US20160340435A1 - Molecular constructs for treating central nervous system diseases - Google Patents

Molecular constructs for treating central nervous system diseases Download PDF

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Publication number
US20160340435A1
US20160340435A1 US15/159,862 US201615159862A US2016340435A1 US 20160340435 A1 US20160340435 A1 US 20160340435A1 US 201615159862 A US201615159862 A US 201615159862A US 2016340435 A1 US2016340435 A1 US 2016340435A1
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pair
scfv
targeting
effector
elements
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Tse-Wen Chang
Hsing-Mao Chu
Wei-Ting TIAN
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Immunwork Inc
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Immunwork Inc
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Priority claimed from US14/997,849 external-priority patent/US20170152323A1/en
Application filed by Immunwork Inc filed Critical Immunwork Inc
Priority to US15/159,862 priority Critical patent/US20160340435A1/en
Assigned to Immunwork Inc. reassignment Immunwork Inc. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHANG, TSE-WEN, CHU, Hsing-Mao, TIAN, Wei-Ting
Publication of US20160340435A1 publication Critical patent/US20160340435A1/en
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    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
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Definitions

  • the present disclosure relates to the field of pharmaceuticals; more particularly, to multi-functional molecular constructs, e.g., those having targeting and effector elements for delivering the effector (e.g., therapeutic drug) to targeted sites.
  • multi-functional molecular constructs e.g., those having targeting and effector elements for delivering the effector (e.g., therapeutic drug) to targeted sites.
  • antibodies can neutralize or trap disease-causing mediators, which may be cytokines or immune components present in the blood circulation, interstitial space, or in the lymph nodes.
  • the neutralizing activity inhibits the interaction of the disease-causing mediators with their receptors.
  • fusion proteins of the soluble receptors or the extracellular portions of receptors of cytokines and the Fc portion of IgG which act by neutralizing the cytokines or immune factors in a similar fashion as neutralizing antibodies, have also been developed as therapeutic agents.
  • Fc-mediated mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytolysis (CMC), are not the intended mechanisms for the antibodies.
  • Some therapeutic antibodies bind to certain surface antigens on target cells and render Fc-mediated functions and other mechanisms on the target cells.
  • the most important Fc-mediated mechanisms are antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytolysis (CMC), which both will cause the lysis of the antibody-bound target cells.
  • ADCC antibody-dependent cellular cytotoxicity
  • CMC complement-mediated cytolysis
  • Some antibodies binding to certain cell surface antigens can induce apoptosis of the bound target cells.
  • the bi-valent or multivalent antibodies may contain two or more antigen-binding sites.
  • a number of methods have been reported for preparing multivalent antibodies by covalently linking three or four Fab fragments via a connecting structure.
  • antibodies have been engineered to express tandem three or four Fab repeats.
  • the present disclosure is directed to a fragment crystallizable (Fc)-based molecular construct that has at least one targeting element and at least one effector element linked, directly or indirectly, to a CH2-CH3 domain of an immunoglobulin.
  • Targeting and effector elements of the present Fc-based molecular constructs are specifically selected such that these Fc-based molecular constructs are suitable for use in the treatment of central nervous system (CNS) diseases, or for use in the manufacture of a medicament for treating CNS diseases.
  • CNS central nervous system
  • methods for treating CNS diseases using such Fc-based molecular constructs also fall within the aspect of the present disclosure.
  • the Fc-based molecular construct comprises a pair of CH2-CH3 segments of an IgG.Fc, a pair of effector elements, and a pair of targeting elements.
  • the pair of effector element is interferon ⁇ 1a (INF- ⁇ 1a) or INF- ⁇ 1b, or an antibody fragment specific for integrin ⁇ 4 or ⁇ -amyloid, while the pair targeting elements is an antibody fragment specific for human transferrin receptor or human insulin receptor.
  • the pair of targeting elements is linked to the C-termini of the pair of CH2-CH3 segments, and vice versa.
  • the pair of effectors elements and the pair of targeting elements is both in the form of single-chain variable fragments (scFvs)
  • the pair of targeting elements is linked to the N-termini of the pair of effector elements in a tandem or diabody configuration, thereby forming a pair of bispecific scFvs that are linked to the N-termini of the pair of CH2-CH3 segments.
  • the pair of CH2-CH3 segments is derived from human IgG heavy chain ⁇ 4 or human IgG heavy chain ⁇ 1.
