Patent Application
20220193233
Implementations of the present disclosure are generally directed to focused cancer immunotherapy and related treatment methods. Particularly, implementations of the present disclosure are directed to stabilized artificial immunocomplexes—comprising immunoglobulin protein molecules (antibody) genetically fused to oncogenic protein molecules (oncogen) with linker peptides. A general implementation of the present disclosure involves the deployment of said stabilized artificial immunocomplexes to program B Lymphocytes (B cells) and dendritic cells effective to activate endogenous immune response to oncogenic protein molecules expressed by cancerous tumor cells. [Engeroff et al, (2017)].
In some embodiments, the disclosed treatment method is employed to treat cancers that overexpress the oncogenic protein Her-2/neu using a stabilized artificial immune complex active immunization strategy that supports B cell programming of epitope specific immunoglobulins, which leads to the creation of Her-2/neu specific B memory cells. In some embodiments, the disclosed treatment method supports dendritic cell programming of epitope specific immunoglobulins leading to Her-2/neu T Cell activation and immune response. [Leon, et al (2014)].
In some embodiments, B cells programmed by the stabilized artificial immunocomplexes of the disclosed treatment method have the capacity to express tumor-specific oncogen on its cell surface; and further, produce and secrete the antibody portion of the programming immunocomplexes into a fluid of a subject. In some embodiments, expressed tumor-specific oncogen is available to activate T Cell-based immune response in a subject. In some embodiments, secreted antibodies are available to bind proteins that match the oncogen portion of the immunocomplexes with specificity when expressed by cancerous tumor cells or when encountered in fluid or in tissue—thereby neutralizing their oncogenic properties. Moreover, in some embodiments, programmed B cells can proliferate into B cells with memory when an immune response is triggered by the presence and detection of the oncogen portion of the immunocomplexes. [Daniels, et al. (2003), Engeroff et al. (2017), Lapointe et al. (2003)].
In some embodiments, dendritic cells programmed by the stabilized artificial immunocomplexes of the disclosed treatment method have the capacity to express tumor-specific oncogen on its cell surface. In some embodiments, expressed tumor-specific oncogen is then available to activate T Cell-based immune response in a subject. [Leon, et al (2014)].
In some embodiments, the stabilized artificial immune complexes (ICs) of the present disclosure comprise direct and stable products of immunological recognition by humoral immunity. In some embodiments, these ICs support humoral immune response to cancer in a subject by programming activate B cells and dendritic cells.
In general, ICs can initiate B cell activation by binding to low affinity receptor sites on the B cell surface. Interleukins or helper T Lymphocytes (T cells) costimulate B cells to complete B cell activation. In most cases, both oncogen and a costimulator are required to activate a B cell and initiate B cell proliferation. B cells proliferate and differentiate into plasma cells. The plasma cells produce antibody with the identical oncogen specificity as the oncogen expressed by activated B cells. Antibody is released from the plasma cells and circulates throughout the subject, binding to specific oncogen that may be circulating freely or expressed on cell surfaces. Activated B cells produce memory B cells that provide future immunity.
In general, ICs can activate dendritic cells by binding its antibody portion to high affinity receptor sites on the dendritic cell surface. Activated dendritic cells release interleukins that costimulate B cells and initiate T cell activation. Once activated, dendritic cells express oncogen on its surface to activate T cell immune response.
