to a composition as a cancer treatment comprising albumin (alb) coated tannic acid-Fe (pTA) nanoparticles (NPs) comprising a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said therapeutic compound that induces immunogenic cell death (ICD) is an anthracycline or a proteasome inhibitor.
In some other illustrative embodiments, this present disclosure relates to a composition as a cancer treatment comprising albumin (alb) coated tannic acid-Fe (pTA) nanoparticles (NPs) comprising a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said therapeutic compound is doxorubicin, idarubicin, epirubicin, mitoxantrones, oxaliplatin, cyclophosphamide, bortezomib, paclitaxel, or carfilzomib (CFZ).
In some other illustrative embodiments, this present disclosure relates to a composition as a cancer treatment comprising albumin (alb) coated tannic acid-Fe (pTA) nanoparticles (NPs) comprising a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said therapeutic compound is carfilzomib (CFZ).
In some other illustrative embodiments, this present disclosure relates to a composition as a cancer treatment comprising albumin (alb) coated tannic acid-Fe (pTA) nanoparticles (NPs) comprising a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said composition is administered locally at the site of a tumor (intratumorally).
In some other illustrative embodiments, this present disclosure relates to a composition as a cancer treatment comprising albumin (alb) coated tannic acid-Fe (pTA) nanoparticles (NPs) comprising a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said composition as a cancer therapy is administered systemically.
In some other illustrative embodiments, this present disclosure relates to a method for treating a patient of cancer comprising the step of administering to a patient in need of relief from said cancer a therapeutically effective amount of a pharmaceutical composition comprising albumin (alb) coated tannic acid-Fe (pTA) nanoparticles (NPs) incorporating a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers, wherein said albumin coated tannic acid-Fe nanoparticles comprising said therapeutic compound are manufactured according to the process of:
In some other illustrative embodiments, this present disclosure relates to a method for treating a patient of cancer comprising the step of administering to a patient in need of relief from said cancer a therapeutically effective amount of a pharmaceutical composition comprising albumin (alb) coated tannic acid-Fe (pTA) nanoparticles (NPs) incorporating a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said therapeutic compound that induces immunogenic cell death (ICD) is an anthracycline or a proteasome inhibitor.
In some other illustrative embodiments, this present disclosure relates to a method for treating a patient of cancer comprising the step of administering to a patient in need of relief from said cancer a therapeutically effective amount of a pharmaceutical composition comprising albumin (alb) coated tannic acid-Fe (pTA) nanoparticles (NPs) incorporating a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said therapeutic compound is doxorubicin, idarubicin, epirubicin, mitoxantrones, oxaliplatin, cyclophosphamide, bortezomib, paclitaxel, or carfilzomib (CFZ).
In some other illustrative embodiments, this present disclosure relates to a method for treating a patient of cancer comprising the step of administering to a patient in need of relief from said cancer a therapeutically effective amount of a pharmaceutical composition comprising albumin (alb) coated tannic acid-Fe (pTA) nanoparticles (NPs) incorporating a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said therapeutic compound is carfilzomib (CFZ).
In some other illustrative embodiments, this present disclosure relates to a method for treating a patient of cancer comprising the step of administering to a patient in need of relief from said cancer a therapeutically effective amount of a pharmaceutical composition comprising albumin (alb) coated tannic acid-Fe (pTA) nanoparticles (NPs) incorporating a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said composition is administered locally at the site of a tumor (intratumorally).
In some other illustrative embodiments, this present disclosure relates to a method for treating a patient of cancer comprising the step of administering to a patient in need of relief from said cancer a therapeutically effective amount of a pharmaceutical composition comprising albumin (alb) coated tannic acid-Fe (pTA) nanoparticles (NPs) incorporating a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said composition as a cancer therapy is administered systemically.
In some other illustrative embodiments, this present disclosure relates to a use of a pharmaceutical composition in the preparation of a medicament for treating a cancer, wherein said pharmaceutical composition comprising albumin (alb) coated tannic acid-Fe nanoparticles (NPs) incorporating a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers, wherein said albumin coated tannic acid-Fe nanoparticles comprising said therapeutic compound are manufactured according to the process of:
In some other illustrative embodiments, this present disclosure relates to a use of a pharmaceutical composition in the preparation of a medicament for treating a cancer, wherein said pharmaceutical composition comprising albumin (alb) coated tannic acid-Fe nanoparticles (NPs) incorporating a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said therapeutic compound that induces immunogenic cell death (ICD) is an anthracycline or a proteasome inhibitor.
In some other illustrative embodiments, this present disclosure relates to a use of a pharmaceutical composition in the preparation of a medicament for treating a cancer, wherein said pharmaceutical composition comprising albumin (alb) coated tannic acid-Fe nanoparticles (NPs) incorporating a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said therapeutic compound that induces immunogenic cell death (ICD) comprises doxorubicin, idarubicin, epirubicin, mitoxantrones, oxaliplatin, cyclophosphamide, bortezomib, paclitaxel, and carfilzomib (CFZ).
In some other illustrative embodiments, this present disclosure relates to a use of a pharmaceutical composition in the preparation of a medicament for treating a cancer, wherein said pharmaceutical composition comprising albumin (alb) coated tannic acid-Fe nanoparticles (NPs) incorporating a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said therapeutic compound that induces immunogenic cell death (ICD) is carfilzomib (CFZ).