  • the pair of effector elements or the pair of the targeting elements takes a Fab configuration (i.e., consisting of the V H -CH1 domain and the V L -C ⁇ domain); this Fab fragment is linked to the N-termini of the first and second heavy chains, so that the Fc-based molecular construct adopts an IgG configuration.
  • the pair of elements that is not in the Fab configuration is linked to the C-termini of the pair of CH2-CH3 segments.
  • the effector element is INF- ⁇ 1a, INF- ⁇ 1b, or an scFv specific for integrin ⁇ 4, while the targeting element is an scFv specific for human transferrin receptor.
  • this molecular construct is suitable for treating multiple sclerosis.
  • the effector element is an scFv specific for ⁇ -amyloid
  • the targeting element is an scFv specific for human transferrin receptor.
  • this molecular construct is suitable for treating Alzheimer's disease.
  • Methods for treating CNS diseases in a subject in need thereof comprise the step of administering to the subject an effective amount of the molecular construct of this aspect.
  • CNS diseases treatable by this method include multiple sclerosis and Alzheimer's disease.
  • the present disclosure is directed to a fragment crystallizable (Fc)-based molecular construct that has at least one targeting element and at least one effector element linked, directly or indirectly, to a CH2-CH3 domain of an immunoglobulin.
  • Targeting and effector elements of the present Fc-based molecular constructs are specifically selected such that these Fc-based molecular constructs are suitable for use in the treatment of diseases/conditions associated with viral or bacterial infection, or for use in the manufacture of a medicament for treating such diseases/conditions.
  • methods for treating diseases/conditions associated with viral or bacterial infection using such Fc-based molecular constructs also fall within the aspect of the present disclosure.
  • the Fc-based molecular construct comprises a pair of CH2-CH3 segments of an IgG.Fc, a pair of effector elements, and a pair of targeting elements.
  • the pair of effector element is an antibody fragment specific for CD32 or CD16b
  • the pair targeting elements is an antibody fragment specific for a viral protein or a bacterial protein.
  • the pair of targeting elements is linked to the C-termini of the pair of CH2-CH3 segments, and vice versa.
  • the pair of effectors elements and the pair of targeting elements is both in the form of single-chain variable fragments (scFvs)
  • the pair of targeting elements is linked to the N-termini of the pair of effector elements in a tandem or diabody configuration, thereby forming a pair of bispecific scFvs that are linked to the N-termini of the pair of CH2-CH3 segments.
  • the pair of CH2-CH3 segments is derived from human IgG heavy chain ⁇ 4 or human IgG heavy chain ⁇ 1.
  • the pair of effector elements or the pair of the targeting elements takes a Fab configuration (i.e., consisting of the V H -CH1 domain and the V L -C ⁇ domain); this Fab fragment is linked to the N-termini of the first and second heavy chains, so that the Fc-based molecular construct adopts an IgG configuration.
  • the pair of elements that is not in the Fab configuration is linked to the C-termini of the pair of CH2-CH3 segments.
  • the effector element is an scFv specific for CD32 or CD16b
  • the targeting element is an scFv specific for a viral protein.
  • the viral protein can be F protein of respiratory syncytia virus (RSV), gp120 protein of human immunodeficiency virus type 1 (HIV-1), hemagglutinin A (HA) protein of influenza A virus, or glycoprotein of cytomegalovirus.
  • RSV respiratory syncytia virus
  • HAV-1 human immunodeficiency virus type 1
  • HA hemagglutinin A
  • this molecular construct is suitable for treating viral infections.
  • the effector element is an scFv specific for Cd35 or C16b
  • the targeting element is an scFv specific for a bacterial protein.
  • the bacterial protein include, but are not limited to, the endotoxin of Gram( ⁇ ) bacteria, the surface antigen of Clostridium difficile , the lipoteichoic acid of Staphylococcus aureus , the anthrax toxin of Bacillus anthracis , or the Shiga-like toxin type I or II of Escherichia coli .
  • such molecular construct is suitable for treating bacterial infections.
  • Methods for treating diseases/conditions associated with infections comprise the step of administering to the subject an effective amount of the molecular construct of this aspect.
  • FIGS. 1A to 1C are schematic diagrams illustrating Fc-based molecular constructs according to various embodiments of the present disclosure.
  • FIG. 2 is a schematic diagram illustrating an Fc-based molecular construct according to various embodiments of the present disclosure.
  • FIGS. 3A and 3B are schematic diagrams illustrating an Fc-based molecular constructs according to various embodiments of the present disclosure.
