Patent Application
20140147411
A BRAF V600E mutation is associated with malignant melanomas. See Davies et al., Nature, 2002, 417, 949-954. Chapman et al., report an improved survival with vemurafenib in melanoma in patients with a BRAF V600E mutation. N Engl J Med 2011; 364:2507-164. While many patients whose tumors harbor the BRAFV600E mutation respond initially to kinase inhibitors such as vemurafenib, the development of resistance is common, and long-term complete responses (CR) are less than optimal. As such, additional approaches to treat those with advanced melanoma are still needed.
INTRON® A is recombinant Interferon alfa-2b. It is adjuvant to surgical treatment in patients with melanoma at high risk for systemic recurrence. See FDA Package Insert.
A clinical trial of the combination of drugs vemurafenib and aldesleukin (IL-2) is contemplated. See http://clinicaltrials.gov/show/NCT01754376.
A clinical trial for treating melanoma by lymphodepletion plus adoptive cell transfer and IL-2 in combination with ipilimumab is contemplated.
See http://clinicaltrials.gov/show/NCT01701674.
Rubinstein et al. report incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032. J Trans Med, 2010, 8:67. See also Sigalotti et al., Br J Cancer. 2011; 105:327-8; Sapkota et al., Oncolmmunology, 2013, 2:1, e22890; Importa et al., Oncolmmunology, 2013, 2:8, e25594; WO2012075327; WO2007002811; WO2013044169; WO2012109329; WO2011093606; CA 2761253; and US 20120244151.
The Cobas® 4800 BRAF V600 Mutation Test is a real-time PCR based method to detect BRAF V600E mutations in DNA isolated from formalin-fixed, paraffin-embedded human melanoma tissue. Other techniques include bidirectional direct Sanger sequencing (“Sanger”) and the Applied Biosystems BRAF Mutation Analysis Reagents kit (“FA test”) for the detection of BRAF V600 mutations in formalin fixed paraffin embedded (FFPE) specimens. See Lopez-Rios et al., PLOS ONE available at www.plosone.org, 2013, 8(1):e53733, Machnicki et al., Acta Biochim Pol., 2013, 60(1):57-64.
Liu et al. report stat3-targeted therapies overcome the acquired resistance to vemurafenib in melanomas. J Inv Derma, 2013, 133, 2041-2049.
References cited herein are not an admission of prior art.
In certain embodiments, the disclosure relates to methods of treating cancer comprising administering an effective amount a tyrosine kinase inhibitor in combination with an immunotherapy to a subject in need thereof. In certain embodiments, the disclosure relates to methods of treating cancer comprising: i) analyzing both chromosomes in a cell from a subject for the a V600E mutation of BRAF; and ii) determining if both of the chromosomes contain the V600E mutation, then treating the subject comprising the step of administering an effective amount a tyrosine kinase inhibitor in combination with an immunotherapy to the subject.
In certain embodiments, the cancer is melanoma.
In certain embodiments, the tyrosine kinase inhibitor is vemurafenib, PLX4720, sorafenib, temozolomide, or dabrafenib.
In certain embodiments, the immunotherapy is administration of an interferon, administration of an interleukin, administration of an anti-PD1 or PD-L1 antibody, administration of an anti-CTLA-4 antibody, a cancer vaccine, adoptive cell transfer, and combinations thereof.
In certain embodiments, the interferon is human recombinant IFN-α, IFN-α2b or IFN-γ or the interleukin is human recombinant IL-2.
In certain embodiments, methods disclosed herein comprise the step of recording whether the subject is homozygous for the V600E mutation, heterozygous for the V600E mutation, heterozygous for the wild-type and V at 600, or homozygous for the wild-type V at 600.
In certain embodiments, the recording is on a computer readable medium.
In certain embodiments, methods disclosed herein comprise the step of reporting whether the subject is homozygous for the V600E mutation, heterozygous for the V600E mutation, heterozygous for the wild-type V at 600, or homozygous for the wild-type V at 600 to a medical professional, subject, or representative thereof.
Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
As used herein, the term “combination with” when used to describe administration with an additional treatment means that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof.
As used herein, the terms “prevent” and “preventing” include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity is reduced.
As used herein, the terms “treat” and “treating” are not limited to the case where the subject (e.g., patient) is cured and the condition or disease is eradicated. Rather, embodiments, of the present disclosure also contemplate treatment that merely reduces symptoms, and/or delays conditions or disease progression.