In some other illustrative embodiments, this present disclosure relates to a use of a pharmaceutical composition in the preparation of a medicament for treating a cancer, wherein said pharmaceutical composition comprising albumin (alb) coated tannic acid-Fe nanoparticles (NPs) incorporating a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said therapeutic compound that induces immunogenic cell death (ICD) is administered locally at the site of a tumor (intratumorally).
In some other illustrative embodiments, this present disclosure relates to a use of a pharmaceutical composition in the preparation of a medicament for treating a cancer, wherein said pharmaceutical composition comprising albumin (alb) coated tannic acid-Fe nanoparticles (NPs) incorporating a therapeutic compound that induces immunogenic cell death (ICD), and one or more diluents, excipients or carriers as disclosed herein, wherein said composition as a cancer therapy is administered systemically.
The following examples and detailed embodiments are provided for explanation only, not mean to limit the scope of this disclosure.
Preparation and Characterization of CFZ-pTA and CFZ-pTA-Alb
CFZ-pTA nanocapsules were prepared by interfacial assembly of iron-tannic acid complexes26 (
CFZ-pTA was further modified with albumin by 4 h incubation in 2 mg/mL albumin solution. The albumin-coated CFZ-pTA nanocapsules (CFZ-pTA-alb) was similar in size to CFZ-pTA (
Albumin Layer of Nanocapsules Attenuates Release and Metabolic Degradation of CFZ
To evaluate the effect of albumin coating on CFZ release control, CFZ-pTA and CFZ-pTA-alb were compared with respect to their CFZ release kinetics in vitro. The particles were first housed in photocrosslinkable PEGDA hydrogel and incubated in 10% FBS-supplemented RPMI 1640 medium, which was sampled at regular intervals to quantify the released CFZ. The hydrogel method was used in lieu of common centrifugation or dialysis methods to avoid the risk of pressurizing and destroying nanocapsules during the repeated centrifugation or excessively diluting the drug to below the detection limit27. The hydrogel confines particles and helps separate them from the medium and does not require a large volume of medium for incubation28. However, the drug release rate measured by this method does not necessarily reflect the actual rate because the hydrogel also functions as a barrier to drug diffusion; thus, the results are only meaningful for rank ordering the two particles. CFZ-pTA-alb showed slower drug release than CFZ-pTA: in 24 h, 5% and 10% of CFZ was released from CFZ-pTA-alb and CFZ-pTA, respectively (
The stable CFZ encapsulation also enhanced the metabolic stability of CFZ, an epoxyketone peptide, which degrades quickly by peptide cleavage and epoxide ring opening29. Metabolic stability of CFZ in different formulations was compared in vitro after 30 min incubation in whole blood, which contain epoxide hydrolases/peptidases29. With cyclodextrin-solubilized CFZ (CFZ-CD), 66.0±4.0% of total CFZ survived the incubation. CFZ-pTA and CFZ-pTA-alb displayed much improved metabolic stability, with 74.1±7.8% and 91.8±6.6% of CFZ, respectively, remaining under the same conditions (
The sustained CFZ release from CFZ-pTA(-alb) also manifested as attenuated cytotoxicity in cancer cell lines. B16F10 (melanoma) cells and HCC-1937 (triple negative breast cancer) were treated with CFZ solubilized in DMSO (CFZ-DMSO), CFZ-pTA, and CFZ-pTA-alb in different concentrations. With 2 h exposure, both CFZ-pTA and CFZ-pTA-alb showed minimal toxicity compared to CFZ-DMSO (
Sustained Release of CFZ Spares Immune Cells
To estimate whether the sustained supply of CFZ is beneficial to sparing immune cells in the tumor microenvironment, we tested the cytotoxic effect of CFZ on splenocytes in two treatment regimens (
Nanocapsules Capture DAMPs from Dying Tumor Cells and Enhance DC Uptake of Tumor-Associated Antigens
DCs play a central role in development of antitumor immunity. DCs take up tumor antigens, deliver them to draining lymph nodes (DLNs), where they cross-present them to activate T-lymphocytes. A previous study reported that polymeric NPs captured neoantigens and DAMPs of dying tumor cells and delivered them to DCs in DLNs21. CFZ-pTA-alb may provide a similar function via the underlying pTA layer, which can interact with proteins via hydrogen bonding and hydrophobic interactions36, 37. To test if CFZ-pTA-alb captures tumor-associated antigens and/or DAMPs, we incubated the nanocapsules in the medium containing soluble proteins released from CFZ-killed B16F10 cells and analyzed the surface-bound proteins. LC-MS/MS analysis showed a large amount of mouse proteins, several of which were identified as DAMPs (Calreticulin, Histone H2A.Z, Histone H1.5, Histone H1.3, Histone H1.4, Histone H3.1, Histone H4, Heat shock 70 kDa protein 4, Heat shock protein HSP 90-α, Heat shock protein HSP 90-β)21, captured by CFZ-pTA-alb (