  • FIG. 4A shows the SDS-PAGE analysis result of purified recombinant 2-chain (scFv ⁇ RSV)-hIgG1.Fc-(scFv ⁇ CD32) fusion protein
  • FIG. 4B and FIG. 4C provide the results of ELISA analyses that respectively illustrate the binding activities of the purified recombinant fusion protein of FIG. 4A to Protein F of RSV ( FIG. 30B ) and to ectodomain of CD32a ( FIG. 30C ).
  • FIG. 5A shows the SDS-PAGE analysis result of the purified recombinant 2-chain (scFv ⁇ endotoxin)-hIgG1.Fc-(scFv ⁇ CD32) fusion protein; and FIG. 5B and FIG. 31C provides the results of ELISA analyses that respectively illustrate the binding affinity of the purified recombinant fusion protein of FIG. 5A to endotoxin ( FIG. 5B ) and to ectodomain of CD32a ( FIG. 5C ).
  • FIG. 6A and FIG. 6B respectively show the SDS-PAGE analysis result and the ELISA results of the purified recombinant 2-chain (Interferon- ⁇ -1a)-hIgG4.Fc-(scFv ⁇ TfR1) fusion protein.
  • FIG. 7A and FIG. 7B respectively shows the SDS-PAGE analysis result and staining result of the purified recombinant 2-chain (scFv ⁇ integrin ⁇ 4)-hIgG4.Fc-(scFv ⁇ TfR1) fusion protein.
  • FIG. 8 shows the ELISA analysis result of the effect of the purified recombinant 2-chain (scFv ⁇ endotoxin)-hIgG1.Fc-(scFv ⁇ CD32a) fusion protein on inhibiting TNF- ⁇ secretion.
  • phrases “at least one of A, B, and C”, “at least one of A, B, or C” and “at least one of A, B and/or C,” as use throughout this specification and the appended claims, are intended to cover A alone, B alone, C alone, A and B together, B and C together, A and C together, as well as A, B, and C together.
  • each molecular construct comprises a targeting element (T) and an effector element (E), and these molecular constructs are sometimes referred to as “T-E molecules”, “T-E pharmaceuticals” or “T-E drugs” in this document.
  • the term “targeting element” refers to the portion of a molecular construct that directly or indirectly binds to a target of interest (e.g., a receptor on a cell surface or a protein in a tissue) thereby facilitates the transportation of the present molecular construct into the interested target.
  • the targeting element may direct the molecular construct to the proximity of the target cell.
  • the targeting element specifically binds to a molecule present on the target cell surface or to a second molecule that specifically binds a molecule present on the cell surface.
  • the targeting element may be internalized along with the present molecular construct once it is bound to the interested target, hence is relocated into the cytosol of the target cell.
  • a targeting element may be an antibody or a ligand for a cell surface receptor, or it may be a molecule that binds such antibody or ligand, thereby indirectly targeting the present molecular construct to the target site (e.g., the surface of the cell of choice).
  • the localization of the effector (therapeutic agent) in the diseased site will be enhanced or favored with the present molecular constructs as compared to the therapeutic without a targeting function.
  • the localization is a matter of degree or relative proportion; it is not meant for absolute or total localization of the effector to the diseased site.
  • the term “effector element” refers to the portion of a molecular construct that elicits a biological activity (e.g., inducing immune responses, exerting cytotoxic effects and the like) or other functional activity (e.g., recruiting other hapten tagged therapeutic molecules), once the molecular construct is directed to its target site.
  • the “effect” can be therapeutic or diagnostic.
  • the effector elements encompass those that bind to cells and/or extracellular immunoregulatory factors.
  • the effector element comprises agents such as proteins, nucleic acids, lipids, carbohydrates, glycopeptides, drug moieties (both small molecule drug and biologics), compounds, elements, and isotopes, and fragments thereof.
  • first, second, third, etc. may be used herein to describe various elements, components, regions, and/or sections, these elements (as well as components, regions, and/or sections) are not to be limited by these terms. Also, the use of such ordinal numbers does not imply a sequence or order unless clearly indicated by the context. Rather, these terms are simply used to distinguish one element from another. Thus, a first element, discussed below, could be termed a second element without departing from the teachings of the exemplary embodiments.
  • link refers to any means of connecting two components either via direct linkage or via indirect linkage between two components.
