Patent Application
20250222030
This application claims priority to Norwegian Application NO20240016 filed on Jan. 4, 2024, which is incorporated herein by reference in its entirety.
The Instant Application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Dec. 19, 2024, is named “NK-TCR with BiTE Sequence listing.xml” and is 14,991 bytes in size. The Sequence Listing does not go beyond the disclosure in the application as filed.
The present disclosure broadly relates to immunotherapy, in particular to cell therapy involving NK cells.
T cells have been used in cell therapy. Naturally occurring T cells express a T cell receptor (TCR). Such cells can be engineered to express other receptors. One example is chimeric antigen receptors (CARs) comprising a targeting unit from antibodies, usually a single-chain Fv (scFv). Several drugs have been approved in USA based on T cells expressing CARs, for example Yescarta®.
In contrast to most conventional antibodies and conventional CARs, TCRs can recognize and bind intracellular target epitopes because proteins are degraded in the cells and peptides from such degradation are presented on the major histocompatibility complex (MHC) on the cell surface.
Considering that all the proteins expressed by a given cell may be degraded into peptides presented on an MHC, TCRs can potentially recognize the whole proteome. This represents a striking numerical advantage over conventional CARs in terms of possible targets. In addition, TCRs can be specifically directed against a mutant variant of a protein while sparing the wild type form. Accordingly, the TCR can distinguish cancer cells expressing the mutated protein from healthy cells expressing the non-mutated protein.
On the other hand, TCRs are complicated molecules to manipulate: they are heterodimers composed of an α- and a β-chain, they do not signal by themselves but require a battery of signalling proteins associated to recruit all the components to create an immune synapse. In addition, their localization in the plasma membrane is believed to depend on co-expression of CD3-chains.
Consequently, TCR-based cell therapy has primarily relied on T cells. However, recombinant expression of TCRs may compete with the endogenous TCR for the “use” of the signalling proteins. Another issue with the introduction of a second TCR into T-cell, is the risk associated with the possibility of forming mixed dimers, thus reducing the number of correct or therapeutic TCR molecules expressed in the cell. In addition, mispairing might generate novel TCRs which could potentially be toxic. Although such “mispairing” of TCRs has yet to be documented in a clinical setting, an important number of innovations has been developed in order to prevent this.
Natural Killer (NK) cells were discovered almost 50 years ago and identified by their ability to recognize and kill tumor cells without the requirement of prior antigen exposure. Since their discovery, NK cells have grown as promising agents for cell-based cancer immunotherapies. However, naturally occurring NK cells do not express TCRs.
The present disclosure provides NK cells expressing a TCR-complex and secreting a bispecific protein (see
The present disclosure provides a Natural Killer cell comprising nucleic acids for expressing an αβ T-cell Receptor, CD3ζ, CD3γ, CD3δ and CD3ε in its cell membrane, and nucleic acid for expressing and secreting a bispecific protein, wherein the bispecific protein comprises a first Fv for binding to a first target epitope and a second Fv for binding to a second target epitope, wherein the first Fv specifically binds, under physiological conditions, to an epitope located on the extracellular part of a CD3-chain, and wherein the second Fv specifically binds, under physiological conditions, to an epitope located on target cells.
In one aspect, the TCR and the bispecific protein recognize different epitopes of the same target protein from the same target cells.
In one aspect, the TCR and the bispecific protein recognize epitopes of different target proteins from the same target cells.
In one aspect, the TCR specifically binds, under physiological conditions, to a peptide from Melanoma-associated antigen 4 presented on an HLA, and the second Fv specifically binds, under physiological conditions, to an extracellular target epitope located on a human cancer cell selected from a group consisting of non-small cell lung cancer, ovarian cancer, melanoma cancer, synovial sarcoma and gastroesophageal cancers.
In one aspect, the TCR specifically binds, under physiological conditions, to a peptide from Kita-Kyushu Lung Cancer Antigen-1 presented on an HLA, and the second Fv specifically binds, under physiological conditions, to an extracellular target epitope located on a human cancer cell selected from a group consisting of breast cancer, gastric cancer, lung cancer, pancreatic cancer and cervix cancer.