Accordingly in some embodiments, the present disclosure comprises a stabilized artificial immunocomplex comprising a protein molecule genetically fused to an isolated antibody. In some embodiments, the protein molecule comprises a tumor-specific oncogen derived from a tumor sample extracted from a subject. In some embodiments, the immunocomplex comprises SEQ ID NO. 1 (see sequence listing). In some embodiments, the immunocomplex comprises SEQ ID NO. 2 (see sequence listing). In some embodiments the immunocomplex comprises a gene N-terminal signal peptide such as SEQ ID NO. 3 (see sequence listing). In some embodiments, the protein molecule is a Her-2/neu molecule with a full ectodomain comprising SEQ ID NO. 4 (see sequence listing). In some embodiments, the protein molecule is a Her-2/neu molecule with a truncated ectodomain such as SEQ ID NO. 5 (see sequence listing). In some embodiments, Her-2/neu is genetically fused to an isolated antibody with an amino acid tether comprising SEQ ID NO. 6 (see sequence listing). In some embodiments, the Her-2/neu protein molecule is genetically fused, at its domain III or at its domain IV, to the isolated antibody. In some embodiments, the Her-2/neu molecule is genetically fused to the isolated antibody ex vivo. In some embodiments, the Her-2/neu is genetically fused, at a C-terminus of its domain III or its domain IV, to an N-terminus of the isolated antibody. In some embodiments, the isolated antibody comprises an IgE class antibody. In some embodiments, the isolated antibody comprises an ε-heavy chain variable region comprising SEQ ID NO. 7 (see sequence listing) and a light chain variable region comprising SEQ ID NO. 8 (see sequence listing), both of which are similar to those found in the monoclonal antibody trastuzumab. In some embodiments, the isolated antibody comprises an Fc region that binds to a FcεRI (CD23) receptor on B cell, or a FcεRI receptor on a dendritic cell, or a FcεRII receptor on a dendritic cell. In some embodiments, the isolated antibody is a recombinant monoclonal antibody comprising ε-heavy chain variable region SEQ ID NO. 7 and light chain variable region SEQ ID NO. 8. In some embodiments, the isolated antibody is a humanized or chimeric antibody. In some embodiments, the isolated antibody is an antibody fragment. In some embodiments, the immunocomplex is mono-epitopic. In some embodiments, the immunocomplex has similar binding specificity in each arm of the N-terminus of the isolated antibody of the immunocomplex. In some embodiments, the immunocomplex has similar tumor-specific oncogen linked to the complimentary determining region (CDR) of each arm of the N-terminus of the isolated antibody of the immune complex. Additionally, in some embodiments present disclosure comprises a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises the stabilized artificial immunocomplex. In some embodiments the stabilized artificial immunocomplex of the pharmaceutical composition comprises a protein molecule genetically fused to an isolated antibody. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. Additionally, the present disclosure comprises a method for treating a malignant breast cancer tumor in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition. In some embodiments, the malignant breast cancer tumor overexpresses Her-2/neu.
In a general implementation of the present disclosure, systems, apparatus, and methods to program B cells with a stabilized artificial immunocomplex (IC) include: designing and administering a stabilized, artificial IC into a subject to promote the binding of the stabilized, artificial IC to a B cell at the B Cell's low affinity receptor site; designing and administering a stabilized, artificial IC into a subject to promote endocytosis of the full stabilized, artificial IC intact into the B cell; designing and administering a stabilized, artificial IC into a subject to promote transport of the oncogenic portions of the stabilized artificial IC to B cell sites that initiate B cell activation; designing and administering a stabilized, artificial IC into a subject to promote the transformation of B cells into effective agents that stimulate the subject's endogenous immune response to overexpressed Her-2/neu without triggering autoimmunity.
In a general implementation of the present disclosure, systems, apparatus, and methods to program dendritic cells with stabilized artificial ICs include: designing and administering a stabilized, artificial IC into a subject to promote the binding of the stabilized, artificial IC to a dendritic cell high affinity receptor site; designing and administering a stabilized, artificial IC into a subject to promote endocytosis of the stabilized, artificial IC into a dendritic cell; designing and administering a stabilized, artificial IC into a subject to promote interleukin secretion from a dendritic cell into the fluid of a subject to support B cell costimulation and T cell activation; designing and administering a stabilized, artificial IC into a subject to promote transport of oncogenic portions of the stabilized artificial IC to dendritic cell sites that initiate T cell immune response; designing and administering a stabilized, artificial IC into a subject to promote the transformation of dendritic cells into effective agents that stimulate the subject's endogenous immune response to overexpressed Her-2/neu without triggering autoimmunity.
An aspect combinable with the general implementation further includes stabilized artificial ICs programming B cells that differentiate into plasmid cells specialized to generate IC-comprising antibody tailored to bind and neutralize IC-comprising oncogen expressed by cancerous tumor cells with binding specificity.
In an aspect combinable with any of the previous aspects, stabilized artificial ICs program B cells that produce memory B cells with the capacity to differentiate into specialized plasmid cells upon re-exposure to the IC-comprising oncogen.
