Patent Application
20240018205
The present invention relates to a composition of a fusion protein comprising interleukin-7 for preventing or treating a human papillomavirus-derived disease.
Interleukin-7 (hereinafter ‘IL-7’) is an immune-stimulating cytokine that stimulates immune responses mediated by B cell and T cell, and plays an important role in the adaptive immune system. IL-7 is mainly secreted from stromal cells of bone marrow and thymus, but also produced in keratinocytes, dendritic cells, hepatocytes, nerve cells, and epithelial cells (Haufler C et al., 1993, J. Exp. Med. 178 (3)): 1109-14).
Specifically, interleukin-7 activates immune function through stimulation Of the survival and differentiation of cells and B cells, the survival of lymphoid cells, and the activation of NK (natural killer) cells, and is especially important for the development of T cells and B cells, It is bound with HGF (hepatocyte growth factor) and functions as pre-pro-B cell growth-stimulating factor or a cofactor for V(D)J rearrangement of T cell receptor beta (TCRβ) (Muegge K, 1993, Science 261 (5117): 93-5). In addition, interleukin-7 regulates lymph node development through lymphoid tissue inducer (LTi) cells and promotes the expansion and survival of naive T cells or memory T cells. It is also known that IL-7 stimulates the secretion of IL-2 and interferon-gamma (interferon-γ), thereby enhancing the human immune response.
Meanwhile, papillomavirus is a DNA-based virus with a diameter of 52 to 55 nm, which infects skin and subcutaneous tissue of humans and other animals. Human papillomavirus (HPV) is usually transmitted through skin keratinocytes or mucous membranes. More than 100 human papillomaviruses (HPV) have been found so far, most of which do not show any symptoms, but in some cases they can cause papillomas in humans, Some HPVs cause the development of warts, and some cause precancerous lesions. In particular, high-risk viruses such as human papilloma virus 16 (HPV 16) and human papilloma virus 18 (HPV 18) can cause cancer such as cervical cancer and testicular cancer.
Cervical cancer is one of the most common causes of cancer-related deaths in women worldwide. Almost all of the cases are caused by infection with human papillomavirus (HPV). Among them, HPV16 and HPV18 account for about 70-75% of cervical cancer patients. Continuous proliferation of infected cells leads to a pre-malignant cervical intraepithelial neoplasia (CIN), which then gradually transform into invasive cancer.
While the prophylactic HPV vaccines can efficiently prevent HPV infection, they do not have therapeutic effects against pre-existing infection and HPV-induced lesions. The most common treatment for CIN2 and CIN3 is surgical excision, which is associated with pregnancy-related complications and a 10% recurrence rate. More seriously, the mortality rate of cervical cancer after conventional treatment is more than 50%.
Meanwhile, recently, therapies to treat HPV infection have been developed by inducing immune enhancement. It has been reported that local administration of toll-like receptor (TLR) agent 7 and 9, imiquimod and CpG after administration of vaccine including HPV16 E7 antigen induced accumulation of E7-specific CD8 T cells in the genital tract and regression of genital tumors (Soong R-S et al, 2014, Clin. Cancer Res. 20:5456-67). However, in humans, imiquitnod usage can induce side effects such as acute and severe local inflammation and ulceration, and administration of CpG requires repeated injections due to its short-lived efficacy. The ability of cytokines, such as IL-2 and IL-15, which function as vaccine adjuvants in animal models, were studied in order to enhance the therapeutic efficacy (Abraham E et al., 1992, J Immunol 149:3719-26). However, such cytokines also require repeated injections and may induce adverse effects, e.g., capillary leakage syndrome in case of IL-2.
Therefore, there still exists a need to develop effective and non-surgical therapy for the prevention and treatment of diseases caused by HPV infection.
The object of the present invention is to provide a composition for preventing or treating a human papillomavirus-derived disease.
Another object of the present invention is to provide a method for preventing or treating a human papillomavirus-derived disease.
In accordance with one aspect of the present invention, there is provided a pharmaceutical composition comprising a fusion protein of immunoglobulin Fc region and IL-7. Also, there is provided a method for preventing or treating a human papillomavirus-derived disease by mucosal administration of the pharmaceutical composition comprising the fusion protein.