In one aspect, the TCR specifically binds, under physiological conditions, to a peptide from PRAME presented on an HLA, and the second Fv specifically binds, under physiological conditions, to an extracellular target epitope located on a human melanoma cell.
In one aspect, the first Fv specifically binds, under physiological conditions, to an extracellular epitope located on a human CD3ε protein.
In one aspect, the second Fv specifically binds, under physiological conditions, to an extracellular epitope located on human cancer cells of solid tumor origin. One example of a BiTE comprising such Fv is provided by SEQ ID NO: 3 (the TP3 BiTE).
In one aspect, the first and/or second Fv is a single-chain Fv.
In one aspect, the NK cell is a human cell line or human primary cell.
The present disclosure also provides a cell population comprising the cells disclosed above.
The present disclosure also provides an aqueous pharmaceutical composition for intravenous or intratumoral administration comprising the cells disclosed above or the cell population disclosed above, wherein the composition is sterile and/or isotonic.
The present disclosure also provides a method of treatment of solid tumors comprising the step of administering the pharmaceutical composition disclosed above to a human patient in need thereof.
Natural killer cells, also known as NK cells, are a type of cytotoxic lymphocyte within the innate immune system. NK cells generally provide rapid responses to virus-infected cell and other intracellular pathogens compared to T cells in the vertebrate adaptive immune response. Accordingly, NK cells may provide a first-line defense against tumor formation.
While T cells detect the antigen presented on major histocompatibility complex (MHC) on infected cell surfaces, NK cells can recognize and kill target cells in the absence of antibodies and MHC, allowing a faster immune reaction.
NK cells were named “natural killers” because they do not require activation to kill cells that are missing “self” markers of MHC class I. This is important because harmful cells that are missing MHC I markers cannot easily be detected and destroyed by T cells.
NK cells differentiate from CD127+ common innate lymphoid progenitors, which are downstream of the common lymphoid progenitor from which B and T lymphocytes are also derived. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, where they then enter into the circulation.
Naturally occurring NK cells express CD56 in their cell membrane, but they are CD3ε-negative in antibody-assays.
NK cells in the present disclosure differ from natural killer T cells (NKTs) phenotypically, by origin and by respective effector functions. NKT-cell activity promotes NK-cell activity by secreting interferon gamma. In contrast to NKT cells, naturally occurring NK cells do not express TCRs or CD3ε in their cell membrane. However, they usually express the surface markers CD16 and CD57 in humans, NK1.1 or NK1.2 in C57BL/6 mice. The NKp46 is also an NK cell marker.
The term “NK T cells” was first used in mice to define a subset of T cells that expressed the NK-cell-associated marker NK1.1 (CD161). It is now generally accepted that the term “NKT cells” refers to CD1d-restricted T cells, present in mice and humans, some of which co-express a heavily biased, semi-invariant T-cell receptor and NK-cell markers.
In the last decade, several NK-cell based anti-cancer products have undergone clinical trials yielding promising results. However, in order to develop alternative NK-cell products for therapy, novel strategies for increasing safety, efficiency, and specificity are needed.
Introduction of a functional TCR complex to NK cells that can inherently detect and eliminate virally infected and/or tumor cells enhances the efficiency of identification and killing of target cells as well as circumventing potential risk of TCR mispairing. The recombinant technology for expressing TCR-complexes in NK cells by co-expression of TCRs and CD3-chains is thoroughly described in Mensali et al 2019, EBioMedicine. 2019 February:40:106-117. doi: 10.1016/j.ebiom.2019.01.031. As disclosed in WO2018129199A1, recombinant expression of CD3γ, CD3δ and CD3ε together with a TCR is sufficient for expressing TCR-complexes in the cell membrane of NK cells.
The present disclosure primarily concerns human cells as a starting material. Such cells may be isolated from a healthy subject by leukapheresis or other suitable methods. Cells isolated from a healthy subject are primary cells. However, other non-human mammalian cells can also be used.