Ipilimumab has been approved by the Food and Drug Administration (FDA) for the treatment of those with metastatic melanoma. It is a human monoclonal antibody that activates the immune system by targeting CTLA-4. Other immune-based approaches are under investigation for the treatment of cancer. These methods attempt to enhance the development of anti-tumor CD8+ cytotoxic lymphocytes (CTLs) and CD4+ T lymphocytes to generate a therapeutic cell-mediated anti-tumor immune response. Immune cell-mediated responses are believed to involve the processing and presentation of antigenic peptides bound to MHC molecules which allow their recognition by CTLs and/or CD4+ T cells. Enhancing MHC expression on tumor cells is believed to be a means of improving tumor cell immune recognition. See Lampen & Hall T, Cur Opin Immunol, 2011; 23:293-8. Rosenberg et al. report adoptive cell transfer is a clinical path to effective cancer immunotherapy. Nat Rev Cancer 2008; 8:299-308.
IFNs are potent inducers of MHC expression. The influence BRAF V600E inhibitors on the induction of MHC molecules by interferons (IFNs) were explored. It has been discovered that vemurafenib can enhance the induction of MHCI and MHCII molecules by IFN-γ and IFN-α2b in A375 melanoma cells. Vemurafenib could enhance MHC induction by IFN-γ in melanoma cells harboring a homozygous BRAFV600E mutation but not in cell lines heterozygous for BRAFV600E or those wild-type at BRAF codon 600. These data suggest that inhibition of BRAFV600E can enhance MHC induction by IFNs in some cellular contexts and supports the notion that the impact of vemurafenib on immune gene expression can be influenced by the zygosity of the BRAFV600E mutation.
The data presented herein demonstrate that BRAFV600E has a repressive effect on MHC expression and that in some cellular contexts, inhibition of BRAFV600E activity can augment the induction of MHCI and MHCII molecules by IFN-γ, a cytokine that is likely to be present in the tumor microenvironment, and IFN-α2b. This finding is relevant for several reasons. Most notably, the expression of MHCI molecules in melanoma has been shown to correlated to the clinical response to immune-based therapies. See Carretero et al., Immunogenetics 2008; 60:439-47 and Carretero et al., Int J Cancer 2012; 131:387-95. These results indicate that BRAFV600E inhibition alone or combined with IFN-α2b is a promising pharmacologic approach to enhance the expression of MHCI molecules on melanoma cells. It supports the notion that combining IFN-α2b and BRAFV600E inhibition may offer an approach to augment tumor cell immune recognition by CTLs in the adjuvant setting. Data herein indicates that in addition to providing a growth advantage to tumor cells, the genetic amplification of BRAFV600E may also promote tumor cell immune escape by attenuating basal MHCI levels.
Because of its pivotal role in regulating cell proliferation, the prevailing paradigm regarding the BRAFV600E mutation in melanoma has centered on whether the mutation is there or not. If the BRAFV600E mutation is present, as measured via sequencing or mutation-specific PCR-based assays, patients are eligible to be placed on a BRAFV600E-selective inhibitor such as vemurafenib or dabrafenib. However, less attention has been given to the zygosity of the BRAFV600E mutation. This is primarily because there has been no clinical need to differentiate BRAFV600E heterozygous tumors from BRAFV600E homozygous tumors or understand how the zygosity of the mutation influences tumor biology.
At present, molecular testing for the use of inhibitors that are selective for the BRAFV600E mutation centers on the detection of BRAFV600E in DNA isolated from patient tumor samples. Typically, this is biopsy material that has been formalin-fixed and paraffin embedded. Indeed, a test for this purpose has been approved by the FDA. However, in these assays, the zygosity of the BRAFV600E mutation is not routinely assessed. Our data raise the possibility that the zygosity of BRAFV600E mutation may influence how melanoma cells respond to vemurafenib (or other targeted inhibitors of BRAFV600E) with regards to the expression of MHC molecules, immune system genes directly relevant to anti-tumor immune responses in melanoma. This is particularly important as combination therapies utilizing both targeted kinase inhibitors and immune-based approaches in patients with advanced melanoma. BRAFV600E zygosity is a relevant biomarker for therapies using BRAFV600E-specific kinase inhibitors alone or in combination with an immune-based therapeutic (such as IL-2 or ipilimumab).