In case where a fusion protein comprising immunoglobulin Fc region and IL-7 according to the present invention is administered via a mucosal route, the number of antigen-specific T cells is increased to prevent or treat a human papillomavirus-derived disease. Also, such administration is easy to conduct. Therefore, the fusion protein comprising immunoglobulin Fc region and IL-7 according to the present invention can be utilized as a new pharmaceutical composition which can replace the conventional HPV preventive vaccine.
Hereinafter, the present invention is explained in detail.
In one aspect for achieving the object, the present invention provides a pharmaceutical composition for preventing or treating a genital disease comprising an interleukin-7 (IL-7) fusion protein in which immunoglobulin Fc region is fused.
The genital disease may be a human papillomavirus-derived disease.
As used herein, the term “human papillomavirus-derived disease” or “human papillomavirus infection disease” refers to a disease caused by human papilloma virus (HPV) infection. Human papilloma virus-derived diseases can be classified into CIN1, CIN2, CIN3, LSIL (low grade squamous intraepithelial lesion), HSIL (high grade squamous intraepithelial lesion) or cancer, etc., depending on the degree of infection or status of a lesion.
As used herein, the term “interleukin-7”may be a protein having the same amino acid sequence as interleukin-7 derived from an animal or a human. Further, the term “interleukin-7”may be a polypeptide or a protein having an activity similar to the interleukin-7 derived in vivo. Specifically, the IL-7 may be a protein comprising an IL-7 protein or a fragment thereof. Also, the IL-7 may be derived from a human, a rat, a mouse, a monkey, cattle or sheep.
The IL-7 comprises a polypeptide consisting of the amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 6. In addition, the IL-7 may have homology of about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more to the sequences of SEQ ID NO: 1 to SEQ ID NO: 6.
Specifically, human IL-7 may have an amino acid sequence represented by SEQ ID NO: 1 (Genbank Accession No. P13232); rat IL-7 may have an amino acid sequence represented by SEQ ID NO: 2 (Genbank Accession No. P56478); mouse IL-7 may have an amino acid sequence represented by SEQ ID NO: 3 (Genbank Accession No. P10168); monkey IL-7 may have an amino acid sequence represented by SEQ ID NO: 4 (Genbank Accession No. NP_001279008); bovine IL-7 may have an amino acid sequence represented by SEQ ID NO: 5 (Genbank Accession No. P26895); and sheep IL-7 may have an amino acid sequence represented by SEQ ID NO: 6 (Genbank Accession No. Q28540).
In addition, the IL-7 protein or a fragment thereof may comprise a variety of modified proteins or peptides, i.e., variants. Such modification may be carried out by substitution, deletion or addition of one or more proteins of wild-type IL-7, which does not alter the function of IL-7. These various proteins or peptides may have homology of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to a wild-type protein.
In general, substitution of a wild-type amino acid residue can be accomplished by substituting alanine or a conservative amino acid that does not affect the charge, polarity, or hydrophobicity of the entire protein.
The term “IL-7 protein” as used in the specification may be used as a concept including “IL-7 protein” and a fragment thereof. The terms “protein,” “polypeptide,” and “peptide” may be used interchangeably, unless otherwise specified.
In addition, the IL-7 may be a modified IL-7 having the following structure:
Herein, said A may be directly linked to the N-terminus of the IL-7 or may be linked through a linker.
Said A may increase the productivity of IL-7 and may be prepared according to the method disclosed in Korean Patent Application No. 10-2016-0072769.
As used herein, said A may be linked, to the N-terminus of IL-7. In the above formula, said A is characterized by containing 1 to 10 amino acids, which may be preferably selected from the group consisting of methionine, glycine, serine, and a combination thereof.
It is known that methionine and glycine do not induce an immune response in the human body. Although various protein therapeutic agents produced from E. coli necessarily contain methionine at the N-terminus thereof, no adverse immune effect has been reported. In the meantime, glycine is widely used in GS linker, and it is known that a commercial product such as Dulaglutide does not induce an immune response.
