Autologous tumor lysate-loaded dendritic cell vaccine for treatment of liver cancer
10238723 ยท 2019-03-26
Assignee
Inventors
Cpc classification
C12N2501/22
CHEMISTRY; METALLURGY
A61K35/15
HUMAN NECESSITIES
A61K2039/5154
HUMAN NECESSITIES
A61K39/464499
HUMAN NECESSITIES
C12N5/0639
CHEMISTRY; METALLURGY
A61K2039/545
HUMAN NECESSITIES
International classification
A61K35/15
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
Abstract
The present disclosure provides compositions comprising mature dendritic cells loaded with autologous tumor cell lysates for the treatment of liver cancers, such as hepatocellular carcinoma. Hepatocellular carcinoma (HCC) is the fifth leading cancer and third leading cause of cancer-related mortality worldwide. Surgical resection and liver transplantation remain the mainstay of effective therapy for patients with early disease. However, a prevalent problem with HCC is the high likelihood of initial diagnosis at an advanced stage.
Claims
1. An immunogenic composition consisting of dendritic cells (DCs) (i) autologous to a subject with a liver tumor, (ii) matured in the presence of lipopolysaccharide as the only maturation agent, (iii) loaded with hepatocellular (HCC) tumor cell lysate prepared by performing repeated freeze-thaw cycles of liver tumor cells obtained from the subject, and (iv) a pharmaceutically acceptable carrier or excipient; and wherein the DCs selectively reduce T regulatory cells (Tregs) in the liver tumor when administered to the subject.
2. The composition of claim 1, wherein the immunogenic composition is a vaccine.
3. The composition of claim 1, wherein the Tregs are FoxP3+.
4. The composition of claim 1, wherein the DCs are prepared from peripheral blood mononuclear cells (PBMCs).
5. The composition of claim 1, wherein the lipopolysaccharide is present at a concentration of 1 g/ml.
6. The composition of claim 1, wherein the composition consists of 110.sup.7 DCs.
7. The composition of claim 1, wherein the subject is human.
8. The composition of claim 1, wherein the HCC tumor cells are obtained from the subject by surgical resection, percutaneous needle biopsy, or laparoscopic tumor biopsy/excision.
9. The composition of claim 4, wherein the PBMCs are cultured in the presence of granulocyte-macrophage colony-stimulating factor.
10. The composition of claim 4, wherein the PBMCs are cultured in the presence of IL-4.
Description
DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION
(19) The following descriptions are provided for clarity and illustrative purposes only, and are not intended to limit the scope of the invention.
(20) As described above, there is a need in the art for improved compositions and methods for treating liver cancer. The present disclosure describes such compositions and methods, in part by providing a dendritic cell (DC)-based immunogenic composition that reduces (e.g., selectively reduces) the number of T regulatory cells (Tregs) in liver tumors when administered to a subject with liver cancer.
(21) In particular, as described in the Examples below, it is presently discovered that the gene FoxP3 is upregulated in human tissue obtained from hepatitis B virus (HBV)-associated HCC tumors and that FoxP3 expression in the tumors was associated with mortality. Further, in a mouse model of HCC, it is discovered that the DC-based immunogenic compositions described herein selectively reduce the number of Tregs in liver tumors and are highly effective for the treatment of liver cancers, such as HCC.
(22) Isolation of PBMCs and Preparation of Dendritic Cell Vaccine
(23) The present disclosure provides improved DC-based immunogenic compositions (e.g., vaccines) for treating liver cancer in a subject, for preventing the recurrence of liver cancer in a subject, and for reducing the frequency of Tregs in the liver of a subject with a liver tumor. In certain embodiments, methods of preparing a DC-based immunogenic composition for the treatment of a liver tumor in a subject are provided. The methods can include (e.g., comprise, consist essentially of, consist of): (a) loading DCs with a tumor cell lysate prepared from autologous tumor cells obtained from the subject; and (b) incubating the DCs in the presence of LPS; wherein, the immunogenic composition selectively reduces Tregs in the liver tumor when administered to the subject. Preferably, the DCs are autologous to the subject to be treated with the DC-based immunogenic composition.
(24) For the preparation of a DC-based immunogenic composition (e.g., vaccine) for administration to humans (though the method can also be applied and/or adapted to other mammals), DCs can be prepared from peripheral blood mononuclear cells (PBMCs). Preferably the PBMCs are obtained from the subject to be treated with the composition.
(25) PBMCs can be isolated according to any suitable method known in the art. Standard operating procedures for the collection of human PBMC are known in the art and described, e.g., in Phuphanich S, et al. Cancer Immunol Immunother. 2013 January; 62(1):125-3. An exemplary method for the isolation of PBMCs is also described, e.g., in U.S. Patent Application Publication No. 2007/0281352 by Dietz et al. See, also, e.g., Fuss et al. Unit 7.1. Guidelines for the Collection of Mononuclear Cells (MNC) Products for the Elutra Cell Separator System Monocyte Enrichment Protocol Current Protocol in Immunology (2009). Typically, blood is obtained from a subject, e.g., by collecting whole blood from the subject with a syringe into 100 IU preservative free heparin for each 10 ml of blood on a COBE spectrum apheresis system, and PBMCs are then isolated from the blood using a Ficoll gradient.
(26) Preferably, the PBMC collection collects a product that meets the following specifications:
(27) White Blood Cell (WBC) content: >510.sup.9 to 3010.sup.9;
(28) Monocyte content: >110.sup.9;
(29) Granulocyte content: <3%;
(30) Red Blood Cell (RBC) content: <7.5 mL.