Culturing of primary NK cells can be challenging, but it is disclosed in Mu et al., BMC Biotechnology, 19, Article number 80 (2019), Klingmann, Cytotherapy, vol. 17, Issue 3, March 2015, p. 245-249, Koepsell et al., Transfusion, vol. 53, issue 2, February 2013, p. 404-410. However, culturing of “immortalized” cells i.e. cell-lines, is generally easier for consistently obtaining larger cell populations. The human cells in the present disclosure can for example be cell-lines like NK-92 or YTS. Such cell lines are commercially available from the American Type Culture Collection (ATCC).
WO2015054299A1 discloses a protocol for culturing as well as media for storage and transport of NK-92 cell line. Said disclosure relates generally to a cell storage medium for transport and/or delivery of cells. For example, it relates to a sterile, isotonic NK-92 cell storage medium for transport and delivery of NK-92 cells, the storage medium comprising: human serum or human serum albumin; an initial density of non-irradiated, substantially non-aggregated NK-92 cells sufficient to provide a therapeutic amount of NK-92 cells at the time of delivery to a treatment facility; and an IL-2 concentration of about 200 IU/mL wherein said medium is maintained within +/−5° C. of a temperature selected for transport, and further wherein said temperature selected for transport is between about 20° C. to about 40° C. in the presence of sufficient oxygen to maintain viability of the cells, such that the NK-92 cells remain viable for administration to a patient up to a period of at least 24 hours after placement into the storage medium.
TCRs are well-known receptors. Their variable domains, either Vα and Vβ or Vγ and Vδ, form a single antigen binding unit based on six complementarity determining regions, CDRs. The CDRs are flanked by framework sequences. The first framework sequence is N-terminal to the CDR1, the second framework sequence is located between CDR1 and CDR2, while the third framework sequence is located between CDR2 and CDR3. Accordingly, both a Vα and a Vβ can be roughly visualized as follows, with the CDRs boxed and the N-terminal indicated as N-:
However, the detailed mechanism of action of TCRs is not completely understood. As visualized in
The TCR target epitopes are peptides presented by MHC. TCRs can specifically bind their target epitopes when the cognate peptide is presented by MHC, but they will show little or no binding to MHC when no peptide is presented, or a different peptide is presented.
MHC class I presents peptides comprising 8 to 10 amino acid residues from inside the cell. MHC class II presents peptides comprising 13 to 25 amino acid residues from outside the cell. The human MHC is called HLA (human leukocyte antigen complex).
In one aspect, the NK cells disclosed herein can express TCRs that specifically bind, under physiological conditions to cognate peptides when presented by HLA-A, HLA-B or HLA-B (i.e. MHC class I). These cognate peptides can represent “intracellular target epitopes”.
In one aspect, the NK cells disclosed herein can express TCRs that specifically bind, under physiological conditions to cognate peptides when presented by HLA-DP, HLA-DM, HLA-DQ, HLA-DR (i.e. MHC class II).
Accordingly, in one aspect, the NK-cell disclosed herein expresses a TCR-complex in the cell membrane, wherein the TCR specifically binds, under physiological conditions to a cognate peptide when presented by MHC class I on cancer cells, and wherein the NK-cell secretes a bispecific protein comprising a first targeting unit for specific binding, under physiological conditions, to a target epitope located on the extracellular part of a CD3-chain, and a second targeting unit for specific binding, under physiological conditions, to a target epitope located on cancer cells.
Accordingly, in one aspect, the NK-cell disclosed herein expresses a TCR-complex in the cell membrane, wherein the TCR specifically binds, under physiological conditions to a cognate peptide when presented by MHC class I on cancer cells, and wherein the NK-cell secretes a bispecific protein comprising a first Fv for binding to a first target epitope and a second Fv for binding to a second target epitope, wherein the first Fv specifically binds, under physiological conditions, to an epitope located on the extracellular part of a CD3ε-chain, and wherein the second Fv specifically binds, under physiological conditions, to an epitope located on cancer cells.