BRAFV600E can influence basal MHCI expression and that inhibitors of BRAFV600E can potentiate the induction of MHC molecules by IFN-γ and IFN-α2b. This effect is believed to be mediated via a mechanism that is influenced by the zygosity of the BRAFV600E mutation.
Analyzing chromosomes for the V600E mutation may be performed by any current method known to the skilled artisan. One method is to isolate a malignant melanocyte, optionally separating the chromosomes within the cell, and sequencing the target DNA region, e.g., using PCR, to determine the presence of the mutation or wild-type sequence.
Genomic DNA was isolated from the cell lines and BRAF codon 600 was amplified using PCR and sequenced. The wild-type BRAF codon 600 sequence is GTG whereas the BRAFV600E codon sequence is GAG. A431 cells are wild-type as are melanoma cell lines MeWo and SKMEL-2. Cell lines heterozygous for BRAFV600E (SKMEL-3, SKMEL-5, and MALME-3M) have a mixture of T and A at the second nucleotide position of codon 600 whereas cell lines harboring only BRAFV600E (A375, SKMEL-28, and HT-144) have only the GAG sequence at codon 600.
The term “polymerase chain reaction” (“PCR”) refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, that describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one “cycle”; there can be numerous “cycles”) to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the “polymerase chain reaction” (hereinafter “PCR”). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified.”
With PCR, it is possible to amplify a single copy of a specific target sequence to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). Any oligonucleotide or polynucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.
The terms “PCR product,” “PCR fragment,” and “amplification product” refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.
The term “amplification reagents” refers to those reagents (deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification except for primers, nucleic acid template, and the amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).
In certain embodiments, the disclosure relates to methods of treating cancer comprising: i) analyzing both chromosomes in a cell from a subject for the a V600E mutation of BRAF; and ii) determining if both of the chromosomes contain the V600E mutation, then treating the subject comprising the step of administering an effective amount a tyrosine kinase inhibitor in combination with an immunotherapy to the subject.
In certain embodiments, the tyrosine kinase inhibitor is vemurafenib, PLX4720, sorafenib, temozolomide, trametinib, or dabrafenib.
In certain embodiments, the immunotherapy is administration of an interferon, administration of an interleukin, administration of an anti-PD1 or PD-L1 antibody, administration of an anti-CTLA-4 antibody, a cancer vaccine, adoptive cell transfer, and combinations thereof.
In certain embodiments, the interferon is human recombinant IFN-α, IFN-α2b or IFN-γ or the interleukin is human recombinant IL-2 or IL-12 or fragments thereof.
In certain embodiments, the disclosure relates to methods of treating the subject comprising the step of administering an effective amount a tyrosine kinase inhibitor in combination with a cancer vaccine. In certain embodiments, the cancer vaccine comprises cancer antigens, MAGE-A3, MART-1, gp100, TRP-2, NY-ESO-1, costimulatory molecules, cytokines, and combinations thereof.
In certain embodiments, the cancer antigen or combinations of antigens are expressed on the surface of virus particle or virus-like particles. The virus particles may be the result of genetically engineered attenuated or weakened viruses that produce virus or virus-like particles containing the antigen.
In certain embodiments, the cancer vaccine is a recombinant virus that expresses cytokines or cancer antigens. Willomann et al., report expression of IFN-beta enhances oncolytic vesicular stomatitis virus for therapy of mesothelioma. Cancer Res. 2009 Oct. 1; 69(19):7713-20. In certain embodiments, the cancer vaccine in is a recombinant vesicular stomatitis virus that expresses IFN-beta. Wollmann et al. report vesicular stomatitis virus variants selectively infect and kill human melanomas but not normal melanocytes. J Virol., 2013, 87(12):6644-59. In certain embodiments, the cancer antigen containing virus like particles may also contain additional adjuvants, e.g., flagellin, GM-CSF, on the surface of the particle. Wang et al., report incorporation of membrane-anchored flagellin into influenza virus-like particles enhances the breadth of immune responses. J Virol., 2008, 82(23): 11813-11823.