(31) For the growth and culture of DCs from PBMCs, a variety of growth and culture media can be used, and the composition of such media can be readily determined by a person having ordinary skill in the art. Suitable growth media are solutions containing nutrients or metabolic additives, and include those that are serum-depleted or serum-based. Representative examples of growth media are RPMI, TC 199, Iscoves modified Dulbecco's medium [Iscove, et al., (1978) J. Exp. Med. 147:923], DMEM, Fischer's, alpha medium, NCTC, F-10, Leibovitz's L-15, MEM and McCoy's. Particular examples of nutrients that will be readily apparent to the skilled artisan include, serum albumin, transferrin, lipids, cholesterol, a reducing agent such as 2-mercaptoethanol or monothioglycerol, pyruvate, butyrate, and a glucocorticoid such as hydrocortisone 2-hemisuccinate. More particularly, the standard media includes an energy source, vitamins or other cell-supporting organic compounds, a buffer such as HEPES or Tris, which acts to stabilize the pH of the media, and various inorganic salts. Particular reference is made to PCT Publication No. WO 95/00632, wherein a variety of serum-free cellular growth media is described.
(32) In a preferred embodiment, DCs are derived from PBMCs according to the following 8-day procedure: On Day 0, the PBMCs are thawed and plated in medium containing 1% autologous plasma onto tissue culture flasks to select for monocytes, which adhere to the plastic surface after a one hour incubation step. Lymphocytes are washed off the flasks, and the monocytes (adherent CD14+ cells) are then cultured for 5 days in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) (with or without interleukin (IL)-4). During this period, the monocytes differentiate into immature DCs (non-adherent, CD14 CD83 cells). On Day 5, the immature DCs are harvested, washed, and transferred to 6-well plates. On Day 6, the DCs are treated with autologous tumor cell lysate (at a ratio of 5 DCs to 1 tumor cell) generated by surgical resection, percutaneous needle biopsy, or laparoscopic tumor biopsy/excision and 5 cycles of freeze/thaw cycles. One hour later, DCs are stimulated to mature by incubating with a 1 g/ml LPS for 24 hours.
(33) GM-CSF (with or without IL-4) induces the differentiation of monocytes (large CD14+ cells) into immature DCs (which are large, non-adherent or loosely adherent, CD14 CD83 cells). GM-CSF is required to keep the cells alive in culture and to induce DC differentiation (whereas IL-4 may prevent monocyte differentiation into macrophages). Macrophages are easily identified as large, adherent cells with a fried egg appearance. Typically, 20 ng/ml GM-CSF is sufficient to induce monocyte differentiation into immature DCs (with or without 200 IU/ml IL-4). Increasing the concentration of these cytokines has no adverse effects, but tends not to improve the yield of immature DCs on Day 5. The skilled artisan will appreciate that the amount of GM-CSF (and/or IL-4) can be adjusted according to the specific culture protocol being used. GM-CSF is commercially available, e.g., from Schering-Plough, Kenilworth, N.J.), and Bayer Healthcare Pharmaceuticals. IL-4 is commercially available, e.g., from Schering Plough and Cellgenix.
(34) While not intending to be bound by any one particular theory or mechanism of action, maturation of DCs is believed to be required because injection of antigen-loaded immature DCs into patients can result in ineffective immunization or even immune tolerance. On the final day of culture (day 7), the mature DCs (CD14 CD83+ cells) are loaded with tumor cell lysates and then washed and frozen in aliquots in a controlled-rate freezer. Quality control (QC) testing is performed afterwards, usually the following day, on frozen control aliquots. If release criteria are met, frozen aliquots designated for injection may be thawed as needed and administered to the patient.
(35) Release criteria can include, e.g., a suitable physical appearance of the DCs. For example, by day 7 of the culture, approximately 80% of the cells in the culture should be large, non-adherent or loosely adherent cells with clearly visible cytoplasmic processes and/or veils. Most of the remaining cells will be lymphocytes (small round cells). There will be some cell debris and evidence of cell death, but this should not be a predominant feature. It is reasonable to expect a 5 to 10% yield of mature DCs (relative to the number of PBMCs plated) on day 7. In a preferred embodiment, the release criteria include >70% viable cells, negative results for all sterility and endotoxin tests, and >50% of the cells having the characteristics of mature DCs by flow cytometry (large, CD14 CD83+ cells).
(36) Procedures for performing flow cytometry for the detection of cell marker expression are well known in the art. Fluorescently labeled anti-CD83 and anti-CD14 antibodies for detecting marker expression on DCs are available, e.g., from Abcam (Cambridge, Mass.), Caltag Laboratories (Burlingame, Calif.) and/or PharMingen (San Diego, Calif.). Other markers that can be detected using labeled antibodies include, e.g., CD45, CD86, CD40 and HLA-DR. For flow cytometry, by way of non-limiting example, cells can be washed with FACS buffer (PBS+2% fetal bovine serum, 0.1% sodium azide) and counted. One million cells in 100 l are then added to culture tubes containing 1 g of each labeled antibody. Cells are then incubated on ice for 40 min, washed two times with FACS buffer, and then suspended in PBS+1% paraformaldehyde and stored at 4 C. before FACS analysis.
(37) It is presently discovered that lipopolysaccharide (LPS) (available, e.g., from Sigma-Aldrich) is particularly effective for maturing DCs for preparing the immunogenic compositions described herein. LPS is a ligand for Toll-like receptor (TLR)-4, which is expressed on mammalian DCs, including human DCs. Activation of signal transduction pathways by signaling through TLRs such as TLR4 leads to the induction of various genes including inflammatory cytokines, chemokines, major histocompatability complex, and upregulation of costimulatory molecules on DCs (i.e., leads to DC maturation). In certain embodiments, DCs are matured in the presence of 1 g/ml LPS. However, it is to be appreciated that other concentrations of LPS may also be used to achieve comparable results (e.g., maturation of DCs, as determined, e.g., by the expression of CD83 or other maturation marker(s)). Such LPS concentrations include, without limitation, 0.001 g/ml, 0.005 g/ml, 0.01 g/ml, 0.05 g/ml, 0.1 g/ml, 0.5 g/ml, 1 g/ml, 1.5 g/ml, 2 g/ml, 2.5 g/ml, 3 g/ml, 3.5 g/ml, 4 g/ml, 4.5 g/ml, 5 g/ml, 10 g/ml, 15 g/ml, 20 g/ml, etc.