It is appreciated that methods in accordance with the present disclosure can include any combination of the aspects and features described herein. That is, methods in accordance with the present disclosure are not limited to the combinations of aspects and features specifically described herein, but also may include any combination of the aspects and features provided.
The details of one or more implementations of the present disclosure are set forth in the accompanying drawings and the description below. Other features and advantages of the present disclosure will be apparent from the description and drawings, and from the claims.
Cancer immunotherapies such as antibodies targeting T cell checkpoints, or adaptive tumor-infiltrating lymphocyte (TIL) transfer, can be used to boost immune response against human malignancies. However, activation of T cells by such antibodies can lead to the risk of autoimmune diseases. Moreover, the selection of tumor-reactive T cells for TIL relies on information regarding mutated oncogen in tumors and does not reflect other factors involved in protein oncogenicity. Therefore, the disclosed treatment method incorporates therapeutic interventions by which T cell reactivity against tumor cells is selectively enhanced (i.e., focused cancer immunotherapy) based on tumor oncogen that are specifically expressed in the tumor of a certain cancer and in many patients with that cancer.
The humanized monoclonal IgG1 antibody trastuzumab (Herceptin®) binds to the extracellular domain of HER-2/neu. Trastuzumab was initially approved in 1998 by the Food and Drug Administration for the treatment of HER-2/neu overexpressing in advanced breast cancers. [Ahn and Vogel (2012)]. Since then, trastuzumab has shown efficacy against breast cancer both as an adjuvant therapy and as a treatment of metastatic disease. However, most patients with advanced breast cancer that are treated with trastuzumab alone or combined with chemotherapeutic agents eventually relapse and the median time to progression is less than 1 year. Additionally, a significant number of breast cancer patients do not respond to trastuzumab-based therapies despite the high level of HER-2/neu expression. Furthermore, in a Phase II clinical trial in patients with HER-2/neu overexpressing recurrent or refractory ovarian or primary peritoneal carcinoma that were treated with trastuzumab alone, a low rate of objective response (7.3%) was observed. [Bookman et al. (2003)]. While trastuzumab has shown efficacy in a subset of patients with either breast or ovarian cancer, additional strategies to target HER-2/neu overexpressing tumors are still needed.
Like trastuzumab, most antibody therapies for the treatment of cancer utilize antibodies that are of the IgG class. However, antibodies of the IgE class may also be potential cancer therapeutics since they have several potential advantages over their IgG counterparts. IgE mediates allergic reactions, which is due to the presence of effector cells in the tissue that are sensitized by IgE bound to Fc epsilon receptor I (FcεRI). These effector cells are degranulated after crosslinking of the IgE that is triggered by a multi-epitope oncogen interaction. IgE can also mediate oncogen presentation via the interaction with FcεRs expressed on oncogen-presenting cells (APC) such as dendritic cells (DC). IgE has been suggested to provide protection against parasitic infections, although this function is controversial. Research on cancer and IgE belongs to the new field of onco-immunology. This field has two aims: (1) to reveal the function of IgE-mediated immune responses against cancer cells to elucidate the understanding of its biology and (2) to develop novel IgE-based treatment options against malignant diseases. [Jensen-Jarolim et al. (2019)]. A key advantage associated with IgE is its exceptionally high affinity for the FcεRs. There are two FcεRs, the FcεRI which binds IgE with high affinity (with an association constant on the order of 1010 M−1) and is expressed on human dendritic cells; and the FcεRII (CD23) which binds IgE with lower affinity (with an association constant on the order of 108 M−1) and is expressed on human B cells. [Chauhan et al. (2020)]. Thus, the affinity of IgE for FcεRI is at least two orders of magnitude higher than that of IgG for the FcγRs (FcγRI-III) and in the case of FcεRII is as high as that of IgG for its high-affinity receptor FcγRI (CD64). [Chauhan et al. (2020)]. Another advantage of the IgE molecule is the low endogenous serum concentration in humans, which is only 0.02% of total circulating immunoglobulins, whereas IgG is the most abundant at 85%. [Chauhan et al. (2020)]. Thus, the competition for FcR occupancy is much lower for IgE. Another potential advantage is that there is no known inhibitory FcεR as there is for FcγR.