We next examined whether the adsorption of DAMPs to nanocapsules increased DC activation. BMDCs were incubated with B16F10 cells pretreated with CFZ, CFZ-pTA-alb or blank-pTA-alb (vehicle) to assess their expression of CD40 and CD86 (DC activation markers) upon their interaction. Untreated or blank pTA-alb-treated (i.e., healthy) B16F10 cells induced no increase in CD40 or CD86 expression in BMDCs. CFZ and CFZ-pTA-alb-treated B16F10 cells increased the expression of the two activation markers, with CFZ-pTA-alb showing a more prominent effect than CFZ (
Although tumor antigen adsorption was not as evident as DAMP binding, we also examined if the CFZ-pTA-alb helped deliver tumor-associated antigens to DCs by serving as their temporary reservoir. B16F10 cells, stained with DiI (a lipophilic dye) and pretreated with CFZ, CFZ-pTA-alb or blank-pTA-alb (vehicle) for 24 h, were incubated with BMDCs for 4 or 24 h. An excess dose of CFZ (10 μM CFZ equivalent) was used to maximize the effect on B16F10 cells, and the cells were rinsed to remove the extra drug prior to the incubation with BMDCs. BMDCs were identified by anti-CD11c antibody and analyzed by flow cytometry. Cells positive for both DiI and CD11c (DiI+CD11c+ cells) were considered the BMDCs taking up DiI-stained B16F10 cells and fragments of dying cells. With no apparent toxicity to B16F10 cells, blank-pTA-alb induced no increase in the BMDCs uptake of the treated B16F10 cells (
These in vitro results suggest that CFZ-pTA-alb can contribute to the generation of tumor-specific immunity at least in three complementary mechanisms: (i) killing tumor cells to generate tumor-associated antigens by CFZ; (ii) sparing immune cells by sustained release of CFZ; and (iii) serving as a reservoir of DAMPs to enhance the activation and antigen uptake of DCs. On the basis of these results, the effect of intratumorally injected CFZ-pTA-alb on tumor growth and its environment was evaluated by two syngeneic tumor models (B16F10@C57BL/6, CT26@Balb/c).
Local Administration of CFZ-pTA-Alb Provides Better Anti-Tumor Effect than CFZ-CD
The antitumor effect of CFZ-pTA-alb was evaluated in B16F10 melanoma-bearing C57BL/6 mice and compared with the effect of CFZ-CD (CFZ dissolved in 2-hydroxypropyl-β-cyclodextrin). Subcutaneous B16F10 tumors were injected intratumorally once with PBS, CFZ-CD or CFZ-pTA-alb at a dose equivalent to CFZ 1.2 μg per mouse. With this regimen, CFZ-CD had no significant suppression of tumor growth as compared to PBS control (
The superior effect of CFZ-pTA-lab may be attributed to the prolonged tumor retention of CFZ in the form of nanocapsules, thereby increased local availability of the drug. To test this, another group of identically treated animals were sacrificed 2 h after the treatment, and CFZ in tumors and plasma were quantified. The tumors and plasma were treated with Triton-X 100 to disassemble CFZ-pTA-alb and release CFZ. Therefore, the measured CFZ represents the total detectable amount of CFZ, including both free/released and encapsulated in CFZ-pTA-alb. The amount of CFZ retrieved from CFZ-pTA-alb-treated tumors was 3 times higher than that of CFZ-CD group (
Given the benefit of sustained CFZ release in protecting immune cells predicted in vitro (
Local Administration of CFZ-pTA-Alb Helps Develop T-Cell Immunity to Tumors
Considering the potential to spare and activate antitumor immune cells in the tumor microenvironment (
To verify the generation of tumor-specific T-cell immunity, we performed immunophenotyping of the treated tumors and tumor DLNs and tested antigen-specific production of IFN-γ from splenocytes of the treated animals. B16F10 tumors are known to be poorly immunogenic with few tumor-infiltrating cytotoxic T cells39 and, thus, difficult to obtain substantial readouts (
Systemic Administration of CFZ-pTA-Alb Provides Better Anti-Tumor Effect than CFZ-CD with Limitation
The antitumor effect of CFZ-pTA-alb was also evaluated by systemic administration in C57BL/6 mice bearing B16F10 tumors. Animals were administered with treatments equivalent to CFZ 6 mg/kg/dose49 via tail vein on two consecutive days per week (similar to the clinical CFZ regimen for multiple myeloma therapy50). With this regimen, CFZ-CD did not differ from the PBS control over 7 days, but CFZ-pTA-alb delayed tumor growth significantly (
Despite the initial delay in tumor growth, the animals treated with CFZ-pTA-alb ultimately succumbed to death with additional injections. The mortality did not correlate with the tumor size or body weight loss. We speculate that the metabolic stability offered by CFZ-pTA-alb enhanced the effect of CFZ not only on tumors but also on off-targets such as the MPS organs, which free drug that is rapidly metabolized may not reach but stable nanocapsules may. Blood chemistry measured at 48 h after intravenous (IV) administration of two consecutive doses did not change significantly. However, splenomegaly was observed in healthy mice treated with CFZ-pTA-alb multiple times (eq. to CFZ 6 or 12 mg/kg/dose two consecutive days per week for 2 weeks), which was not seen with blank pTA at an even higher dose. This suggests acute toxicity of CFZ reaching the spleen as NPs.