(38) While LPS is the preferred agent for inducing DC maturation to prepare the immunogenic DC-based compositions described herein, the skilled artisan will appreciate that it is possible to use other maturation agents. Non-limiting examples include, e.g., other TLR-4 ligands (e.g., heat shock proteins), as well as ligands for other TLRs.
(39) In mammalian organisms, TLRs have been shown to recognize the bacterial products LPS, lipoteichoic acid, peptidoglycan, lipoprotein, CpG-DNA, and flagellin, as well as the viral product double stranded RNA, and the yeast product zymosan. TLR2 can recognize bacterial lipoproteins, peptidoglycan, and lipoteichoic acids. TLR3 may recognize virus-derived double-stranded RNA. TLR4 is predominantly activated by lipopolysaccharide. TLR9 is required for response to unmethylated CpG DNA. TLR7 and TLR8 have been shown to recognize single stranded RNA molecules, and small synthetic antiviral molecules and TLR11 detects profilin-like protein (PLP). Furthermore, TLR5 detects bacterial flagellin. Synthetic TLR ligands, such as polyinosine-polycytidylic acid (poly(I:C)), a synthetic analog of dsRNA, is a TLR3 ligand, and is available, e.g., from InvivoGen (San Diego, Calif.).
(40) Human DCs have been shown to express many different TLRs, depending on their maturation stage (see Kokkinopoulos et al. Molecular Immunology 42 (2005) 957-968). Thus, many of the above-described TLR ligands and/or combinations thereof may be used to induce DC maturation. Suitable concentration of TLR ligands for stimulating DC maturation are known in the art and are readily determined by one of ordinary skill in the art. In one embodiment, DCs are matured using LPS in combination with at least one other TLR ligand, e.g., such as one described above.
(41) Typically, DCs are loaded with tumor cell lysate one hour prior to the addition of LPS to prepare the DC vaccine. In other embodiments, DCs are loaded with tumor cell lysate 0.5 hour, 1.5 hours, 2 hours, 2.5 hour, 3 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours 21 hours, 22 hours, 23 hours, 24 hours, or longer prior to the addition of LPS. In a particularly preferred embodiment, the tumor cell lysate is autologous to the subject to be treated with the DC vaccine. It is presently discovered that DCs loaded with whole tumor cell lysate (in lieu of, e.g., only specific tumor antigens) are highly effective for reducing the frequency of Tregs in liver tumors and for treating liver cancer.
(42) In other embodiments, DCs are loaded with tumor cell lysate one hour after the addition of LPS to prepare the DC vaccine. In other embodiments, DCs are loaded with tumor cell lysate 0.5 hour, 1.5 hours, 2 hours, 2.5 hour, 3 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours 21 hours, 22 hours, 23 hours, 24 hours, or longer, after the addition of LPS.
(43) In a preferred embodiment, tumor cell lysate is prepared by repeated freeze thaw cycles. For example fresh resected tumor tissues can be dissociated, digested in a protease mixture (e.g., containing HBSS, 2.5 U/ml hyaluronidase type V, 0.5 mg/ml collagenase type IV, and 0.05 mg/ml deoxyribonuclease type I) followed by filtration. After a washing step, the cells are frozen in liquid nitrogen. For thawing, the frozen cells are immersed in a 37 C. water bath for, e.g., 2 minutes (though longer or shorter times are possible). The thawed cells are then placed in liquid nitrogen to quickly freeze the cells again. This freeze/thaw cycle can be repeated at least once, twice, 3 times, 4 times, 5 times, 6 times, 7 times or more. In a preferred embodiment, the cells are frozen and thawed 5 times to prepare the tumor cell lysate. It is to be appreciate that any suitable method may be used for freeze/thawing the tumor cells.
(44) Tumor cell lysates may also be prepared from tumor tissue according to other suitable methods known in the art, e.g., using a suitable cell lysis buffer, such as, but not limited to, NP-40 or Triton-X, or using mechanical means by a (mechanical) tissue homogenizer. Methods for preparing cell lysates are known in the art.
(45) Typically, DCs are incubated in the presence of the tumor cell lysate (e.g. autologous tumor cell lysate) for a predetermined incubation time, e.g., 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours or longer. In a preferred embodiment, the incubation time is 1 to 2 hours.
(46) Typically, DCs are incubated with the tumor cell lysate at a ratio of: lysate of 1 tumor cell to 5 DCs (i.e., 1:5). The skilled artisan will appreciate that other ratios of tumor cell (lysate of tumor cell) to DC are possible, e.g., 1:1, 1:2, 1:3, 1:4, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, etc. The effective and/or desired ratio can be readily determined by one of ordinary skill in the art.
(47) While, in a preferred embodiment, DCs are first matured (e.g., with LPS), and then loaded with tumor cell lysate, in other embodiments of the presently disclosed compositions and methods, DCs are first loaded with tumor cell lysate and then matured (e.g., with LPS or another suitable agent).
(48) Compositions and Formulations
(49) While it is possible to use a composition disclosed herein (a composition comprising DCs matured with LPS and loaded with autologous tumor cell lysate) for therapy as is, it may be preferable to formulate the composition in a pharmaceutical formulation, e.g., in admixture with a suitable pharmaceutical excipient, diluent, or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Accordingly, in one aspect, the present invention provides a pharmaceutical composition or formulation comprising at least one active composition disclosed herein (e.g., the autologous DCs prepared as disclosed herein) in association with a pharmaceutically acceptable excipient, diluent, and/or carrier. The excipient, diluent and/or carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
(50) The compositions can be formulated for administration in any convenient way for use in human or veterinary medicine. For in vivo administration to humans, the compositions disclosed herein can be formulated according to known methods used to prepare pharmaceutically useful compositions. The DCs can be combined in admixture, either as the sole active material or with other known active materials, (e.g., one or more chemotherapeutic agents) with pharmaceutically suitable diluents (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/or carriers. Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Co.