  • polypeptide refers to a polymer having at least two amino acid residues. Typically, the polypeptide comprises amino acid residues ranging in length from 2 to about 200 residues; preferably, 2 to 50 residues. Where an amino acid sequence is provided herein, L-, D-, or beta amino acid versions of the sequence are also contemplated. Polypeptides also include amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • the term applies to amino acids joined by a peptide linkage or by other, “modified linkages,” e.g., where the peptide bond is replaced by an ⁇ -ester, a ⁇ -ester, a thioamide, phosphoramide, carbomate, hydroxylate, and the like.
  • conservative substitutions of the amino acids comprising any of the sequences described herein are contemplated.
  • one, two, three, four, or five different residues are substituted.
  • the term “conservative substitution” is used to reflect amino acid substitutions that do not substantially alter the activity (e.g., biological or functional activity and/or specificity) of the molecule.
  • conservative amino acid substitutions involve substitution one amino acid for another amino acid with similar chemical properties (e.g., charge or hydrophobicity).
  • Certain conservative substitutions include “analog substitutions” where a standard amino acid is replaced by a non-standard (e.g., rare, synthetic, etc.) amino acid differing minimally from the parental residue. Amino acid analogs are considered to be derived synthetically from the standard amino acids without sufficient change to the structure of the parent, are isomers, or are metabolite precursors.
  • polypeptides comprising at least 80%, preferably at least 85% or 90%, and more preferably at least 95% or 98% sequence identity with any of the sequences described herein are also contemplated.
  • Percentage (%) amino acid sequence identity with respect to the polypeptide sequences identified herein is defined as the percentage of polypeptide residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percentage sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • sequence comparison between two polypeptide sequences was carried out by computer program Blastp (protein-protein BLAST) provided online by National Center for Biotechnology Information (NCBI).
  • Blastp protein-protein BLAST
  • NCBI National Center for Biotechnology Information
  • X is the number of amino acid residues scored as identical matches by the sequence alignment program BLAST in that program's alignment of A and B, and where Y is the total number of amino acid residues in A or B, whichever is shorter.
  • PEGylated amino acid refers to a polyethylene glycol (PEG) chain with one amino group and one carboxyl group.
  • PEG polyethylene glycol
  • the PEGylated amino acid has the formula of NH 2 —(CH 2 CH 2 O) n —COOH.
  • n ranges from 1 to 20; preferably, ranging from 2 to 12.
  • terminal refers to an amino acid residue at the N- or C-end of the polypeptide.
  • terminal refers to a constitutional unit of the polymer (e.g., the polyethylene glycol of the present disclosure) that is positioned at the end of the polymeric backbone.
  • free terminus is used to mean the terminal amino acid residue or constitutional unit is not chemically bound to any other molecular.
  • antigen or “Ag” as used herein is defined as a molecule that elicits an immune response. This immune response may involve a secretory, humoral and/or cellular antigen-specific response.
  • the term “antigen” can be any of a protein, a polypeptide (including mutants or biologically active fragments thereof), a polysaccharide, a glycoprotein, a glycolipid, a nucleic acid, or a combination thereof.
  • antibody is used in the broadest sense and covers fully assembled antibodies, antibody fragments that bind with antigens, such as antigen-binding fragment (Fab/Fab′), F(ab′) 2 fragment (having two antigen-binding Fab portions linked together by disulfide bonds), variable fragment (Fv), single chain variable fragment (scFv), bi-specific single-chain variable fragment (bi-scFv), nanobodies, unibodies and diabodies.
  • Fab/Fab′ antigen-binding fragment
  • F(ab′) 2 fragment having two antigen-binding Fab portions linked together by disulfide bonds
  • variable fragment Fv
  • scFv single chain variable fragment
  • bi-specific single-chain variable fragment bi-scFv
  • nanobodies unibodies and diabodies.
  • an “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • the well-known immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.
  • a typical immunoglobulin (antibody) structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, with each pair having one “light” chain (about 25 kDa) and one “heavy” chain (about 50-70 kDa).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains, respectively.
  • the antibody fragment can be produced by modifying the nature antibody or by de novo synthesis using recombinant DNA methodologies.
  • the antibody and/or antibody fragment can be bispecific, and can be in various configurations.
  • bispecific antibodies may comprise two different antigen binding sites (variable regions).
  • bispecific antibodies can be produced by hybridoma technique or recombinant DNA technique.
  • bispecific antibodies have binding specificities for at least two different epitopes.