According to one embodiment, the IL-7 may be an oligopeptide comprising 1 to 10 amino acids selected from the group consisting of methionine (Met, M), glycine (Gly, G) and a combination thereof. Preferably, the IL-7 may be an oligopeptide consisting of 1 to 5 amino acids. For example, said A may be represented by the amino acid sequence selected from the group consisting of methionine, glycine, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine-methionine, methionine-glycine-glycine, glycine-methionine-glycine, glycine-glycine-methionine, and glycine-glycine-glycine. Herein, the modified IL-7 may have any one of the amino acid sequences selected from SEQ ID NOS: 15 to 20.
Further, immunoglobulin Fc region may comprise an animal or human immunoglobulin Fc region, or a modified immunoglobulin Fc region thereof.
The IL-7 may be linked to the N-terminus Or the C-terminus of the Fc region. It is known that even when IL-7 is fused to the C-terminus of the Fc region, IL-7 activity is maintained (U.S. Pat. No. 8,338,575 B2). Herein, the IL-7 may be linked to Fc region through a linker.
As used herein, the term “Fc region,” “Fc fragment” or “Fc” refers to a protein which comprises heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of immunoglobulin but does not comprise variable regions of heavy or light chain and light chain constant region 1 (CL1). It may further comprise a hinge region of the heavy chain constant region. Hybrid Fc or a hybrid Fc fragment may herein also be referred to as “hFc” or “hyFc.” Also, as used herein, the term “a modified immunoglobulin Fc region” or “Fc region variant” refers to a Fc region in which one or more amino acids in the Fc region are substituted or a Fc region which is prepared by combining different Fc regions. Preferably, it refers to a Fc region whose binding force. with a Fc receptor and/or a complement has been modified so as to exhibit weakened antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) compared to the wild-type Fc region. The modified immunoglobulin Fc region may be a combination sequence of two or more of IgG1, IgG2, IgG3, IgD, and IgG4.
In particular, the modified it immunoglobulin Fc region comprises CH2 domain and CH3 domain in the N-terminus to C-terminus direction, wherein the CH2 domain comprises a portion of an amino acid residue of CH2 domain of IgD and human IgG4, and the CH3 domain comprises a portion of an amino acid residue of human IgG4 CH3 domain.
The Fc region variant can be modified so as to prevent the cleavage at the hinge region. Specifically, the 144th amino acid and/or the 145th amino acid of SEQ ID NO: 9 can be modified. Preferably, the variant may be a mutant in which K, the 144th amino acid of SEQ ID NO: 9, is substituted by G or S, and E, the 145th amino acid, is substituted by G or S.
In particular, the Fc region of the modified immunoglobulin comprises CH2 domain and CH3 domain in the N-terminus to C-terminus direction, wherein the CH2 domain comprises a portion of an amino acid residue of CH2 domain of human IgD and human IgG4, and the CH3 domain comprises a portion of an amino acid residue of human IgG4 CH3 domain.
As used herein, the term “Fc region”, “Fc fragment” or “Fc” refers to a protein which comprises heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of immunoglobulin but does not comprise variable regions of heavy or light chain light chain and constant region 1 (CL1). It may further comprise a hinge region of the heavy chain constant region. Hybrid Fc Or a hybrid Fc fragment may herein also be referred to as “hFc” or “hyFc”. Also, as used. herein, the term “Fc region variant” refers to a Fc, region in which one or more amino acids in the Fc region are substituted or which is produced by combining different Fc regions. The Fc region variant can be modified so as to prevent severing at the hinge region. Specifically, the 144th amino acid and/or the 145th amino acid of SEQ ID NO: 9 can be modified, Preferably, the variant may be a mutant in which K, the 144th amino acid of SEQ ID NO: 9, is substituted by C or S, and E, the 145th amino acid, is substituted by G or S.