(51) In certain aspects, the DC-based immunogenic compositions described herein may be administered as part of a combination therapy with other active agents. While liver tumors, such as HCC, are typically relatively insensitive to systemic chemotherapy, it can be advantageous to administer such chemotherapeutic agents or other cancer therapy in combination with the DC-based immunogenic compositions described herein. Thus, non-limiting examples of chemotherapeutic agents which may be administered in a combination therapy with the compositions described herein (either in the same composition or as a separate composition), include without limitation: taxanes such as taxol, taxotere or their analogues; alkylating agents such as cyclophosphamide, isosfamide, melphalan, hexamethylmelamine, thiotepa or dacarbazine; antimetabolites such as pyrimidine analogues, for instance 5-fluorouracil, cytarabine, capecitabine, and gemcitabine or its analogues such as 2-fluorodeoxycytidine; folic acid analogues such as methotrexate, idatrexate or trimetrexate; spindle poisons including vinca alkaloids such as vinblastine, vincristine, vinorelbine and vindesine, or their synthetic analogues such as navelbine, or estramustine and a taxoid; platinum compounds such as cisplatin; epipodophyllotoxins such as etoposide or teniposide; antibiotics such as daunorubicin, doxorubicin, bleomycin or mitomycin, enzymes such as L-asparaginase, topoisomerase inhibitors such as topotecan or pyridobenzoindole derivatives; and various agents such as procarbazine, mitoxantrone, and biological response modifiers or growth factor inhibitors such as interferons or interleukins. Other chemotherapeutic agents include, though are not limited to, a p38/JAK kinase inhibitor, e.g., SB203580; a phospatidyl inositol-3 kinase (PI3K) inhibitor, e.g., LY294002; a MAPK inhibitor, e.g. PD98059; a JAK inhibitor, e.g., AG490; preferred chemotherapeutics such as UCN-01, NCS, mitomycin C (MMC), NCS, and anisomycin; taxoids in addition to those describe above (e.g., as disclosed in U.S. Pat. Nos. 4,857,653; 4,814,470; 4,924,011, 5,290,957; 5,292,921; 5,438,072; 5,587,493; European Patent No. 0 253 738; and PCT Publication Nos. WO 91/17976, WO 93/00928, WO 93/00929, and WO 96/01815. In other embodiments, a cancer therapy can include but is not limited to administration of cytokines and growth factors such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, TNF-beta, and/or similar cytokines, or an antagonist of a tumor growth factor (e.g., TGF- and IL-10). Antiangiogenic agents, include, e.g., endostatin, angiostatin, TNP-470, Caplostatin (Stachi-Fainaro et al., Cancer Cell 7(3), 251 (2005)). Drugs that interfere with intracellular protein synthesis can also be used in the methods of the present invention; such drugs are known to those skilled in the art and include puromycin, cycloheximide, and ribonuclease.
(52) The effective amounts of compounds, compositions including pharmaceutical formulations of the present invention include doses that partially or completely achieve the desired therapeutic, prophylactic, and/or biological effect. In a specific embodiment, an effective amount of dendritic cells administered to a patient having a liver tumor is effective for reducing the size or inhibiting the growth of the liver tumor in the patient. The actual amount effective for a particular application depends on the condition being treated and the route of administration. The effective amount for use in humans can be determined from animal models. For example, a dose for humans can be formulated to achieve circulating and/or gastrointestinal concentrations that have been found to be effective in animals.
(53) When formulated in a pharmaceutical composition or formulation, a therapeutic compound disclosed herein can be admixed with a pharmaceutically acceptable carrier or excipient. The term carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
(54) In a preferred embodiment, the DCs are formulated for administration to humans in 0.9% NaCl saline. The formulation may also contain DMSO (e.g., 10% DMSO).
(55) Administration and Dosage
(56) The DCs may be administered in any suitable preparation. For in vivo administration to a patient, such as a mammal, e.g., a human patient, dendritic cells of the present invention may be administered by a parenteral route. The term parenteral includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques. Also included are intra-tumoral injection, and direct intra-organ injection (e.g., intra-splenic or intra-hepatic injection). For injection or infusion techniques, the DCs may be suspended in any suitable injection buffer, such as, but not limited to PBS or PBS containing anti-coagulants.
(57) The compositions described herein will typically contain an effective amount of DCs, alone, or in combination with an effective amount of any other active material, e.g., a chemotherapeutic agent. Effective amounts, or dosages, and desired concentrations of DCs contained in the compositions may vary depending upon many factors, including the intended use, patient's body weight and age, and route of administration.
(58) The dosage of the compositions and formulations disclosed herein may vary widely, depending upon the nature of the disease, the patient's medical history, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. The initial dose may be larger, followed by smaller maintenance doses. The dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi-weekly, biweekly, quarterly, etc., to maintain an effective dosage level. Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration can be performed according to art-accepted practices. In certain embodiments, a subject may be administered 1 dose, 2 doses, 3 doses, 4 doses, 5 doses, 6 doses or more of a DC-based composition described herein. An exemplary dosing schedule of the DC-based immunogenic compositions described herein is illustrated in
(59) Keeping the above description in mind, typical dosages (effective amounts) of DCs for administration to a patient may range from 110.sup.3 to 110.sup.8 cells per dose, although more or less cells may be used. Preferably the number of dendritic cells ranges from 110.sup.4 to 110.sup.8, more preferably from 110.sup.5 to 110.sup.8, still more preferably from 110.sup.6 to 110.sup.8, and most preferably from 110.sup.6 to 110.sup.7. In a preferred embodiment, the DCs are administered in an amount of 110.sup.7. However, other ranges are possible, depending on the patient's response to the treatment Moreover, an initial dose may be the same as, or lower or higher than subsequently administered doses of the DCs.