In certain embodiments, the cancer antigen or combinations of antigens are expressed on the surface of cells. The cells may be the result of genetically engineering cells to express the antigen on the surface. In certain embodiments, the antigen is anchored on the surface of the cell through glycosyl phosphatidylinositol (GPI), e.g., as a result of mixing cells with an antigen conjugate with glycosyl phosphatidylinositol. The cancer antigen containing particles may also contain additional adjuvants, cytokines, or costimulatory molecules. See Bozeman et al., Vaccine, 2013, 31(20):2449.
In certain embodiments, the disclosure relates to methods of treating a subject comprising the step of administering an effective amount a tyrosine kinase inhibitor in combination with adoptive cell transfer. Adoptive cell transfer relates to the isolation, amplification, optionally modification, and reinfusion of cells of the immune system cells, e.g., T lymphocytes, NK cells. In certain embodiments, the disclosure relates to methods using adoptive cell transfer (ACT) with autologous tumor-infiltrating lymphocytes (TILs) or using tumor-infiltrating lymphocytes (TILs) or T lymphocytes obtained from bone marrow or peripheral blood and expanded ex vivo. In certain embodiments, the disclosure relates to using autologous TILs expanded ex vivo from tumor fragments or single cell enzymatic digests of melanoma metastases.
In certain embodiments, the disclosure relates to methods wherein the subject undergoes chemotherapy causing lymphodepletion, e.g., by administering chemotherapy agents such as cyclophamide and flubarabine.
In certain embodiments, the disclosure relates to methods using peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. In certain embodiments, the disclosure relates to methods infusing tumor-specific CD4+ and CD8+ T cells generated in vitro with repeated stimulation of irradiated autologous tumor cells. In certain embodiments, the disclosure relates to methods using PBMC stimulated with artificial antigen-presenting cells, costimulatory molecules, cytokines, and combinations thereof.
In certain embodiments, the disclosure relates to methods using T cells engineered to stably express transgenes by vector based transduction. Vector mediated gene transfer approaches may use vectors that are derived from viruses, e.g., gamma retroviruses, lentiviruses or foamy virus vectors, that have the ability to integrate into the cell genome and provide transgene expression. Transduction may be through the use of replicating cells for viral integration into genomic DNA or nondividing cells. Bauer et al., report a foamy virus vector. Nature Medicine, 2008, 14, 93-97 (2008).
In certain embodiments, the cell therapy comprises modifying autologous cells, e.g., dendritic cells, genetically modified to produce IL-2, IL-12, or interleukin-12p70 (IL-12p70). Carreno et al. report genetically modified dendritic cells producing IL-12p70 elicit Tcl-polarized immunity. J Clin Invest. 2013, 123(8):3383-94
In certain embodiments, the methods disclosed herein comprise using a tumor antigen expressing recombinant vesicular stomatitis virus in combination with an adoptive cell transfer therapy. Rommelfanger et al. report a systemic combination virotherapy for melanoma with tumor antigen-expressing vesicular stomatitis virus and adoptive T-cell transfer. Cancer Res., 2012, 15; 72(18):4753-64.
In certain embodiment, methods disclosed herein comprise viral gene transfer into autologous cells of a drug-resistant enzyme such as P140KMGMT for reinfusion and subsequent treatment with temozolomide. Dasgupta et al. report engineered drug-resistant immunocompetent cells enhance tumor cell killing during a chemotherapy challenge. Biochem Biophys Res Commun., 2010, 391(1):170-5.
In certain embodiments, the disclosure relates to methods of treating cancer comprising: i) analyzing both chromosomes in a cell from a subject for the a V600E mutation; and ii) determining if both of the chromosomes contain the V600E mutation, then treating the subject comprising the step of administering an effective amount a tyrosine kinase inhibitor and an immune therapy in combination with a chemotherapy agent, e.g., imatinib, nilotinib, orafenib, bevacizumab, pazopanib, everolimus, and combinations thereof.