Discussion In this study we demonstrated the potential to translate local chemotherapy to systemic antitumor immunity via sustained induction of ICD. ICD inducers can generate tumor-associated antigens in situ, thereby activating immune responses against tumors without a prior knowledge of tumor antigens. CFZ, an irreversible proteasome inhibitor, was used as a chemotherapeutic ICD inducer on the basis of its mechanism of action leading to ER stress10 and the evidence of immunogenic apoptosis52. CFZ was better tolerated by immune cells when used at a low dose for an extended period rather than as a high dose bolus (
We observed that intratumoral injection of CFZ-pTA-alb to B16F10 tumors grown in immunocompetent C57BL/6 mice delayed the development of the tumor inoculated on the contralateral side, whereas PBS and CFZ-CD had no effect (
This study has focused on the advantage of controlling CFZ release by CFZ-pTA-alb in sparing immune cells in the tumor microenvironment. The literature suggests that the sustained CFZ release in the tumor may have additional benefits. Chemotherapy based on MTDs tend to selectively target chemosensitive cancer cells, leaving behind chemoresistant cell populations that may lead to tumor relapse and emergence of drug resistance57. On the contrary, metronomic chemotherapy targets the tumor microenvironment and disengages tumor cells from its support system, resulting in long lasting tumor regression16, 18, 57. In addition, sustained low doses of chemotherapeutics show antiangiogenic effects58, 59 and avoid the induction of tumor-initiating cancer stem cells57. Bortezomib (the first-generation proteasome inhibitor) has shown to induce endothelial cell apoptosis and downregulate proteins responsible for the angiogenic phenotype in endothelial cells, such as vascular endothelial cell growth factor, interleukin-6, insulin-like growth factor-I, angiopoietin 1 and angiopoietin 260, 61. The antiangiogenic effect was also reported with oprozomib, an orally active second-generation proteasome inhibitor62. As a proteasome inhibitor sharing a similar mechanism of action, CFZ may have an antiangiogenic effect likewise, which would manifest better with sustained release. The contribution of sustained CFZ delivery by CFZ-pTA-alb to antiangiogenesis is worth investigation.
CFZ-pTA-alb was administered as a local (IT) injection in this study. IT injection is clinically feasible for solid tumors that can be located by palpation, imaging or ultrasonography63, 64. Given the specificity and mobility of the activated T cells, the local induction of ICD by IT chemotherapy may be an effective way of inducing systemic antitumor responses with a minimal risk of toxic side effects inherent to systemic chemotherapy65, 66, 67. Nevertheless, it would be ideal if CFZ-pTA-alb can be administered systemically without the need of locating tumors. The IV-injected CFZ-pTA-alb attenuated the growth of B16F10 tumors in C57BL/6 mice (
To summarize, a nanocapsule formulation of CFZ, CFZ-pTA-alb, was developed for the sustained CFZ delivery and extended ICD induction in solid tumors. Tannic acid/iron assemblies and albumin coating helped control the drug release, enhancing metabolic stability and prolonging tumor retention of CFZ. Moreover, CFZ-pTA-alb helped activate antitumor immunity by selectively sparing immune cells in the tumor microenvironment from CFZ toxicity and serving as a reservoir of immunostimulatory DAMPs released from dying tumor cells. Consequently, CFZ-pTA-alb outperformed CFZ-CD (cyclodextrin-solubilized CFZ) at the equivalent dose in controlling tumor growth and developing local and systemic antitumor immunity in mouse models of B16F10 and CT26 tumors. CFZ-pTA-alb is an effective formulation to translate local ICD induction to systemic antitumor immunity.
Materials and Methods
Carfilzomib (CFZ) was purchased from Shenzhen Chemical Co. LTD. (Shanghai, China). Tannic acid, iron chloride, 2-hydroxypropyl-β-cyclodextrin, human serum albumin (≥96% agarose gel electrophoresis) and Irgacure 2959 were purchased from Sigma-Aldrich (St. Louis, Mo., USA). Polyethyleneglycol dimethylacrylate (PEGDA, MW: 3400 Da) was purchased from Alfa Aesar (Haverhill, Mass., USA). Mouse interferon-gamma (IFN-γ) ELISA kit was purchased from Invitrogen (Eugene, Oreg., USA). GM-CSF was purchased from PeproTech (Rock Hill, N.J., USA). Purified rat anti-mouse CD16/CD32 (Fc block), APC anti-mouse CD11c, FITC anti-mouse CD40, PE anti-mouse CD86, FITC anti-mouse CD4, APC anti-mouse CD8a, and Zombie Violet were purchased from BioLegend (San Diego, Calif., USA). AH1 peptide was purchased from Anaspec (Fremont, Calif., USA). All other materials were of analytical grade and purchased from Thermo Fisher Scientific (Waltham, Mass., USA).
TA/Fe3+-Stabilized CFZ Nanocore Preparation and Albumin Coating
CFZ was mixed with TA in 30 μL ethanol at a CFZ to TA molar ratio of 2.4. To this mixture, 10 mL water containing 100 μg FeCl3 (molar ratio of TA to Fe3+: 1.1) was added and mixed briefly with simple agitation to form nanocapsules. The particles were centrifugated at 43,400 rcf for 20 min at 4° C. to remove excess TA and FeCl3. The formed nanocapsules were called CFZ-pTA, where pTA stands for polymerized tannic acid crosslinked via Fe3+. The CFZ-pTA nanocapsules were further incubated in albumin solution (2 mg/mL) in water at CFZ-pTA to albumin weight ratio of 1:2, with mild rotation at room temperature (RT) for 4 h. The particles were centrifuged at 43,400 rcf for 20 min at 4° C. to remove unadsorbed albumin and washed twice by repeated centrifugation. The albumin-coated CFZ-pTA nanocapsules were called CFZ-pTA-alb. As controls, pTA (TA/Fe3+ nanoassemblies) and pTA-alb (albumin-coated pTA) were prepared in the same method as above omitting CFZ. Bare CFZ particles (with no pTA) were prepared by adding water to ethanolic solution of CFZ. The particles were suspended in water and stored at 4° C.