(60) The number and frequency of doses may also be determined based on the patient's response to administration of the composition, e.g., if the patient's symptoms improve and/or if the patient tolerates administration of the composition without adverse reaction; in some patients, a single dose is sufficient, other patients may receive a weekly, biweekly, or monthly administration of the DC-containing composition. The duration and frequency of treatment will depend upon the patient's response to treatment, i.e., if the patient's condition improves. For example, if the patient has a liver tumor, tumor size and/or rate of regression can be determined, e.g., by CT or MRI, or other routine methods in the art, and dosing and duration of treatment may be scaled based on the patient's individual response to treatment. Similar diagnostic tools can also be used to determine whether recurrence of a tumor has been prevented. By way of non-limiting example, response to treatment in patients with measurable disease, can be determined, e.g., by one or more of the following: the absence of tumor recurrence, absence of tumor metastasis, absence of tumor progression, increase or preservation of progression-free survival, increase or preservation of survival. Clinical response to therapy can be determined by axial imaging (e.g., computerized tomography (CT)), magnetic resonance imaging (MRI), or ultrasound, physiologic imaging (e.g., positron emission tomography (PET)), AFP levels, and/or physical examination.
(61) Uses of the DC-Based Immunogenic Compositions
(62) In certain aspects, the present disclosure provides methods for selectively reducing the number of Tregs in a liver tumor of a subject. In other aspects, the present disclosure describes the use of the DC-based immunogenic compositions described herein in a method and/or in a medicament for reducing the number of Tregs in a liver tumor of a subject. These methods and/or uses can include (e.g., comprises, consists essentially of, consists of): (a) loading DCs with a tumor cell lysate prepared from autologous tumor cells obtained from the subject; (b) incubating the DCs in the presence of LPS; and (c) following steps (a) and (b), administering the DCs to the subject, thereby selectively reducing Tregs in the liver tumor.
(63) Tregs are a component of the immune system that suppress immune responses of other cells. Tregs as described herein express CD4 and Foxp3 (CD4+Foxp3+ regulatory T cells). FOXP3 is an excellent marker for Tregs, and can be detected at the mRNA and/or protein level. The nucleic acid sequence for human Foxp3 mRNA is known and has GenBank Accession No. NM_014009 (SEQ ID NO: 1). The amino acid sequence for human FOXP3 is known and has GenBank Accession No. ABQ15210 (SEQ ID NO: 2). Exemplary primer sequences that can be used to detect Foxp3 mRNA expression are forward 5-AGAAGCAGCGTCAGTACCCCT-3 (SEQ ID NO: 3) and reverse: 5-CTGCACGGGACTCAAGAGAC-3(SEQ ID NO: 4). Antibodies that can be used for the detection of Tregs (e.g., CD4, FOXP3) by flow cytometry are commercially available, e.g., from Abcam.
(64) The compositions and methods described herein may reduce the frequency of Tregs (e.g., absolute number of Tregs or percent relative to total number of tumor infiltrating lymphocytes) in the liver tumor of a subject treated with the composition by at least 0.5-fold, at least 1.5-fold, at least 2-fold, at least 2.5 fold, at least 3-fold, at least 3.5 fold, at least 4-fold, at least 5-fold, or more. In other embodiments, the compositions and methods described herein may reduce the frequency of Tregs in the liver tumor of a subject treated with the composition by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or greater
(65) In other aspects, the present disclosure provides a method of treating a liver tumor in a subject. In other aspects, the present disclosure describes the use of the DC-based immunogenic compositions described herein in a method and/or in a medicament for treating a liver tumor in a subject. These methods and/or uses can include (e.g., comprise, consist essentially of, consist of): (a) loading DCs with a tumor cell lysate prepared from autologous tumor cells obtained from the subject; (b) incubating the DCs in the presence of LPS; and (c) following steps (a) and (b), administering the DCs to the subject, wherein the administration of the DCs selectively reduces Tregs in the liver tumor. In some aspects, these method and/or uses include: (a) loading DCs autologous to the subject with a tumor cell lysate prepared by repeated freeze-thaw cycles of tumor cells obtained from the subject to obtain tumor-cell-lysate-loaded DCs; (b) incubating the DCs in the presence of LPS to obtain mature DCs; and, (c) intravenously administering the mature, tumor-cell-lysate-loaded DCs to the subject; wherein the administration of the DCs selectively reduces Tregs in the subject's liver tumor.
(66) In other aspects, the present disclosure provides a method for preventing the recurrence of liver cancer in a subject. These methods and/or uses can include (e.g., comprise, consist essentially of, consist of): (a) loading DCs with a tumor cell lysate prepared from autologous tumor cells obtained from the subject; (b) incubating the DCs in the presence of LPS; and (c) following steps (a) and (b), administering the DCs to the subject, wherein the administration of the DCs selectively reduces Tregs in the liver tumor. In some aspects, these method and/or uses include: (a) loading DCs autologous to the subject with a tumor cell lysate prepared by repeated freeze-thaw cycles of tumor cells obtained from the subject to obtain tumor-cell-lysate-loaded DCs; (b) incubating the DCs in the presence of LPS to obtain mature DCs; and, (c) intravenously administering the mature, tumor-cell-lysate-loaded DCs to the subject; wherein the administration of the DCs selectively reduces Tregs in the subject's liver tumor.
(67) The presently disclosed methods include using autologous tumor cell lysate to prepare the DC-based immunogenic compositions. Thus, for preventing recurrence of liver cancer in a patient who does not have a liver tumor, but previously was diagnosed with a liver tumor (and treated such that the liver tumor was no longer detectable), a patient's tumor cells obtained from the patient before treatment of the liver tumor can be used to prepare the DC vaccine. The DC vaccine may then be administered to the patient to prevent recurrence of the liver tumor.