Accordingly, in one aspect, the NK-cell disclosed herein expresses a TCR-complex in the cell membrane, wherein the TCR specifically binds, under physiological conditions to a cognate peptide when presented by MHC class I on cancer cells, and wherein the NK-cell secretes a bispecific protein comprising a first Fv for binding to a first target epitope and a second Fv for binding to a second target epitope, wherein the first Fv specifically binds, under physiological conditions, to an epitope located on the extracellular part of a CD3δ-chain, and wherein the second Fv specifically binds, under physiological conditions, to an epitope located on cancer cells.
Accordingly, in one aspect, the NK-cell disclosed herein expresses a TCR-complex in the cell membrane, wherein the TCR specifically binds, under physiological conditions to a cognate peptide when presented by MHC class I on cancer cells, and wherein the NK-cell secretes a bispecific protein comprising a first Fv for binding to a first target epitope and a second Fv for binding to a second target epitope, wherein the first Fv specifically binds, under physiological conditions, to an epitope located on the extracellular part of a CD3γ-chain, and wherein the second Fv specifically binds, under physiological conditions, to an epitope located on cancer cells.
Accordingly, in one aspect, the NK-cell disclosed herein expresses a TCR-complex in the cell membrane, wherein the TCR specifically binds, under physiological conditions an “intracellular target epitope” when the peptide presented by MHC class I on cancer cells, and wherein the NK-cell secretes a bispecific protein comprising a first Fv for binding to a first target epitope and a second Fv for binding to a second target epitope, wherein the first Fv specifically binds, under physiological conditions, to an epitope located on the extracellular part of a CD3ε-chain, and wherein the second Fv specifically binds, under physiological conditions, to an extracellular target epitope located on the same cancer cells.
Accordingly, the modified NK cells disclosed herein can display an MHC-restricted antigen-specific cytotoxicity and they may be useful in therapy of cancer, viral infections, autoimmunity, and graft versus host disease (GvHD).
In contrast to TCRs, most naturally occurring antibodies comprise two identical Fv's (see
An Fv is a protein moiety able to bind a target epitope under physiological conditions. The Fv comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH). Such variable domains are well-known for skilled persons.
Provided herein are NK cells expressing and secreting a protein comprising a first targeting unit for binding to a first target epitope and a second targeting unit for binding to a second target epitope. Such proteins are bispecific.
The first and second targeting unit may have any format as long as they provide specific binding, under physiological conditions, to their target epitopes. Such binding units may be obtained by phage display libraries by conventional screening.
They can also be obtained from TCRs. However, more conventionally, such targeting units may be obtained from antibodies of any type including single-domain antibodies.
In particular, provided herein are NK cells expressing and secreting proteins comprising a first Fv for binding to a first target epitope and a second Fv for binding to a second target epitope. Of course, the first and second target epitope are not identical.
In particular, provided herein are NK cells expressing and secreting proteins comprising a first and a second Fv for binding to a first and a second target epitope, wherein each Fv comprises a VL and a VH, and the VL comprises three CDRs flanked by framework sequences, and the VH comprises three CDRs flanked by framework sequences, wherein the first Fv specifically binds, under physiological conditions, to an epitope located on the extracellular part of a CD3-chain, and wherein the second Fv specifically binds, under physiological conditions, to an epitope located on target cells. The target cells can by any malignant cells such as cancer cells.
Each VL and VH herein comprises three complementarity determining regions (CDRs) flanked by framework sequences. Framework sequences are structurally conserved regions that normally tend to form a β-sheet structure positioning the CDRs for specific binding to the target epitope under physiological conditions.
The CDR sequences herein have been determined using the well-known Kabat system.
The first Framework sequence is N-terminal to the CDR1, the second Framework sequence is located between CDR1 and CDR2, while the third Framework sequence is located between CDR2 and CDR3. Accordingly, both a VL and VH can be roughly visualized as follows, with the CDRs boxed and the N-terminal indicated as N-:
In the bispecific proteins herein, each of the framework sequences in the first Fv may independently be human, or murine or of any other suitable source.
A framework sequence is human if it is present in a human antibody, and it is flanking a CDR in a human VL or human VH, or it is encoded by a human genomic sequence flanking a CDR in a human VL-sequence or human VH-sequence.
Antibodies obtained from humans will have human framework sequences. However, it is well known that murine framework sequences can be completely or partially replaced by human framework sequences. This process is called humanization, and the resulting Fv is thus humanized.