Nanocapsule Characterization
Particle Size and Surface Charge
The hydrodynamic diameter (z-ave), polydispersity index (PDI) and zeta potential of particles were measured by dynamic light scattering (DLS) in sodium phosphate buffer (10 mM, pH 7.4) using a Zetasizer Nano-ZS90 (Malvern Instruments, Worcestershire, UK).
Morphology
Particle morphology was examined by transmission electron microscopy (TEM). An aqueous suspension of freshly prepared particles was placed on a carbon coated copper grid (400 mesh), negatively stained with 1% uranyl acetate and allowed to dry in air. The dried grid was examined under an FEI Tecnai T20 transmission electron microscope (Hillsboro, Oreg., USA). To visualize the capsule structure of CFZ-pTA, the core part of the particles was dissolved in PBS containing 0.2% Tween 80 for 6 h. The particles were collected by centrifugation, resuspended in water, put on the grid and processed as above.
CFZ and TA Contents in CFZ-pTA
The CFZ content in CFZ-pTA was determined by C18 reverse phase HPLC (25 cm×4.6 mm, particle size: 5 μm). CFZ-pTA with a premeasured mass was added to an aqueous solution (pH 7.4) containing ethylenediaminetetraacetic acid (EDTA) (100 mM, to remove Fe3+) and urea solution (5M, to disrupt hydrogen bonding between TA and CFZ), mixed briefly by vortexing, and mixed with additional acetonitrile (to dissolve CFZ), making the final particle concentration ˜50 μg/mL. The resulting solution was filtered on 0.45 μm syringe filter prior to analysis. As reported previously68 with slight modifications, the mobile phase was composed of water and acetonitrile containing 0.05% trifluoroacetic acid and run in an acetonitrile gradient of 40-80% over 22 min at 0.7 mL/min. CFZ was detected with a UV detector at a wavelength of 210 nm.
The TA content in CFZ-pTA was estimated by bicinchoninic acid (BCA) assay (Pierce® BCA assay kit). CFZ-pTA 0.1 mg was suspended in 200 μL of 0.05 N HCl to dissolve TA. The BCA reagent was mixed with the sample in 8:1 v/v ratio at 37° C. for 30 min. The absorbance of the solution was read at 570 nm by a SpectraMax M3 microplate reader (Molecular Device, Sunnyvale, Calif., USA). CFZ solution at an equivalent concentration was treated same way, and the absorbance was subtracted from the reading of CFZ-pTA. The difference was compared to a calibration curve drawn with TA to determine the TA concentration in the sample.
Albumin Content in CFZ-pTA-Alb
The albumin content in CFZ-pTA-alb was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). CFZ-pTA-alb with a premeasured mass or standard albumin solutions were prepared in Laemmli buffer containing β-mercaptoethanol and heated at 95° C. for 5 min. The samples were resolved on a 12% SDS-PAGE run at 120 V for 30 min. After staining with QC Colloidal Coomassie Stain and imaged with Azure c300 (Dublin, Calif., USA), the band intensity was analyzed by densitometry (AzureSpot Analysis Software). The albumin content was determined by comparing the band intensities of CFZ-pTA-alb samples and standard albumin solutions.
Size Stability
CFZ-pTA or CFZ-pTA-alb (at a concentration equivalent to CFZ 60 μg/mL, n=3 per group) were incubated in 50% fetal bovine serum (FBS) at 37° C. for 24 h. The suspensions were sampled periodically to measure their particle size by the Zetasizer.
In Vitro CFZ Release Kinetics
CFZ-pTA or CFZ-pTA equivalent to 10 μg of CFZ were suspended in 0.25 mL of 10% PEG dimethylacrylate (PEGDA, 3400 Da) solution in PBS. The suspensions were illuminated under UV (365 nm) for 10 min in the presence of 25 μL of irgacure solution (20% w/v in methanol) to crosslink PEGDA. The crosslinked PEGDA matrix was briefly rinsed with water to remove free particles and immersed in 1 mL of RPMI-1640 medium supplemented with 10% FBS as a release medium (n=3 per group). The matrix was then incubated at 37° C. on an orbital shaker, and the release medium was completely removed for HPLC analysis and replaced with 1 mL of fresh medium at predetermined time points.
Cytotoxicity of CFZ
B16F10 (derived from murine melanoma; ATCC, Manassas, Va., USA) or HCC-1937 (derived from human triple negative breast cancer; ATCC, Manassas, Va., USA) were grown in RPMI 1640 or DMEM medium, respectively, supplemented with 10% FBS and penicillin (100 IU/mL) and streptomycin (100 μg/mL). Cells were seeded in a 96 well plate at a density of 4,000 cells per well. After 24 h incubation, the culture medium was replaced with fresh medium containing CFZ (as a stock solution in DMSO), CFZ-pTA or CFZ-pTA-alb. The cells were subject to either continuous treatment (in drug-containing media at concentrations equivalent to CFZ 10-400 nM for 72 h) or pulse treatment (in drug-containing media at concentrations equivalent to CFZ 50-800 nM for 2-24 h, followed by washout and additional incubation in drug-free medium up to 72 h). Cell viability was estimated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Culture medium was replaced with 100 μL of fresh medium and 15 μL of MTT solution (5 mg/mL), and cells were incubated for 4 h. The stop/solubilizing solution was added to dissolve formazan crystals, and the absorbance was read at the wavelength of 562 nm by the SpectraMax M3 microplate reader. The cell viability was calculated by normalizing the measured absorbance to that of control cells that did not receive treatments.