(68) Liver cancers that may be treated using the compositions and methods disclosed herein include but are not limited to hepatocellular carcinoma (HCC) or hepatoma, cholangiocarcinoma or bile duct cancer (BDC), and other primary or secondary liver cancers. Cancers of other organs or tissues may also be treated using the compositions and methods disclosed herein, including but not limited to primary or secondary cancers of the aero-digestive tract, pharynx, esophagus, stomach, pancreas, small bowel, colon, rectum, anus, kidney, bladder, prostate, breast, ovary, uterus, bone, muscle, connective tissue, lungs, tracheal-bronchial tree, brain, ocular, skin, lymph nodes, and bone marrow.
(69) In some embodiments, a subject with liver cancer, who is to be treated with a DC-based immunogenic composition described herein, has undergone surgical resection of the liver tumor. In other embodiments, the subject has undergone percutaneous needle biopsy, or laparoscopic tumor biopsy/excision. The patient may have undergone any suitable procedure for obtaining a tumor tissue sample for the preparation of autologous tumor cell lysate according to the methods described herein.
(70) In some aspects, the DC-based immunogenic compositions described herein can be administered as a combination therapy. For example, a subject may either before, simultaneously, or after treatment with a DC-based immunogenic composition described herein, undergo one or more procedures or therapies, including, e.g., surgical resection, liver transplantation, cryosurgery, hepatic artery chemoembolization, percutaneous ethanol, radiofrequency ablation (RFA) (surgical and percutaneous), and/or cisplatin gel injection. As described above, in certain embodiments the DC-based immunogenic compositions may also be administered as a combination therapy with a chemotherapeutic agent.
(71) For example, surgery can be performed in non-cirrhotic patients with hepatocellular carcinomaand in cirrhotic patients with well-preserved synthetic functions. However, only 20% of patients are potentially resectable at the time of presentation. In noncirrhotic patients, surgical mortality is less than 3% in experienced hands, but increases to 8% in patients with cirrhosis. Not all patients are eligible for liver resection. Resection is not indicated when: 1) the tumor has spread to other parts of the liver or the body, 2) the size or location of the tumor (near major blood vessels) precludes it from being safely removed without compromising function of the remainder of the liver, 3) the associated cirrhosis or disease limits the ability to safely operate upon or remove part of the liver, and 4) other medical conditions make surgery unsafe.
(72) RFA makes use of a heating probe to destroy tumors within the liver. A thin probe is placed within the tumor, typically under ultrasound guidance. After deploying the tip array, an electrical current is applied, generating heat (80-100 C.) that destroys the tumor. RFA is generally indicated for small tumors within the liver and can be applied with minimal side effects. The advantage of this technique is that it can be used either in the operating room with an open or laparoscopic approach, or directly through the skin (percutaneous approach). As with cryotherapy, RFA can be used in conjunction with liver resection. Some of the tumor may be surgically removed, while remaining disease is treated with RFA.
(73) Hepatic artery chemoradiation is a commonly performed procedure in the treatment of unresectable liver tumors (i.e., those that are inoperable). Most hepatic tumors are supplied by the hepatic arterial system, as opposed to normal liver tissue, in which most of the blood supply comes from the portal venous system. Chemoembolization has several theoretical advantages over intravenous pump infusion therapy because it delivers highly concentrated drugs to the tumor itself and arrests blood flow, the latter prolonging contact time within the tumor. This technique deprives the tumor of its oxygen supply while achieving a drug concentration in the tumor 10-25 times greater than that which can be achieved by infusion alone. In addition, the dwell time for the drug is markedly prolonged, with measurable drug levels present as long as a month after chemoembolization. Up to 85% of the administered drug is trapped in the liver, minimizing systemic toxicity.
(74) Percutaneous radio frequency ablation causes local tissue destruction by frictional heat. When the temperature surpasses 90 C., an immediate destructive effect occurs within the tumor.
(75) Percutaneous cisplatin gel infusion is a new and promising therapeutic option for the treatment of unresectable liver tumors. Cisplatin is an anti-neoplastic drug.
(76) In accordance with the present invention there may be employed conventional molecular biology, microbiology, protein expression and purification, antibody, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3.sup.rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, N.J.; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, N.J.; Nucleic Acid Hybridization, Hames & Higgins eds. (1985); Transcription And Translation, Hames & Higgins, eds. (1984); Animal Cell Culture Freshney, ed. (1986); Immobilized Cells And Enzymes, IRL Press (1986); Perbal, A Practical Guide To Molecular Cloning (1984); and Harlow and Lane. Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press: 1988).
EXAMPLES
(77) The present invention is described further below in working examples which are intended to further describe the invention without limiting the scope therein.
Example 1: Orthotopic Mouse Model of Hepatocellular Carcinoma
(78) This Example describes the generation of a murine model for HCC based on hepatic implantation of Hepa 1-6, an AFP-expressing murine HCC cell line, in immunocompetent mice.
(79) Materials and Methods:
(80) 1. Commercial Source of Hepa 1-6, Hepa 1-6-Luc and 3LL cells: Hepa 1-6 and 3LL cells were obtained from ATCC. Hepa 1-6-Luc cells were generated to stably express the firefly luciferase (Luc). The Luc gene was cloned into the pGL4.51 (luc2/CMV/Neo) vector. Hepa 1-6 cells were transfected, and Hepa 1-6 clones stably expressing luciferase (Hepa 1-6-Luc) were selected using G418 Sulfate. The expression of luciferase was confirmed using Luciferase Reporter Assay System (Promega).
(81) 2. Western blot: AFP was detected by Western blot using a goat anti-mouse AFP antibody (R&D Systems).
(82) 3. Bioluminescence imaging and analysis: In vivo bioluminescence imaging of luciferase-expressing Hepa 1-6 tumor was performed using a Xenogen IVIS-200 Series Imaging System (PerkinElmer, Waltham, Mass.). Imaging and measurement of bioluminescence signals were acquired and analyzed using Caliper Living Image, Version 2.50.1 (Caliper Life Sciences, Hopkinton, Mass.).