In one embodiment, the human framework sequences are mature human framework sequences available from known human antibodies. Without being bound by theory, such framework sequences may convey very low risk of triggering unwanted immunogenic responses against the Fv in humans, and at the same time increase the likelihood of obtaining stable binding units which are expressed well in cellular systems.
In one embodiment, the Fv comprises or consists of VL-linker-VH. In another embodiment, the Fv comprises or consists of VH-linker-VL.
Such antigen binding domains are often referred to as single chain Fv-fragments (scFv's). The linker between VL and VH in a scFv generally needs a certain length in order to allow the VH and VL to form a functional antigen binding domain. In one embodiment, the linker comprises 10 to 50 amino acid residues. In one embodiment, the linker comprises 15 to 25 glycine and/or serine residues.
Alternatively, the VH and VL may be connected by a disulphide bridges or alternative linkers. The VL and VH may be embedded in a Fab-fragment of an antibody or an antibody as such.
Bispecific proteins for binding and recruiting T cells to target cells can have a variety of formats. Examples of such T cell-engagers are BiTEs. For example, Blinatumomab is a bispecific CD19-directed CD3 T-cell engager that binds to CD19 expressed on the surface of cells of B-lineage origin and CD3 expressed on the surface of T cells. It activates endogenous T cells by connecting CD3 in the T-cell receptor (TCR) complex with CD19 on benign and malignant B cells. Blinatumomab mediates the formation of a synapse between the T-cell and the tumor cell, upregulation of cell adhesion molecules, production of cytolytic proteins, release of inflammatory cytokines, and proliferation of T cells, which result in redirected lysis of CD19+ cells.
Three suitable BiTEs are disclosed by protein sequences SEQ ID NO: 2 (CD19 BiTE), SEQ ID NO: 3 (TP3 BiTE) and SEQ ID NO: 4 (CD22 BITE).
TP3 is a known murine antibody, and the TP3 Fv including TP3 scFv's are described in WO2020127734 (A1). These Fv's specifically bind, under physiological conditions, to an epitope located on osteosarcoma cells. Accordingly, such BiTEs may be used in treatment of solid tumors like osteosarcoma.
Many types of bispecific T-cell engagers are known. Some examples are described by Goebeler and Bargou (Nat. Rev. Clin. Oncol. 17(7):418-434 (2020); doi: 10.1038/s41571-020-0347-5), or by Zhou et al. (Biomarker Research 9(38) (2021).
Nucleic acids encoding and expressing proteins are well known, for example DNA and RNA. The proteins mentioned herein can be encoded by recombinant nucleic acids. Recombinant nucleic acids are non-naturally occurring nucleic acids.
The recombinant nucleic acids may comprise promoters, enhancers and other well-known elements for providing expression of the bispecific proteins in NK cells.
In order to achieve secretion of the bispecific proteins herein, the nucleic acids encoding them needs to contain a signal sequence (13-30 aa) that will target the BiTE protein to the secretory pathway and optionally a terminal His tag that can be used for either purification or detection (
Pharmaceutical compositions can comprise the KK cells and a pharmaceutically acceptable excipient. The pharmaceutical compositions herein can be a composition suitable for administration of therapeutic cells to a patient. The most common administration route for therapeutic cells is intravenous administration. Accordingly, said pharmaceutical compositions may for example be sterile aqueous solutions with a neutral pH. Said pharmaceutical compositions can contain pharmaceutically acceptable excipients suitable for their intended use. Accordingly, said pharmaceutical compositions may for example be sterile aqueous solutions with a physiological pH. The sterile pharmaceutical compositions may be a cell suspension for infusion comprising from 1×106 NK cells to 1×1010 NK cells, such as 1×107 NK cells to 1×109 NK cells.
Such drugs can be for example be supplied in an infusion bag containing approximately 30 to 100 mL of a frozen suspension of the NK cells in 5% DMSO and 2.5% human serum albumin.