  • the term “specifically binds” as used herein, refers to the ability of an antibody or an antigen-binding fragment thereof, to bind to an antigen with a dissociation constant (Kd) of no more than about 1 ⁇ 10 ⁇ 6 M, 1 ⁇ 10 ⁇ 7 M, 1 ⁇ 10 ⁇ 8 M, 1 ⁇ 10 ⁇ 9 M, 1 ⁇ 10 ⁇ 10 M, 1 ⁇ 10 ⁇ 11 M, 1 ⁇ 10 ⁇ 12 M, and/or to bind to an antigen with an affinity that is at least two-folds greater than its affinity to a nonspecific antigen.
  • Kd dissociation constant
  • treatment includes preventative (e.g., prophylactic), curative or palliative treatment; and “treating” as used herein also includes preventative (e.g., prophylactic), curative or palliative treatment.
  • treating refers to the application or administration of the present molecular construct or a pharmaceutical composition comprising the same to a subject, who has a medical condition a symptom associated with the medical condition, a disease or disorder secondary to the medical condition, or a predisposition toward the medical condition, with the purpose to partially or completely alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of said particular disease, disorder, and/or condition.
  • Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition, and/or to a subject who exhibits only early signs of a disease, disorder and/or condition, for the purpose of decreasing the risk of developing pathology associated with the disease, disorder and/or condition.
  • an effective amount refers to the quantity of the present molecular construct that is sufficient to yield a desired therapeutic response.
  • An effective amount of an agent is not required to cure a disease or condition but will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered or prevented, or the disease or condition symptoms are ameliorated.
  • the effective amount may be divided into one, two, or more doses in a suitable form to be administered at one, two or more times throughout a designated time period.
  • Effective amount will vary with such factors as particular condition being treated, the physical condition of the patient (e.g., the patient's body mass, age, or gender), the type of subject being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives. Effective amount may be expressed, for example, as the total mass of active component (e.g., in grams, milligrams or micrograms) or a ratio of mass of active component to body mass, e.g., as milligrams per kilogram (mg/kg).
  • application and “administration” are used interchangeably herein to mean the application of a molecular construct or a pharmaceutical composition of the present invention to a subject in need of a treatment thereof.
  • subject and patient are used interchangeably herein and are intended to mean an animal including the human species that is treatable by the molecular construct, pharmaceutical composition, and/or method of the present invention.
  • subject or patient intended to refer to both the male and female gender unless one gender is specifically indicated. Accordingly, the term “subject” or “patient” comprises any mammal, which may benefit from the treatment method of the present disclosure.
  • Examples of a “subject” or “patient” include, but are not limited to, a human, rat; mouse; guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and fowl.
  • the patient is a human.
  • mammal refers to all members of the class Mammalia, including humans, primates, domestic and farm animals, such as rabbit, pig, sheep, and cattle; as well as zoo, sports or pet animals; and rodents, such as mouse and rat.
  • non-human mammal refers to all members of the class Mammalis except human.
  • the present disclosure is based, at least on the construction of the T-E pharmaceuticals that can be delivered to target cells, target tissues or organs at increased proportions relative to the blood circulation, lymphoid system, and other cells, tissues or organs.
  • the therapeutic effect of the pharmaceuticals is increased, while the scope and severity of the side effects and toxicity is decreased.
  • a therapeutic effector is administered at a lower dosage in the form of a T-E molecule, than in a form without a targeting component. Therefore, the therapeutic effector can be administered at lower dosages without losing potency, while lowering side effects and toxicity.
  • Drugs used for many diseases can be improved for better efficacy and safety, if they can be targeted to the disease sites, i.e., if they can be localized or partitioned to the disease sites more favorably than the normal tissues or organs.
  • Certain antibody drugs which target infectious microorganisms or their toxic products, can be improved, if they are empowered with the ability to recruit immunocytes, which phagocytose and clear the antibody-bound particles.
  • drugs can be improved if they can be preferentially distributed to the disease sites or cells or if they can recruit phagocytic immunocytes.
  • the therapeutic agents are often required to pass through the blood-brain barrier (BBB) to get into the CNS.
  • BBB blood-brain barrier
  • Some therapeutic agents do not get into the CNS; they regulate certain activities, such as immune activities, in the peripheral, which then modulates the diseased conditions in the CNS.
  • the BBB is formed by the endothelial cells lining the capillaries of blood vessels in the CNS. Unlike the capillaries in the peripheral tissues and organs, the capillary endothelial cells in the BBB are connected by tight junctions formed by occludin, claudins, and junctional adhesion molecules.