(83) 5. Administration of cyclophosphamide: Mice were treated intraperitoneally with or without cyclophosphamide (Bristol-Myers Squibb) at 200 mg/kg three days prior to tumor implantation.
(84) Results:
(85) An orthotopic mouse model of HCC was developed using Hepa 1-6-Luciferase (Hepa 1-6-Luc) expressing murine hepatoma cells. 10.sup.6 Hepa 1-6-Luc cells were injected in a volume of 20 l PBS into the right lobe of the liver of wild type C57L/J mice. Solid tumors developed in the mice within 3 weeks. Histology was performed and revealed that the solid tumors resembled poorly differentiated HCC in humans (
(86) Alpha-fetoprotein (AFP) is a serological marker of HCC in humans. It was thus determined whether Hepa 1-6 cells secrete murine AFP. AFP levels were measured by Western blot using the antibodies described above. As shown in
(87) The levels of murine AFP in vivo were also measured over the course of solid tumor development in the orthotopic mouse model. It was observed that AFP levels increased in circulation as Hepa 1-6 tumors progressed in vivo (
(88) Tumor development was monitored over time using bioluminescence imaging. As shown in
(89) The Hepa 1-6-Luc mice plasma AFP levels were plotted against the bioluminescence measurements. As shown in
(90) Survival studies indicated that all tumor-bearing animals succumbed to progressing liver tumor within 60 days with a median survival of 29 days.
(91) Treatment with high dose cyclophosphamide to compromise immune responses resulted in a much poorer survival for these animals with a median survival of 19.5 days (
Example 2: Preparation of Dendritic Cell-Based Vaccine for Hepatocellular Carcinoma
(92) This Example describes the preparation of the DC-based vaccine.
(93) Bone marrow-derived DCs were cultured with 20 ng/ml Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) for one week, pulsed with hepa 1-6 lysates generated from 5 freeze/thaw cycles, and matured with 1 g/ml LPS (Sigma-Aldrich) overnight.
(94) Hepa 1-6 lysates prepared by freeze/thaw cycles were used, since, whereas it was determined that live Hepa 1-6 cells inhibited LPS-induced maturation as measured by the expression of the maturation marker CD86 on the CD11c+DCs (
Example 3: Dendritic Cell-Based Vaccine Treats and Prevents Treats HCC Tumor Growth
(95) This example demonstrates that the DC-based HCC vaccine described in Example 2 can treat or prevent HCC.
(96) Hepa 1-6 cells were implanted in the liver, according to the method described in Example 1, and a week later, tumor bearing mice were randomly assigned to receive the DC vaccine described in Example 2 (210.sup.6 cells, injected intravenously) (DC vaccine (therapy), n=40) or vehicle (no DC vaccine, n=44) or mice were first administered the DC vaccine (210.sup.6 cells injected intravenously) before implanting the Hepa 1-6 cells (DC vaccine (prevention), n=16).
(97) Mice treated with the DC vaccine after Hepa 1-6 tumors were established (therapy) all survived for up to 60 days (
(98) The DC vaccine also inhibited tumor progression in mice bearing Hepa 1-6 tumors, as determined by bioluminescence imaging (described in Example 1, above) (
Example 4: Role for T Regulatory Cells in HCC
(99) This Example demonstrates that FoxP3, a marker expressed in T regulatory cells, is upregulated within the liver tumors of HCC patients, and that the DC vaccine prevents the accumulation of Foxp3.sup.+ Treg within Hepa 1-6 tumors in the HCC model described in Example 1.
(100) Materials and Methods:
(101) Human FoxP3: Human tumor samples and paired non-neoplastic liver were collected post-operatively. Surgical resection tissue specimens were used for RNA extraction using the RNeasy Mini Kit (Qiagen) according to manufacturers' protocols. cDNA was generated through reverse transcription, and Foxp3 mRNA level was quantify by real-time PCR using the following primer sequence: forward: 5-AGAAGCAGCGTCAGTACCCCT-3 (SEQ ID NO: 3), reverse: 5-CTGCACGGGACTCAAGAGAC-3 (SEQ ID NO: 4).
(102) FoxP3 Tregs in mouse model: Single cell suspension from tumor was prepared by digesting tumor tissue specimens with a mixture of enzymes containing collagenase type I (0.05 mg/ml), collagenase type IV (0.05 mg/ml), hyaluronidase (0.025 mg/ml) (all from Sigma), DNase I (0.01 mg/ml), and soybean trypsin inhibitor (0.2 trypsin soybean inhibitor units/ml) (Boehringer Mannheim) for 15 min at 37 C. Lymphocytes were enriched by layering the single cell suspension on the LSM lymphocyte separation medium (MP), followed by centrifugation at 400g at room temperature for 20 minutes. The lymphocyte layer was collected, washed and stained for cell surface CD4 and CD8 (eBioscience). Intracellular Foxp3 staining was performed according to manufacturers' instructions (eBioscience). Flow cytometry was performed using a FACScan caliber (Becton Dickenson).
(103) Results:
(104) The expression of FoxP3 in tumors of human hepatitis B (HBV)-associated HCC patients was determined and correlated with survival and compared with liver samples of non-neoplastic livers. As shown in
(105) Consistent with the observed decrease in Treg in the tumor, the secretion of TGF-, which is secreted by Tregs and other cell types within the tumor, was inhibited in tumors of mice that had been administered the DC vaccine (
(106) Furthermore, signal transducer and activator of transcription 3 (STAT3), is a transcription factor which is associated with cancer. Interestingly, STAT-3 activation was inhibited in the tumor following administration of the DC vaccine (
Prophetic Example 1: Clinical Trial
(107) This Examples describes a clinical trial with a DC-based vaccine in HCC patients.