In addition, the hFc can be represented by the following formula (I):
N′—(Z1)p-(Y)q-Z2-Z3Z4-C″, [Formula (I)]
wherein,
In addition, the modified immunoglobulin Fc region or Fc region variant can he represented by the following formula (I):
N′—(Z1)p-Y-Z2-Z3Z4-C″, [Formula (I)]
wherein,
In addition, Fc fragment of the present invention may be a wild type sugar chain, an increased sugar chain compared with the wild type, a reduced sugar chain compared with the wild type, or a form in which the sugar chain is removed. The increase, reduction or removal of immunoglobulin Fc sugar chain can be carried out by a conventional method known in the art such as chemical method, enzymatic method and genetic engineering method using microorganisms. The removal of the sugar chain from Fc fragment rapidly reduces the binding affinity of the primary complement component C1 to C1q and results in a decrease or loss of ADCC (antibody-dependent cell-mediated cytotoxicity) or CDC (complement-dependent cytotoxicity), thereby not inducing unnecessary immune responses in vivo, In this regard, immunoglobulin Fc fragment in a deglycosylated or aglycosylated form may be more suitable for the purpose of the present invention as a carrier of a drug. As used herein, the term “deglycosylation” refers to enzymatical elimination of sugar from Fc fragment, and the term “aglycosylation” refers to the production of Fc fragment in an unglycosylated form by a prokaryote, preferably E. coli.
The modified immunoglobulin Fc region may comprise amino acid sequences of SEQ ID NO: 9 (hFc01), SEQ ID NO: 10 (hFc02), SEQ ID NO: 11 (hFc03), SEQ ID NO: 12 (hFc04) or SEQ ID NO: 13 (hFc05). In addition, the modified immunoglobulin Fc region may comprise the non-lytic mouse Fc of SEQ ID NO: 14.
According to the present invention, the modified immunoglobulin Fc region may be one described in U.S. Pat. No. 7,867,491, and the production of the modified immunoglobulin Fc region may be carried out with reference to the disclosure of U.S. Pat. No. 7,867,491.
In addition, the interleukin-7 fusion protein in which immunoglobulin Fc region is fused may have the amino acid sequence of any one of SEQ ID NOS: 21 to 27.
Meanwhile, the interleukin-7 fusion protein in which immunoglobulin Fc region is fused according to the present invention may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any carrier that is suitable for being delivered to a patient and is non-toxic to the patient. Distilled water, alcohol, fats, waxes and inert solids may be included as carriers. Pharmacologically acceptable adjuvant (a buffer or a dispersant) may also be included in the pharmacological composition.
In another aspect of the present invention, there is provided a method for preventing or treating a genital disease comprising administering to an individual an interleukin-7 (IL-7) fusion protein in which immunoglobulin Fc region is fused and a pharmaceutically acceptable carrier.
The genital disease may be a human papillomavirus-derived disease, for example, cervical cancer.
Herein, the method of administration to an individual may be a local administration, preferably mucosal administration. In case of that the composition of the present invention is provided topically, such as intravaginal or aerosol administration, the composition preferably comprises a portion of an aqueous or physiologically compatible body fluid suspension or solution. Accordingly, the carrier or vehicle may be physiologically acceptable, and thus it can be added to the composition and delivered to the patient, which does not adversely affect the electrolyte and/or volume balance of the patient. Thus, a carrier fix a formulation may generally include physiologic saline. Also, it may include a portion of viscous suspension or solution depending on the lesion or physiological condition.
The method for preventing or treating a disease using a fusion protein of the present invention or a composition comprising the same may comprise administering another drug or physiologically active substance having the effect of preventing or treating a disease in combination with the protein or the composition of the present invention, while the route, timing, and dosage of the administration may he determined depending on the type of a disease, the disease condition of a patient, the purpose of treatment or prevention, and other drugs or physiologically active substances co-administered.
The isolated nucleic acid molecule encoding the modified interleukin-7 or a fusion protein comprising the same may encode a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOS: 15 to 25. The nucleic acid molecule may comprise a polynucleotide sequence selected from the group consisting of SEQ ID NOS: 29 to 39. The nucleic acid molecule may further comprise a signal sequence or a leader sequence.
Hereinafter, the present invention is explained in detail. The following Examples are intended to further illustrate the present invention without limiting its scope.