The cytotoxicity of CFZ was also measured on splenocytes. The spleen was freshly harvested from a male C57BL/6 mouse, cut into pieces, and filtered through 70 μm and 40 μm cell strainers sequentially to obtain a single cell suspension. The cell suspension was incubated with 1 mL ammonium-chloride-potassium (ACK) lysis buffer for 1 min to remove red blood cells. The splenocytes, suspended in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 IU/mL) and streptomycin (100 μg/mL), were seeded at 200,000 cells per well in a 96 well plate and treated with CFZ solutions for 24 h or 2.4 h with additional 21.6 h incubation in drug-free medium. Cell viability was measured as described above.
In Vitro Metabolic Stability of CFZ
Metabolic stability of CFZ in different formulations (CFZ-CD, CFZ dissolved in 10 mM citrate buffer (pH 3.1) with 20% (w/v) 2-hydroxypropyl-β-cyclodextrin)69, CFZ-pTA and CFZ-pTA-alb) was measured in whole blood. The blood was collected from C57BL/6 male mice and stored in a heparinized tube on ice. CFZ formulations were added to the cold blood at a final concentration of 67 μM (n=3 per group) and incubated at 37° C. for 30 min. The blood sample was mixed with 200 μL of 5% Triton-X 100 and vortexed for 10 min. To this mixture, 600 μL of acetonitrile was added, bath-sonicated for 10 min, and centrifuged at 4500 rcf for 30 min to remove precipitated proteins. The supernatant was analyzed by HPLC68. A calibration standard curve was prepared with CFZ doped in blood at final concentrations of 4-278 μM and treated in the same way as the samples immediately.
Protein-Capturing Ability of CFZ-pTA-Alb
B16F10 cells were treated with 10 μM CFZ solution in serum-free RPMI 1640 medium (supplemented with penicillin 100 IU/mL and streptomycin 100 μg/mL) for 48 h. The cells were centrifuged at 300 rcf for 5 min to separate soluble protein antigens and DAMPs released from dying cells. CFZ-pTA-alb was incubated in the collected supernatant at a NP concentration of 1 mg/mL for 2 h at 37° C. with rotation. The particles were centrifuged at 16,100 rcf for 20 min at 4° C., washed with water twice, resuspended in water at a concentration of 4 mg/mL and analyzed by SDS-PAGE. Protein bands were excised and analyzed by LC-MS/MS according to the method described previously70.
CFZ-pTA-Alb—Dendritic Cell Interaction
Bone marrow cells were collected from healthy male C57BL/6 as reported previously71. Mice at the age of 6-8 weeks were sacrificed using CO2 asphyxiation. Using a syringe, the cavities of femur bones were flushed with RPMI 1640 medium containing penicillin/streptomycin. The collected bone marrow was pipetted several times and passed through a 40 μm cell strainer to obtain single cell suspension. The cells were collected by centrifugation at 500 rcf for 8 min, treated with ACK buffer, rinsed, and suspended in IMDM medium supplemented with 10% FBS, penicillin/streptomycin, 20 ng/mL GM-CSF, and 10 mM β-mercaptoethanol at a density of 2×106 cells per 10 mL to differentiate into dendritic cells (DCs). Additional medium was supplemented three days later, and the floating and loosely adherent cells were collected by centrifugation on day 6. The DC phenotype was confirmed by CD11c staining. In a 6-well plate, 105 DCs were cocultured for 4 or 24 h with 4×105 DiI-labeled B16F10 cells (denoted as *B16F10 cells), which had been left untreated or treated with blank pTA-alb, CFZ solution or CFZ-pTA-alb (at 10 μM CFZ equivalent) for 24 h and rinsed once. The co-cultured cells were collected, resuspended in staining buffer, incubated with Fc block for 5 min, and stained with APC-labeled anti-mouse CD11c antibody to determine the extent of phagocytic uptake of *B16F10 cells by dendritic cells. Separately, the co-cultured cells (with unlabeled B16F10 cells) were stained with APC-labeled anti-mouse CD11c, FITC-labeled anti-mouse CD40 and PE-labeled anti-mouse CD86 antibodies to determine the activation status of DCs. The stained cells were analyzed by flow cytometry (Accuri C6, BD Biosciences, San Jose, Calif., USA). Fluorescence-minus-one (FMO) controls were used for compensation and gating. The extent of phagocytosis was expressed as the percentage of DiI+CD11c+ cells per total CD11c+ cells. The DC activation was assessed by CD11c+CD40+ or CD11c+CD86+ per total CD11c+ cells.