Fully human anti-HER-2/neu IgE has been developed and evaluated for its potential as a cancer therapeutic. After an immunogenic stimulation in a subject, antibodies are produced by specialized B cells for the purpose of combining with the evoking oncogenic determinants wherever they are encountered, thereby forming immune complexes (ICs). This process is part of a humoral immune response and is usually of benefit to the subject because it leads to the neutralization or elimination of the oncogen.
ICs act as regulatory factors in immune responses because they can interact with oncogen receptor-bearing lymphocytes and subpopulations of T and B cells, as well as unclassified lymphocytes and macrophages having Fc and complement (C) receptors. ICs can modulate humoral and cellular immune responses by interacting with B and T cells having Fc, C, and/or oncogen receptors. Through such interactions, ICs may suppress or augment immune responses, depending on the molar ratio of the oncogen and antibody, the epitope density of the complex, the steric and chemical conformation of oncogene, and the mass, class, and affinity of antibody.
In some embodiments, the formation and function of ICs according to aspects of the disclosure treatment method include two features. First, the immunoglobulin (Ig) class, which determines antibody valence for a specific oncogen as well as its ability to bind to cellular Fc receptors and to activate the C system, is IgE. Second, the association constant for the union of specific antibody and oncogen is sufficient to form a stabilized IC. Generally, B cells have receptors for IgE Fc. Therefore, in some embodiments, the artificial ICs of this disclosure, comprising IgE immunoglobulin bind to B cells at Fc sites. In some embodiments, IgE binding to B cells at its Fc site program B cells, which can act as effective agents to activate endogenous immune response when introduced within a subject without triggering autoimmunity.
Accordingly, implementations of the present disclosure are generally directed to focused cancer immunotherapy, allergo-oncology, and related treatment methods. Particularly, implementations of the present disclosure are directed to stabilized artificial immunocomplexes—comprising immunoglobulin antibody genetically fused to oncogenic protein (oncogen)—that program B Lymphocytes (B cells) and dendritic cells, effective to activate endogenous immune response to oncogenic protein expressed by cancerous tumor cells or encountered in a subject fluid.
In one implementation, the stabilized artificial immune complexes are directed to focused breast cancer immunotherapy and related treatment methods. Approximately 25% of breast cancers demonstrate amplification of the oncogen HER-2/neu, which is associated with more aggressive disease and poor prognosis. In some studies, HER-2/neu overexpression has also been described in 9-32% of ovarian cancer tissue. [Ahn and Vogel (2012)]. As is the case for breast cancer, HER-2/neu overexpression in ovarian cancer is associated with poor prognosis. [Ahn and Vogel (2012)]. HER-2/neu is a member of the epidermal growth factor receptor (EGFR) family that have intrinsic tyrosine kinase activity that leads to the activation of downstream signaling pathways of cell proliferation and survival. [Ahn and Vogel (2012)].
Implementations of the present disclosure are directed to IgE class immunoglobulins as target proteins to be genetically fused to oncogen in the formation of artificial ICs. Traditionally, immunoglobulin E (IgE) has an evolutionary role in mammals as the primary line of defense against parasites and venoms. [Mukai et al. (2016)]. In this role IgE acquire powerful effector functions to expedite adaptive immune sensitization. [Mukai et al. (2016), Sutton et al. (2019)].
IgE's Fc region receptor-binding is unique among other classes of immunoglobulins. The high affinity receptor FcεRI is structurally homologous to other members of the FcR family and is found on multiple effector cells. The second IgE receptor FcεRII (CD23) is unlike all other antibody receptors. It is a member of the C-type (Ca2+-dependent) lectin-like superfamily [Sutton et al. (2015), Griffith (2011)] and functions to endocytose the entire antigen/immunoglobulin immune complex. CD23 is the target receptor for this immune complex. [Sutton et al. (2019), Karagiannis (2001)].
IgE differs from the other sub-classes of immunoglobulins in its domain architecture, glycosylation, conformational dynamics, and allosteric properties. These attributes are favorable for the endocytosis of the entire antigen/immunoglobulin immune complex. IgE is the most heavily glycosylated member of the immunoglobulin family. [Sutton et al. (2019)]. Manipulation of the heavy chain glycosylation profile in this immune complex allows targeting of the FcεRII (CD23) IgE receptor on the surface of B cells and endocytosis of the antigen/immunoglobulin immune complex. [Sutton et al. (2019), Sutton et al. (2015), Griffith (2011), Karagiannis (2001)]. Glycosylation is functionally important for unloading of IgE/antigen complexes by CD23 in endosomes. [Sutton et al. (2019), Karagiannis (2001)].