(108) The targeted enrollment number for this phase I trial is nine HBV-HCC patients with safety being the primary end point, and immunology being the second end point.
(109) Patient tissues are collected following surgical resection and blood is drawn according to the schema below. Blood is processed for plasma, serum, and PBMC isolation. Tissue specimens following liver resection are submitted to the clinical pathology lab for histology, and a small portion of tissues from four different locations (tumor, tumor periphery, non-neoplastic liver adjacent to tumor, and distant non-neoplastic liver) are collected for research purposes. Upon collection, tissues are processed for DNA and RNA isolation. All processed blood and tissue samples as well as un-processed tissue pieces are stored in liquid nitrogen. All sample collection and procession procedures meet the NCI Office of Biorepositories and Biospecimen Research Best Practices. Histologic specimens are assessed for the degree of fibrosis using the Ishak method (0-6) (see, Goodman Z D. Grading and staging systems for inflammation and fibrosis in chronic liver diseases. Journal of Hepatology 2007; 47(4):598-607). Tumor tissues are processed to obtain autologous tumor cell lysates.
(110) For the preparation of autologous tumor cell lysates, fresh resected tumor tissues are dissociated into 5-mm.sup.3 pieces under sterile conditions. 1 gram of tumor tissues is digested in a minimum volume of 40 ml of a protease mixture that consists of HBSS, 2.5 U/ml hyaluronidase type V, 0.5 mg/ml collagenase type IV, and 0.05 mg/ml deoxyribonuclease type I. The digestion is performed at room temperature with constant stirring in a trypsinizing flask for 2 to 6 hours. The resulting cell suspension is filtered through a layer of number 70 nylon mesh. Cells are washed two times in HBSS and resuspended in 0.5 ml HBSS and counted. Cells are resuspended in 90% human AB serum plus 10% DMSO to reach a final concentration of 10.sup.6 cells/ml. 1 ml cell suspension is aliquotted into cryo vials, and the cells are frozen in liquid nitrogen. The frozen autologous tumors are thawed by immersing in a 37 C. water bath for 2 minutes. The thawed cells are then placed in liquid nitrogen to quickly freeze the cells again. This freeze/thaw cycle is carried out a total of 5 times to obtain the tumor cell lysate.
(111) After surgical resection, the patients are scheduled for unmobilized leukapheresis procedure to collect peripheral blood mononuclear cells (MNC). The goal of the MNC collection is to collect a product that meets the following specifications: White Blood Cell (WBC) content: 510.sup.9 to 3010.sup.9; Monocyte content: 110.sup.9; Granulocyte content: <3%; Red Blood Cell (RBC) content: <7.5 mL (otherwise RBC debulking is recommended).
Standard operating procedures for the collection of human PBMC are known in the art and described, e.g., in Phuphanich S, et al. Cancer Immunol Immunother. 2013 January; 62(1):125-35. Next, the PBMCs are cultured according to the following protocol to generate DCs.
(112) The DCs are differentiated from PBMCs in an 8-day procedure. On Day 0, the PBMCs are thawed and plated in RPMI-1640 medium containing 1% autologous plasma onto tissue culture flasks to select for monocytes, which adhere to the plastic surface after a one hour incubation step at 37 C. Lymphocytes are washed off the flasks, and the monocytes (adherent CD14+ cells) are then cultured for 5 days in the presence of 20 ng/ml GM-CSF (Bayer Healthcare Pharmaceuticals), with or without 400 IU/ul [60 ng/ul] IL-4). During this period, the monocytes differentiate into immature DCs (non-adherent, CD14 CD83 cells).
(113) On Day 5, the immature DCs are harvested by vigorously swirling the flasks, and by pipetting up and down to resuspend non-adherent and loosely adherent cells. The DCs are washed, and transferred to 6-well plates. On Day 6, tumor cell lysates are added to the tissue culture wells in a ratio of lysate of 1 tumor cell to 5 DCs. One hour later, 1 g/ml LPS (Sigma-Aldrich) was added to the cells and incubated for 18-24 hours.
(114) Next, the DCs are washed and resuspended in 5 ml sterile 0.9% NaCl, USP and then each tube is brought to 14 ml with more PBS. One batch of the cell is resuspended in the DC freezing media and another batch is resuspended in injection solution (sterile saline). In most cases, the volumes are 2 ml for freezing and 4 ml for injection (sterile saline). The cells can then be cryofrozen for future use or used immediately. Quality control testing is performed afterwards, usually the following day, on frozen control aliquots. If release criteria are met, frozen aliquots designated for injection can be thawed as needed and administered to the patient. This method has been validated to ensure potency and stability of the cells by testing batches of frozen/thawed DCs for immunostimulatory capacity and for viability under transport conditions. Frozen/thawed DCs routinely stimulated antigen-specific T cell clones just as well as freshly prepared DCs using a controlled-rate freezer. Thawed DCs are stable for at least two hours in injection solution when kept on ice.
(115) QC (release) criteria include >70% viable cells, negative results for all sterility and endotoxin tests, and >50% of the cells having the characteristics of mature DCs by flow cytometry (large, CD14 CD83+ cells). Close to 80% mature DCs, are routinely obtained with this method, with most of the remaining cells being lymphocytes. Cell viability is typically >90%.
(116) For immune monitoring purposes, an aliquot of DCs without antigen loading and DCs loaded with autologous tumor lysates are stored. Quality control testing is performed the following day.
(117) The patients are then scheduled for up to six DC vaccine injections. The DC vaccine is administered to the patients intravenously through a transfusion set in five to ten minutes. The patients are observed for 2 hours after vaccination to assess immediate complications. Toxicity is graded according to World Health Organization common toxicity criteria. Safety is monitored via clinical assessment, serial full blood count and biochemistry. Patients are followed after vaccine treatment through imaging and immune monitoring. A schema showing the vaccination protocol is illustrated in
(118) The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
(119) It is further to be understood that all values are approximate, and are provided for description.
(120) Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.