Experiments were performed to determine whether inhibitors of BRAFV600E could potentiate the effects of IFN-γ on MHC expression. A375 cells were selected as a model tumor cell line since A375 cells are known to respond to IFN-γ and harbor the BRAFV600E mutation. To confirm that PLX4720 inhibits BRAFV600E signaling, A375 cells were treated with either vehicle (DMSO) or 10 μM PLX4720 and evaluated levels of ERK phosphorylation (at residues threonine 202 and tyrosine 204) as a read out for activated MAPK signaling. As shown in
Experiments were performed to examine whether PLX4720 could influence the induction of MHCI molecules by IFN-γ in A375 cells. Treatment of A375 cells with IFN-γ lead to an induction of MHCI cell surface expression as measured by flow cytometry (
Experiments were performed to determine whether this effect was influenced by the concentration of IFN-γ because the cellular response to IFN-γ can vary with concentration. While PLX4720 is structurally related to vemurafenib it is not used clinically. Therefore, we repeated these experiments using vemurafenib as it has been approved for use in patients whose tumors are BRAFV600E positive. SKMEL-2 cells were included as a control since these cells are wild-type at BRAF codon 600 and thus should be unaffected by selective inhibitors of BRAFV600E such as vemurafenib. As shown in
In contrast to A375 cells, vemurafenib had no effect on MHC induction in SKMEL-2 cells despite the fact that these cells responded to IFN-γ with increases in cell surface MHCI, MHCII and B2M protein levels (
Since the anti-proliferative effect of vemurafenib on A375 cells is optimal at nanomolar concentrations, these experiments were repeated using serial dilutions of vemurafenib from 10 μM to 100 nM. As observed using 10 μM of vemurafenib, lower concentrations of vemurafenib also enhanced the induction of MHCI, B2M and MHCII molecules in response to IFN-γ (
Experiments were performed to determine whether the over-expression of BRAFV600E would have the opposite effect of vemurafenib on MHC expression. To this end, A375 cells were transfected with a plasmid encoding BRAFV600E and green fluorescent protein (GFP) on the same transcript or the parental plasmid encoding GFP alone (empty vector). As shown in
Experiments were performed to determine whether vemurafenib can influence MHC induction in response to type I interferons since like IFN-γ (a type II interferon) as these cytokines are potent inducers of MHCI. Experiments were performed to determine whether vemurafenib can enhance MHCI induction in response to IFN-α2b, A375 cells were pre-treated with either vehicle (DMSO) or vemurafenib and then exposed them to increasing concentrations of IFN-α2b. Vemurafenib enhanced the induction of MHCI molecules by IFN-α2b at all the doses utilized which ranged from 0.09 to 909 U/ml (
Experiments were performed to determine whether the results obtained with A375 cells could be reproduced in other cellular contexts. These experiments were repeated using additional melanoma cell lines. These included another BRAF wild-type cell line MeWo, as well as cells lines known to harbor the BRAFV600E mutation including MALME-3M, SKMEL-3, SKMEL-5, SKMEL-28, HT-144 and UACC-62. These cell lines were selected since they were all commercially available and because information regarding their mutation status was available using the Wellcome Trust Sanger Institute database which is publically available (http://www.sanger.ac.uk). The BRAF mutation status was confirmed for these cell lines by pyrosequencing BRAF codon 600 and included an additional non-melanoma cell line as a control (A431). For this analysis, the terms wild-type, heterozygous and homozygous only refer to the sequence of BRAF codon 600. Specifically, wild-type cells are those where no mutant sequence is present at codon 600, heterozygous cells are those where both mutant (V600E) and wild-type sequence is detected, and homozygous cells are those where only the mutant (V600E) sequence is detected. Consistent with what was reported in the aforementioned database, MALME-3M, SKMEL-3, and SKMEL-5 cells all harbored both mutant and wild-type sequence at codon 600 of BRAF. In contrast, A375, SKMEL-28 and HT-144 cells possessed only the mutant sequence at codon 600. Experiments were repeated using these cell lines to assess how vemurafenib influenced MHC induction by IFN-γ. Vemurafenib had no impact on the induction of MHCI and MHCII molecules in MeWo cells which are wild-type at BRAF codon 600 (
To determine whether this effect was unique to inhibitors targeting the MAPK pathway, how the induction of MHCI and MHCII molecules by IFN-γ is altered in the presence of an inhibitor of the phosphoinositide 3-kinase (PI3K) pathway was examined. BEZ235 a dual PI3K/mammalian target of rapamycin inhibitor was used. While BEZ235 reduced the phosphorylation of AKT (serine 473) in our model system consistent with its ability to inhibit mTOR and PI3K signaling, it did not enhance the induction of MHC molecules by IFN-γ, rather, it attenuated MHC induction in some of the cell lines examined (
This application claims priority to U.S. Provisional Application No. 61/726,030 filed Nov. 14, 2012, hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61726030 | Nov 2012 | US |