  • At least six antibodies namely, aducanumab, bapinerumab, crenezumab, gantenerumab, ponezumab, and solanezumab, specific for ⁇ -amyloid, which is responsible for causing Alzheimer's disease, have been developed and placed in clinical development. These antibodies generally fall short of satisfactory therapeutic efficacy in improving Alzheimer's disease. A general belief is that if those antibodies are to achieve therapeutic efficacy, a significant portion must get across the BBB to enter the injured sites in the CNS. However, only very minute portions of those antibodies get across the BBB.
  • Interferon- ⁇ -1a IFN- ⁇ -1a
  • IFN- ⁇ -1b interferon- ⁇ -1b
  • MS multiple sclerosis
  • the pharmaceuticals, IFN- ⁇ -1a produced by mammalian cells and IFN- ⁇ -1b produced in E. coli are one-chain protein of 166 amino acid residues containing one disulfide bond. It has been claimed that those therapeutic agents reduce relapse of MS in 18-38% of treated patients.
  • the mechanisms of action of IFN- ⁇ -1a and IFN- ⁇ -1b are very complex and not completely understood, involving the increased generation of anti-inflammatory immune cells and factors and the down-regulation of pro-inflammatory cells and factors.
  • IFN- ⁇ treatment in MS patients also reduces the trafficking of pro-inflammatory T cells across the BBB. It is yet unanswered whether IFN- ⁇ -1a and IFN- ⁇ -1b mediate their pharmacologic effects in part by getting into the injured sites in the CNS.
  • natalizumab specific for the cell adhesion molecule integrin ⁇ 4.
  • the antibody functions by inhibiting inflammatory immune cells to attach to and pass through the epithelial layer lining the BBB. While natalizumab has been shown to be therapeutic efficacious, it has serious immunosuppressive side effect. In particular, it causes progressive multifocal leukoencephalopathy, an opportunistic infection caused by John Cunningham virus (JC virus).
  • JC virus John Cunningham virus
  • the endothelial cells in the capillaries forming the BBB express transferrin receptors and insulin receptors, which mediate the transcytosis of transferrin and insulin molecules, respectively, to the cerebral parenchyma.
  • transferrin receptor As a ferry, only a small proportion gets through while the reaming bulk are trapped or degraded.
  • the transferrin receptors on the endothelial cells in the BBB can serve as site-specific antigen for recruiting administered therapeutics. Once the therapeutic is concentrated in the BBB, an increased proportion of it will pass through the capillaries.
  • anti-inflammatory drugs such as anti-TNF- ⁇ , anti-IL12/IL-23, anti-IL17, and anti-CD3, should be investigated for their therapeutic effects on many types of diseases of the CNS.
  • the transferrin receptor is used as a target site recruiter.
  • the effector moiety can be a few copies of scFv specific for ⁇ -amyloid; for treating multiple sclerosis, the effector moiety can be a few copies of IFN- ⁇ -1a or IFN- ⁇ -1b, or a few copies of scFv specific for integrin ⁇ 4.
  • Embodiments of the present disclosure disclose several T-E molecules respectively exist in single multi-arm linker-units or joint-linker configurations, each contains scFv specific for transferrin receptor as the targeting element and IFN- ⁇ -1a or IFN- ⁇ -1b or scFv specific for integrin ⁇ -4 as the effector element.
  • Alternative embodiments disclose T-E molecules respectively exist in single linker-units or joint-linker configurations, each contains scFv specific for transferrin receptor as the targeting element and scFv specific for ⁇ -amyloid as the effector element.
  • Fingolimod is an immunosuppressive drug that is derived from a natural product myriocin originally isolated from certain fungi. Fingolimod has been approved for reducing the relapse of relapsing-remitting multiple sclerosis. Fingolimod is phosphorylated in vivo to form fingolimod-phosphate, which resembles naturally occurring sphingosine-1-phosphate (S1P), an extracellular lipid mediator, and can bind to 4 of the 5 S1P receptors. The S1P receptors are expressed on lymphocytes and involved in lymphocyte migration. A generally pharmacologic mechanism of fingolimod is that it inhibits lymphocytes egress from the lymphoid tissues to the circulation and hence to the CNS.
  • S1P sphingosine-1-phosphate
  • Fingolimod can cross BBB to enter CNS and many cell types in the CNS express S1P receptors, which play roles in cell proliferation, morphology, and migration. It is believed that fingolimod can have direct on the CNS.
  • the administration of fingolimod causes common side effects of headache and fatigue, and severe side effects of skin cancer, macular edema, and fatal infections, such as hemorrhaging focal encephalitis.