It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a cell”, is understood to represent one or more cells. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein. As any skilled person will understand, an NK-cell expressing a TCR-complex will normally express numerous such TCR-complexes in its cell membrane. Furthermore, an NK-cell expressing and secreting BiTEs will normally express and secrete numerous such BiTEs.
Physiological conditions, as used herein, means the environment encountered or simulated, in a living patient, where the disclosed cells and bispecific proteins are intended to bind an epitope located on target cells. In general, the physiological conditions in most human extracellular fluids are normally ca. 37° C., pH in the range of 6.0 to 7.5. However, in solid tumors, the physiological conditions i.e. the tumor microenvironment (TME) is often hypoxic and acidic.
The BiTE disclosed in the example comprise a well-known C-terminal oligopeptide comprising multiple histidine residues, also known as a “His-tag”, for purification and detection during research and early development. Such “His-tag” will not necessarily be present in a protein drug.
For promoting an understanding of the principles of the present disclosure, reference will now be made to embodiments illustrated herein and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope is thereby intended. Any alterations and further modification in the described NK cells, the described methods for producing/preparing the NK cells, the described therapeutic compositions containing the NK cells, and the described therapeutics use of the NK cells and any further application of the principles of the invention as described herein, are contemplated as would normally occur to one skilled in the art within the present field.
As used herein, NK-TCR means NK cells expressing a TCR-complex.
As used herein, NK-TCR-BiTE means NK cells expressing a TCR-complex and secreting a BiTE.
As used herein, target cells are malignant or otherwise undesired cells, e.g. cancer cells or autoimmune cells, which do not express TCR-complexes in their cell membrane in any significant amount.
BiTE anti-CD19:
METDTLLLWVLLLWVPGSTGDIQMTQTTSSLSASLGDRVTISCRASQDIS
GGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQ
METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVS
SEVQLQQSGAELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWIG
METDTLLLWVLLLWVPGSTGEVQLVESGGGLVKPGGSLKLSCAASGFAFS
GGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDG
GGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSG
METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQDVN
A retrovirus containing a transgene encoding for the anti-CD19 BiTE 2A-linked with GFP was produced into Hek cells. The supernatant of the Hek cells was harvested twice and stocked at 4° C. The supernatant was used to transduce NK-TCR cells. These NK-TCR were produced as previously depicted (Mensali et al. 2019) using a polycistronic CD3 construct and a human TCR (Radium-1, Inderberg et al. 2017). The transduction efficiency was monitored using GFP as a transduction marker.
NK-TCR cells transduced or not with CD19 BiTE were run into a flowcytometer and fluorescence was detected using laser excitement at 488 nm and detected at 510 nm. Expression of BiTE in NK cells by GFP detection is shown in
Expression of BiTE in NK cells by purification in the culture medium. NK-TCR-BiTE and NK-TCR were grown in NK medium at 1M/mL for 2 days and 1 mL of supernatant was harvested. Nickel slurry was used to pull down His-tagged BiTE from the supernatant and each fraction was separated by SDS-PAGE and transferred to a nitrocellulose membrane for Western blot detection using an anti-His antibody. As a control, a validated BiTE solution was used. For each test, the first fraction is the starting material which is not detected for the NK supernatants due to a concentration below detection level. The flowthrough of the slurry represents what is not bound, and here again, NK supernatants are negative whereas the large-scale beads seems to be more concentrated than the slurry's capacity, hence the presence of BiTE in this fraction. Finally, the beads were also run, they should contain the attached fraction, concentrated on the beads. As shown in
In order to validate that the released BiTE is able to redirect CD3 positive effector cells, we have mixed NK-TCR-BiTE or NK-TCR with B lymphoma cell line BL41, which is CD19 positive. As shown and previously published (Walseng et al 2019), NK-92 are not able to kill BL-41, however, when anti-CD19 BiTE is secreted, NK-TCR where able to efficiently kill CD19-positive targets. This teaches us that (i) BiTEs released by NK cells are functional and (ii) NK cells expressing a TCR-CD3 complex can be redirected by BiTE.
The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms first, second etc. as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers. The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.
While the invention has been described with reference to an exemplary embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20240016 | Jan 2024 | NO | national |