Although the FcεRI and FcεRII work in concert and are engaged by the Fc portion of the immunocomplex of the disclosure, the glycosylation sites on the fully constant region are altered to express greater affinity for the unique secondary IgE receptor FcεRII/CD23. The CD23 receptor has the function of transferring immunocomplex from cell surface CD23 receptors on the surface of B cells into non-degradable compartments within the B cell and recycling them as cell surface receptors for immune antigen sensitization interactions with CD4 and CD8 t-cells and dendritic cells. [Karagiannis (2001)]. B cell activation by CD23 endocytosis has also been shown to initiate B cell migration from lymph node and spleen to tumor tissues for sensitization of T cells and production of tumor specific immunoglobulin. [Acharya et al. (2010)].
It has been previously shown that IgE complexed with a mono-epitopic antigen through its interaction with CD23/Fcε RII is capable of mediating antigen presentation in the absence of receptor cross-linking. [Daniels et al. (2012), Bheekha et al. (1995)]. In some embodiments of the instant disclosure, the immunocomplex is mono-epitopic in that it has similar specificity and similar antigen linked to the complimentary determining region (CDR) of each arm of the N-terminus of the immunoglobulin E component of the immunocomplex.
An example artificial recombinant IC 100 comprising an IgE immunoglobulin protein molecule genetically fused to a Her-2/neu oncogenic protein molecule is shown in
An example IgE immunoglobulin molecule 200 comprising In Planta glycosylation of IgE to target low affinity CD23 IgE receptor on B-cells is shown in
An example of a designed scheme to form a Her-2/neu oncogenic protein molecule comprising a truncated domain III and IV 310 from a Her-2/neu protein with a full ectodomain (Domain I-IV) 300 is shown in
An example designed Her-2/neu oncogenic protein molecule interacting with an example IgE immunoglobulin 410 to form 100 is shown in
100 can interact with two immune cell-mediated therapeutic pathways as shown in
Reference is now made to the following examples, which together with the above descriptions, illustrate embodiments of the disclosure in a non-limiting fashion.
The following examples describe materials and methods to produce a recombinant protein (target protein) comprising an IgE molecule specific for receptor tyrosine-protein kinase erbB-2 (also known as Her-2) [Homo sapiens], that is genetically fused to the same Her-2 antigen. One having ordinary skill in the art will recognize that the fusion protein permits binding of the antigenic epitope of Her-2 by the CDR regions of the antibody, thus making an immune complex. Generally, the nomenclature used herein and the laboratory procedures utilized in the present disclosure include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature.
Designing fusion proteins: The target protein comprises an IgE molecule composed of two heavy chains (heavy chain, epsilon type) and two light chains (light chain) assembled in the usual way [see e.g., Diamos et al, (2019)], wherein the antigen combining site is specific for Her-2 antigen, and the heavy chain and light chain variable regions are the same as those found in the monoclonal antibody trastuzumab (Herceptin®) (i.e. PDB: 6OGE_E and 6OGE_D) with a structure similar to that indicated in NCBI Structure Database 6OGE. Further, the target protein comprises Her-2 antigen fused to the variable region of either the heavy chain or light chain of trastuzumab. Using the published structures determined for trastuzumab bound to Her-2 as guides, the instant design discloses several embodiments for genetic fusions including: 1) C-terminus of Her-2 fused to N-terminus of heavy chain or 2) C-terminus of Her-2 fused to N-terminus of light chain, and the use of linker peptides of varying lengths and content of glycine/serine. Other embodiments include 1) use of the full length ectodomain of Her-2 vs. the C-terminal domain IV of Her-2 to which the antibody binds, and 2) use of the native Her-2 ER-targeting signal peptide vs. the barley alpha amylase gene signal peptide that have been used extensively for other recombinant antibodies and antibody fusion proteins.