Local Administration of CFZ-pTA-Alb
Antitumor Effect in B16F10@C57BL/6 Mice
All animal procedures were approved by Purdue Animal Care and Use Committee, in conformity with the NIH guidelines for the care and use of laboratory animals. Male C57BL/6 mice (5-6 week old, ˜20 g) were purchased from Envigo (Indianapolis, Ind., USA) and acclimatized for 1 week prior to tumor inoculation. One million B16F10 melanoma cells were inoculated subcutaneously to each C57BL/6 mouse in the upper flank of right hind limb. When tumors reached 50-100 mm3 on the average, mice were randomly assigned to 3 groups and received an intratumoral injection of 50 μL of PBS, CFZ-CD or CFZ-pTA-alb (1.2 μg CFZ equivalent). The tumor volume and body weight were measured every day. The tumor length (L) and width (W) were measured using a digital caliper and the volume (V) was calculated as: V=(L×W2)/2). The specific growth rate of the tumor was calculated as Δ log V/Δt (t: time in days)38. The mice were sacrificed at 7 days after the treatment.
B16F10 tumors were collected, cut into small pieces, mechanically disrupted, and filtered through 70 μm and 40 μm cell strainers sequentially to obtain a single cell suspension. The cell suspension was incubated with 3 mL of ACK lysis buffer for 1 min to remove red blood cells. The cells were rinsed with PBS, resuspended in cell staining buffer at a density of 106 cells per 100 μL, incubated with Fc block for 5 min, and stained with PE-labeled anti-mouse CD8a antibody for 1 h at 4° C. The stained cells were analyzed by flow cytometry (Accuri C6, BD Biosciences, San Jose, Calif.).
CFZ Retention in B16F10 Tumor
Fifty microliters of CFZ-CD or CFZ-pTA-alb (1.2 μg CFZ equivalent) were injected intratumorally into 100 mm3 B16F10 tumors inoculated in C57BL/6 mice. Two hours later the mice were sacrificed, blood was collected via cardiac puncture and put in a heparinized tube. Tumors were harvested, washed with PBS and homogenized in cold PBS (pH 7.4) at 100 mg tissue per 400 μL with an Omni Tissue Master 125 homogenizer. A 100 μL of the tumor homogenate was vortex-mixed with 200 μL of 5% Triton-X 100 for 2 min and then mixed with tert-Butyl Methyl Ether (TBME) (1800 μL) containing carfilzomib-d8 (Cayman Chemical, Ann Arbor, Mich., USA) as an internal standard (250 ng/mL) by rotation for 40 min to extract CFZ. The mixture was centrifuged at 4,500 rcf for 15 min, and the organic layer was separated, transferred to a glass vial, and dried in vacuum. The dried sample was dissolved in 100 μL of DMSO, filtered through 0.45 μm syringe filter and analyzed by LC-MS/MS (Agilent triple quadrupole mass spectrometer coupled with the Agilent 1200 Rapid Resolution HPLC, operated in a positive ion mode)72. A calibration standard curve was prepared with tumor homogenates doped with CFZ at a concentration of 0.99-500 ng/mL and processed in the same way as the samples. For quantification of CFZ absorbed to the system, 5 μL of plasma was mixed with 40 μL of acetonitrile, containing 250 ng/mL carfilzomib-d8 as an internal standard, and made up to 20 μL with water. The mixture was vortexed for 5 min then centrifuged at 13,000 g for 10 min. The supernatant was analyzed by LC-MS/MS72. A calibration standard curve was prepared with plasma doped with CFZ at a concentration of 3.26 to 416.7 ng/mL and processed in the same way as the samples.
Effects on Remote B16F10 Tumors in C57BL/6 Mice or Athymic Nude Mice
C57BL/6 mice were inoculated with 106 B16F10 cells subcutaneously in the upper flank of right hind limb. Seven days later, the mice were inoculated with 3×105 B16F10 cells on the contralateral side. When the first tumor on the right side reached 50 mm3 on the average, mice were randomly assigned to 3 groups. Each mouse received an intratumoral injection of 20 μL of PBS, CFZ-CD or CFZ-pTA-alb (60 μg CFZ equivalent) in the first tumor. The treated tumor and the second inoculation site on the left were monitored every other day. Animals losing >20% body weight loss or with tumors greater than 10% of the body weight were humanely sacrificed prior to the end of the study. The same procedure was repeated with athymic nude (Foxn1nu) mice.
Anti-Tumor Effect and Tumor Immunophenotyping in CT26@Balb/c Mice
105 CT26 mouse colon carcinoma cells (ATCC, Manassas, Va., USA) were inoculated subcutaneously in the mammary fat pad of each female Balb/c mouse. When tumors reached 50-100 mm3 on the average, mice were randomly assigned to 3 groups and received an intratumoral injection of 50 μL of PBS, CFZ-CD or CFZ-pTA-alb (1.2 μg CFZ equivalent). The tumor volume and body weight were measured every day for 7 days. A separate group of CT26 tumor-bearing Balb/c mice (3×105 CT26 cells inoculated subcutaneously to the upper flank of the right hind limb) were treated with PBS, CFZ-CD or CFZ-pTA-alb (60 μg CFZ equivalent) and sacrificed at 6 days post treatment. Tumors were digested in 10 mL of complete RPMI 1640 containing 1 mg/mL collagenase I for 1-2 h, passed through a 70 μm cell strainer to remove large debris, and then pelleted. The samples were treated with 3 mL ACK buffer for 2 min at room temperature, rinsed with 10 volumes of PBS, passed through a 70 μm cell strainer and incubated with Zombie Violet and Fc block at room temperature in dark for 10 min. BV605-labeled anti-mouse CD45 and APC-labeled anti-mouse CD8a antibodies were added to the samples and incubated for 30 min at 4° C. The stained cells were rinsed with PBS and resuspended in 1 mL of 10% neutral buffered formalin for flow cytometry analysis (BD LSRFortessa, San Jose, Calif., USA).