  • a fingolimod molecule has an NH2 group and thus provides a functional group to couple with a bi-functional linker with an NHS group.
  • One preferred embodiment of the present invention is to prepare a T-E construct, which contains a targeting element for delivery to the BBB and a drug bundle of fingolimod as an effector element.
  • a bundle of fingolimod 5-10 molecules are incorporated to a linker unit, using either a cleavable linker or non-cleavable linker to conjugate fingolimod molecules to the linking arms of a linker unit.
  • fingolimod Since fingolimod, after uptake in a patient, is modified to fingolimod phosphate to resemble sphingosine1-phosphate and become active, the drug bundle is alternatively prepared with fingolimod phosphate.
  • a linker unit with fingolimod or fingolimod phosphate bundle is conjugated with 1 or 2 scFv specific for a transferrin receptor I.
  • Upon administration of the molecular construct a portion of it is carried to the BBB.
  • the fingolimod molecules released from the cleavable linkers pass through the BBB and enter the CNS. Or, a portion of the entire construct enters the CNS.
  • Cleavable linkers can be designed by employing a number of cleaving mechanisms.
  • S—S bond An installment of S—S bond is often used, since S—S disulfide bond can be cleaved by a reduction reaction at the target tissue site.
  • a peptide bond between amino acids which is sensitive to proteases, such as matrix metalloproteinases in many tissues and cathepsins in endosomes in target cells, is also commonly used as a cleavable bond in many linker designs.
  • the T-E molecular design of the present invention can also be applied for the prevention and treatment of infectious diseases.
  • the plurality of the linking arms can enhance the avidity and specificity of binding to target infectious microorganisms or their products and elicit immune functions to facilitate the clearance of the microorganisms and their products.
  • Such improvements should increase the efficacy of the candidate antibodies for the prevention and therapy of infectious diseases.
  • Many antibodies, which have failed to meet expectation in clinical trials, may be configured with the present invention and re-investigated.
  • a preferred set of embodiment of the present invention is to employ joint-linkers configuration with one linker-unit for targeting and one linker-unit for recruiting effector function.
  • An alternative set of preferred embodiment is to employ single linker-units with multiple linking arms for targeting elements and a coupling arm for an effector element.
  • the targeting elements may be one of the two categories: (1) scFv or sdAb specific for a surface component of a microorganism or its product, e.g., envelope protein gp120 of human immunodeficiency virus type 1 (HIV-1), F protein of respiratory syncytia virus (RSV), a surface antigen of Clostridium difficile or Staphylococcus aureus , or endotoxin of Gram-negative bacteria or Shiga-like toxin of Escherichia coli , or (2) the extracellular portions of cell surface receptors of viruses, such as the HIV-1 gp120-binding CD4 domain.
  • HIV-1 human immunodeficiency virus type 1
  • RSV respiratory syncytia virus
  • the effector elements are 1 or 2 scFv or sdAb specific for one Fc receptor of IgG, e.g. Fc ⁇ RIIA (CD32), Fc ⁇ RIIIB (CD16b), or Fc ⁇ RI (CD64).
  • Fc ⁇ RIIA and Fc ⁇ RIIIB bind to IgG with low affinity (Kd in the range of 10 ⁇ 6 to 10 ⁇ 7 ), and Fc ⁇ RI binds to IgG1 and IgG3 with high affinity (Kd 10 ⁇ 9 ).
  • scFv or sdAb specific for Fc ⁇ RIIA or Fc ⁇ RIIIB because they can compete favorably with IgG in binding to the receptors.
  • the antibodies specific for carbohydrate antigens on bacterial surface are usually weak in binding affinity and are expressed in IgM rather than IgG.
  • An IgM molecule has 10 Fv's (antigen-binding sites).
  • an IgM molecule which has a molecular weight of about 1000 kd, cannot cross capillaries and reach to extravascular space.
  • a molecular construct carrying 6 scFv or 10 sdAb will have a molecular weight of about 150 kd.
  • the molecular construct contains 2 or more scFv specific for an Fc ⁇ receptor and can bind to multiple Fc ⁇ receptor molecules on phagocyte cell surface, so that the bound viral particles are destined to phagocytosis pathway.
  • immunoglobulin antibody can serve as the base of a targeting or effector element, and its corresponding effector or targeting element can be incorporated at the C-terminal of its two heavy ⁇ chains in the form of scFv domains.
  • two-chain IgG.Fc is used as the base of the molecular platform.