Female C57BL/6 mice, 8-10 weeks of age used in the following examples were purchased from The Jackson Laboratory (Bar Harbor, USA). All animals were raised under specific pathogen-free conditions in the animal care facility in POSTECH. The procedures of animal experiments were performed in accordance with the National institutes of Health (NIH) guidelines for mouse experiments. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC). Also, female Sprague-Dawley rats at 11 weeks of age were purchased from the Charles River Laboratories (Raleigh, USA). All animals were raised under specific pathogen-free conditions in the animal care facility of MPI research. The procedures of animal experiments were performed in accordance with the regulations outlined in the United States Department of Agriculture (USDA) animal welfare act (9 CFR, parts 1-3).
The codon-optimized human IL-7 and granulocyte colony-stimulating factor (G-CSF) genes were individually fused with a hybrid Fc-fragment. The schematic structure of Fc-fused. IL-7 is shown in
3 mg of medroxyprogesterone acetate (Depo-Provera, Pfizer) was subcutaneously injected to mice in a diestrus state 4 days before treatment. The mice were anesthetized by intraperitoneal injection with 100 mg/kg ketamine (Yuhan) and 10 mg/kg xylazine hydrochloride (Bayer) in PBS. Then, 10 μg of rIL-7, IL-7-Fc or G-CSF-Fc were mixed with PBS and applied (administered) on the vaginal mucosal tissues using a micropipette.
IL-7-Fc was coupled with Cy-5.5 mono-reactive NHS ester, Eluted proteins were desalted and concentrated by using centrifugal filter devices (Merck Millipore) and protein concentration of the dye-labeled IL-7-Fc was measured using an anti-human IL-7 ELISA set (Southern Biotech), Cy-5.5-conjugated IL-7-Fc (1 mg/kg) and Cy-5.5 in PBS were intravaginally administered to anesthetized mice with equivalent signal intensity. At days 1 and 7 after administration, mice were euthanized and their vaginas were washed, and each of the organs was obtained. The fluorescence signal intensity was then quantified using an IVIS spectral machine (Caliper Life Science). Signal intensity was measured quantitatively in the organ by measuring photons per second per centimeter squared per steradian (p/s/cm2/sr).
Blood samples were collected before administration and up to 7 days after administration of IL-7-Fc, and serum IL-7 concentration was measured using a human IL-7 ELISA set (Southern biotech).
After topical administration of IL-7-Fc, for histopathological analysis using a microscope, 0.8, 3 and 8 mg/kg/dose of IL-7-Fc were intravaginally administered to rats once a week for 4 weeks (total dose of 5). The uterine cervix/vaginal tissues were excised and fixed with neutralizing formalin. The fixed tissues were placed in paraffin, cut with a thickness of 4-6 μm and stained with hematoxylin and eosin (H&E, Sigma-Aldrich). To determine the dose-dependence of vaginal inflammation, rats were observed individually at 4 hours and 24 hours after each dose administration and weekly, The following scoring scale was used: 0=no erythema, 1=very slight erythema (barely perceptible), 2=well-defined erythema, 3=moderate erythema, 4=severe erythema (redness) to eschar formation.
Spleen and CV tissues were surgically excised using sterile technique. The splenocytes were obtained by mechanically disrupting the tissues. For the preparation of CV cells, CV tissues were minced and treated with 1 mg/ml collagenase D (Roche) and 0.5 mg/ml DNase (Sigma-Aldrich). The cells were passed through a 40 μm strainer (BD), washed, and re-suspended with RPMI-1640 containing 10% , FBS and antibiotics.
To prevent non-specific binding of immunoglobulins to Fc receptor, the cells used in the following Examples were treated with CD16/32 (2.4G2) and stained with the following monoclonal antibodies: CD4 (RM4-5), CD8 (53-6.7), CD44 (IM7), CD62L (MEL-14), CD11b (M1/70), CD11c (N418), and MHCII (M5/114.15.2), from eBioscience; CD3e (145-2C1), and TCRγδ (GL3), from BD; CXCR3 (CXCR3-173), from Biolegend; and Live/Dead (Life technologies). All samples were analyzed using an LSR Fortessa (BD) and FlowJo software (Tree Star).
A two-tailed paired Student's t-test was used to evaluate the statistical difference between the two experimental groups, For in vivo tumor experiments, differences in survival rates between the groups were determined by a tog-rank test using the Prism 5.0 software (GraphPad).