Tumor-Specific Immunity in CT26@Balb/c Mice
Female Balb/c mice were inoculated with 300,000 CT26 cells subcutaneously in the upper flank of right hind limb, followed by 100,000 CT26 cells on the left hind limb 7 days later. When tumors on the right limb grew to ˜50 mm3, the mice were randomly assigned to 3 groups and treated with an intratumoral injection of 20 μL of PBS, CFZ-CD or CFZ-pTA-alb (60 μg CFZ equivalent). The tumor size was monitored for 22 days. At sacrifice, the spleens were collected to evaluate the response to AH1 peptide, a CT26-related peptide antigen. Splenocytes were prepared as described in [00109], plated at a density of 3×105 cells per well in a 96-well plate, and stimulated with 10 μg/mL of AH1 peptide in the presence of 20 ng/mL of GM-CSF. After 72 h incubation, the cells were centrifuged at 300 rcf for 5 min to separate a supernatant. The concentration of interferon-γ (IFN-γ) in each supernatant was measured by ELISA (Invitrogen, Carlsbad, Calif., USA). The IFN-γ production from the AH1 peptide-challenged splenocytes was compared with that of the non-challenged cells collected from the same mouse.
Systemic Administration of CFZ-pTA-Alb
Antitumor Effect in B16F10@C57BL/6 Mice
One million B16F10 melanoma cells were inoculated subcutaneously in the upper flank of the right hind limb of a male C57BL/6 mouse (5-6 week old). When the tumors reached ˜100 mm3, mice were randomly assigned to 3 groups (n=5 for PBS, n=6 for CFZ-CD and n=9 for CFZ-pTA-alb, at a dose equivalent to CFZ 6 mg/kg). One hundred microliters of each treatment were injected via tail vein two consecutive days a week for 2 weeks. The tumor volume and body weight were measured every day.
Blood Chemistry and Tissue Levels of CFZ at 48 h Post-Treatment
Separate groups of C57BL/6 mice with 100 mm3 B16F10 tumors were treated with PBS, CFZ-CD, CFZ-pTA-alb or blank pTA-alb at a dose equivalent to CFZ 6 mg/kg via tail vein injection on two consecutive days. The mice were sacrificed at 48 h after the second injection and bled via terminal cardiac puncture. The serum was separated for analysis of blood chemistry.
The tumor and major organs were harvested, washed with PBS, snap-frozen in liquid nitrogen and kept at −80° C. until analysis. For quantification of CFZ, tissues were homogenized in cold PBS (pH 7.4) at 100 mg tissue per 400 μL with an Omni Tissue Master 125 homogenizer. A 100 μL of each tissue homogenate was vortex-mixed with 200 μL of 5% Triton-X 100 for 2 min and then mixed with TBME (1800 μL) containing carfilzomib-d8 as an internal standard (250 ng/mL) by rotation for 40 min to extract CFZ. The organic layer was separated by centrifugation at 4,500 ref for 15 min, transferred to glass vials and evaporated under vacuum. The dried films were reconstituted in 100 μL acetonitrile and analyzed by LC-MS/MS (Agilent triple quadrupole mass spectrometer coupled with the Agilent 1200 Rapid Resolution HPLC, operated in a positive ion mode)72. A calibration standard curve was prepared with tissue homogenates doped with CFZ at a concentration of 0.99-500 ng/mL and processed in the same way as the samples.
Statistical Analysis
Statistical analysis was performed with GraphPad Prism 7 (La Jolla, Calif., USA). All in vitro data were analyzed by unpaired two-way t-test, one-way or two-way ANOVA test to determine the difference of means among groups, followed by the recommended multiple comparisons tests. In vivo data were analyzed by one-way ANOVA, followed by Tukey's multiple comparisons test unless specified otherwise. The comparison of survival curves was conducted with the Log-rank (Mantel-Cox) test. The p-value was indicated for each comparison. A value of p<0.005 was considered statistically significant, and p-values between 0.05 and 0.005 were referred as suggestive, according to the recommendation of the American Statistical Association73.
Those skilled in the art will recognize that numerous modifications can be made to the specific implementations described above. The implementations should not be limited to the particular limitations described. Other implementations may be possible.
While the inventions have been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only certain embodiments have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected.
It is intended that that the scope of the present methods and compositions be defined by the following claims. However, it must be understood that this disclosure may be practiced otherwise than is specifically explained and illustrated without departing from its spirit or scope. It should be understood by those skilled in the art that various alternatives to the embodiments described herein may be employed in practicing the claims without departing from the spirit and scope as defined in the following claims.
This present patent application relates to and claims the priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Ser. No. 62/899,978 filed on Sep. 13, 2019, the content of which is hereby incorporated by reference in its entirety into the present disclosure.
This invention was made with government support under a grant CA232419, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/50284 | 9/11/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62899978 | Sep 2019 | US |