Fusion Protein Amino Acid Sequence for IgE/her 2/Neu Immune Complex with Full her 2/Neu Ectodomain (SEQ ID NO. 1):
Fusion Protein Amino Acid Sequence for IgE/her 2/Neu Immune Complex with Truncated her 2/Neu Domains III and IV (SEQ ID NO. 2):
Fusion Protein Amino Acid Sequence for Barley Alpha Amylase Signal Peptide (BASP) (SEQ ID NO. 3)
Fusion Protein Amino Acid Sequence for her 2/Neu with Full Ectodomain (SEQ ID NO. 4)
Fusion Protein Amino Acid Sequence for her 2/Neu with Truncated Ectodomain Comprising Domains III and IV (SEQ ID NO. 5)
Fusion Protein Amino Acid Sequence for Linker Peptide Tether (SEQ ID NO. 6)
Fusion Protein Amino Acid Sequence for Trastuzumab-IgE c-Heavy Chain (PDB:60GE_E) (SEQ ID NO. 7)
Fusion Protein Amino Acid Sequence Trastuzamab-IgE Light Chain (PDB:60GE_E). KEGG Entry D03257 214 Amino Acids 23.47 KDa FASTA (SEQ ID NO. 8)
Constructing plasmid vectors for expression of target protein candidates: In another embodiment, the disclosure enables the design of DNA coding sequences for the proteins with codons are optimized for expression in a plant subject Nicotiana benthamiana, with restriction sites to facilitate gene cloning, given a set of candidates Her-2-IgE fusion proteins (See
Expression and testing of target protein candidates for selection of the optimal construct: In another embodiment, agro-infiltration of plasmid vectors into leaves of N. benthamiana enable recombinant expression of the target proteins. The plants must be monitored daily, and leaves collected every 3, 4, and 5 days after infiltration to optimize timing. In another embodiment, soluble proteins are extracted from leaves, and resolved by SDS-PAGE under either reducing or non-reducing conditions. Reducing conditions show the heavy chain and light chain polypeptides at the expected molecular weights (depending on type of Her-2 fusion), while non-reducing conditions maintain disulfide-bonds of correctly assembled high-molecular weight complexes (H2L2). In another embodiment, gels are stained for total protein, and separate gels are electro-transferred to PVDF membranes for western blotting. The blots are probed with antibodies specific for IgE heavy chain, light chain, and Her-2 to verify that all components are present in the high-MW complexes. Examination of gels and blots provide evidence of proteolytic degradation, especially since the composition or length of the linker peptides affects the stability of the fusion protein in vivo as well as during protein extraction and analysis. One of ordinary skill would recognize that an optimal construct can be selected based on analysis of these data.
Demonstration of correct binding of the IgE variable regions with the Her-2 antigenic site can be achieved through examination of molecular structures by methods such as X-ray crystallography, cryo-electron microscopy, or nuclear magnetic resonance studies. [Hao, (2019)]
Purifying optimal target protein products: Purification of a selected target protein can be achieved using protein A-affinity chromatography after filtration of soluble leaf extracts to remove bacteria-size particles. Following low-pH elution and immediate neutralization, size-exclusion chromatography is used to purify the full-size, correctly assembled complexes. If concentration of the target protein is needed, centrifugal concentrators can be used to obtain the appropriate protein content. One of ordinary skill would recognize that the final product from the centrifuge can be assayed by SDS-PAGE and western blot to confirm molecular integrity.
Using modified IgE to produce an IgE antibody/Her-2 Immune Complex: Targeted oncogen consists of the truncated ectodomain of human Her-2. The truncated ectodomain enhances immune complex stability. In some embodiments, domains III and IV are separated from domains I and II of the full length ectodomain of Human Her-2. In some embodiments, domains III and IV are attached to the complimentary regions of the N-terminus variable regions of the IgE molecule with a fusion sequence of at least 10 amino acids.
The immune complex is designed to ensure stability during processing as an antigen/antibody immune complex capable of negotiating a dysregulated immune environment intact and engaging the targeted CD23 low affinity receptor on B-cells as well as the FcεRI high affinity IgE receptor.
As shown in
Several implementations have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. For example, various forms of the methods described above may be used, with steps re-ordered, added, or removed. Accordingly, other implementations are within the scope of the following claims.
This application claims priority to pending U.S. Provisional Patent Application No. 63/199,056 filed on Dec. 3, 2020, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63199056 | Dec 2020 | US |