Cy-5.5 (Cy-5.5) and Cy-5.5 -conjugated IL-7-Fc (Cy5.5-IL-7-Fc) were intravaginally administered to C57BL/6 wild-type mice (n=3/group), The results are shown in
As shown in
PBS, rIL-7 and IL-7-Fc were intravaginally administered to mice (n=7/group), and serum concentration of IL-7 was measured by human IL-7 ELISA. The results are shown in
These results reveal that the application of the Fc-fused protein on the mucosal epithelium enables genital-epithelial barrier transcytosis.
IL-7-Fc was intravaginally administered to mice (n=3/group) at 0, 3, 7, 14 and 21 days prior to sacrifice, and the number of leukocytes in cervical tissues was calculated using flow cytometry (Table 1). In addition, mice (n=6/group) were treated with PBS, IL-7, IL-7-Fc, IFN-α2a-Fc or G-CSF-Fc, and 7 days later, CD4 and CD8 cells in CV tissues were analyzed by flow cytometry. The results are shown in Tables 1 and 2 and
As shown in Table 1 and
As shown in Table 2 and
These results indicate that IL-7-Fc intravaginal administration induces local accumulation of immune cells such as T cells and DCs. Also, it was found that the effect of the IL-7-Fc intravaginal administration was superior to other immune inducers.
IL-7-Fc was intravaginally administered to SD rats five times, i.e., at day 1, 8, 15, 22, and 29. Sections of the genital tract were microscopically examined at 33 days post-initial treatment (Table 3A). Vaginal inflammation scores were recorded prior to administration and at 4 and 24 hours after administration using the scoring scale (Table 3B). The results are Shown in Tables 3A and 3B.
aMineraliztion: the formation or deposition of minerals in a tissue
bInfiltration: the presence of mixed leukocyte (i.e. lymphocytes, dendritic cells, macrophage)
cMinimal: the amount of change barely exceeds normal limits
dMild: easy identification of the lesion with limited severity and no functional impairment
eWithin normal limits: the condition to be considered normal
apredose
b4 hour postdose
c24 hour postdose
dNumber of mice
eVaginal imitation severity scoring scale: 0 = no erythema, 1 = very slight erythema (barely perceptible), 2 = well-defined erythema, 3 = moderate erythema, 4 = sever erythema (redness) to eschar formation
As shown in Tables 3A and 3B above, pathological evaluation of the degree of inflammation of cervical tissues (Table 3A) and vagina (Table 3B) showed that the local administration of IL-7-Fc was safe and did not induce serious inflammation within genital tract.
IL-7-Fc was administered subcutaneously or intravaginally to mice (n=5/group) and the distribution of T cells in the cervix/vaginal tissues was observed by the method of Preparation Example 6.
As a result, as shown in
The therapeutic efficacy was confirmed using a TC-1 tumor cell line expressing HPV16 E6 and HPV E7 antigens. 1×106 TC-1/fluc cell line (which was manipulated to express the luciferase gene in the TC-1 cell line expressing the HPV16 E6 and E7 gene) was administered intravaginally to the mice (n=7 or 8/group). Four (4) days before administration of the TC-1/fluc cell line, 3 mg of medroxyprogesterone acetate (Depo-Provera, Pfizer) was administered subcutaneously to the Mice in the diestrus state. On the day of TC-1/fluc cell line administration, the mice were anesthetized and a mixture of 10 μl of 20% nonoxynol-9 (USP) and 40 μl of 3% carboxymethyl cellulose (CMC) (Sigma-Aldrich) was administered intravaginally to the mice, and 6 hours later, the mice were anesthetized again and their vaginas were washed with PBS and then TC-1/fluc cell line was administered to the mice.
At 1, 8, and 15 days after TC-1/fluc cell line administration, 1 μg of IL-7-Fc was intravaginally administered to the mice, and the cancer progression was investigated by in vivo Bioluminescence imaging at days 8 and 15. At day 20, the anticancer effect was examined by observing the appearance (
| Number | Date | Country | |
|---|---|---|---|
| 62361170 | Jul 2016 | US | |
| 62360696 | Jul 2016 | US | |
| 62263262 | Dec 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15775182 | May 2018 | US |
| Child | 18327423 | US |