  • Each of the polypeptide chain is fused with one or two targeting and one or two effector elements, for a total of two to three elements on each chain.
  • the T-E molecule with an Fc-based configuration will have a total of four to six elements (e.g., scFv or any other antibody fragments).
  • the Fc portion of the molecular constructs also carries Fc-mediated effector functions, ADCC, and/or complement-mediated activation. While in certain other applications, such Fc-mediated effector functions are avoided.
  • targeting elements are positioned at the N- or C-terminus. If the effector elements function by binding to a cell surface component, they should also be positioned at the terminus. If the effector elements function by binding to and neutralizing soluble factors, they can be positioned between a terminal targeting or effector element and CH2-CH3.
  • the molecular construct can be used to treat central nervous system (CNS) diseases or infectious diseases.
  • CNS central nervous system
  • the present disclosure is also advantageous in that, in some embodiments, it utilizes the linker unit according to the first aspect of the present disclosure, which provides a facile means for controlling the number of the targeting and effector elements of the present Fc-based molecular constructs.
  • the present Fc-based molecular construct may take different configurations, which are discussed below, respectively.
  • both the targeting element and effector element are antibodies or fragments thereof.
  • FIG. 1A is a schematic diagram illustrating an Fc-based molecular construct 800A according to certain embodiments of the present disclosure.
  • the Fc-based molecular construct 800A comprises two identical CH2-CH3 chains 810, a pair of effector elements E1 linked to the N-termini of the CH2-CH3 chains 810, and a pair of targeting elements T1 linked to the C-termini of the CH2-CH3 chains 810.
  • both the targeting element T1 and effector element E1 are scFvs.
  • the Fc-based molecular construct 800B illustrated in FIG. 1B is quite similar to the Fc-based molecular construct 800A of FIG. 1A in structure, except that the two effector elements E1 are respectively linked to the C-termini of the CH2-CH3 chains 810, while the two targeting effectors are respectively linked to the C-termini of the CH2-CH3 chains 810.
  • both the effector elements and targeting elements are linked to the N-termini of the CH2-CH3 chains.
  • the effector element and the targeting element are in the form of single-chain variable fragments (scFvs)
  • the effector element and the targeting element may be linked in a tandem or diabody configuration, thereby forming a bispecific scFv that is linked to the N-terminus of the CH2-CH3 chain.
  • the Fc-based molecular construct 800C ( FIG. 1C ) comprises an Fc portion, and accordingly, each CH2-CH3 chain 810 has a T1-E1 bispecific scFv linked to the N-terminus thereof.
  • the pair of effector elements or the pair of the targeting elements takes a Fab configuration (i.e., consisting of the V H -CH1 domain and the V L -C ⁇ domain); this Fab fragment is linked to the N-termini of the CH2-CH3 chains, so that the Fc-based molecular construct adopts an IgG configuration.
  • the pair of elements that is not in the Fab configuration may be linked to the C-termini of the pair of CH2-CH3 segments.
  • each of the two targeting elements T1 comprises the V H -CH1 domain 820 and the V L -C ⁇ domain 825, thereby forming a Fab configuration 830 that is linked to the N-termini of the CH2-CH3 chains 810, so that the Fc-based molecular construct 900 adopts the IgG configuration.
  • the pair of effector elements E1 is linked to the C-termini of the pair of CH2-CH3 chains 810.
  • the present Fc-based molecular construct has an effector element which is a peptide.
  • the effector element can be a peptide with certain therapeutic effect, while the targeting element is an antibody or a fragment thereof (see, FIGS. 3A and 3B ).
  • the Fc-based molecular construct 1000A of FIG. 3A comprises a pair of targeting elements T1 (as scFvs) linked to the N-termini of the pair of CH2-CH3 segments 1210, and a pair of effector elements E1 (in the form of therapeutic peptides) linked to the C-termini of the pair of CH2-CH3 segments 1210.
  • the pair of targeting elements T1 (as scFvs) is linked to the C-termini of the pair of CH2-CH3 segments 1210
  • the pair of effector elements E1 (in the form of therapeutic peptides) is linked to the C-termini of the pair of CH2-CH3 segments 1210.
  • the targeting element can be constructed into a Fab fragment, so that the molecular constructs take the IgG configuration.
  • the CH2-CH3 chains are adopted from human immunoglobulins ⁇ 1 or ⁇ 4.
  • ⁇ 1 is chosen, when Fc-mediated functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated activity (inflammatory activation or target cell lysis), are desired.
  • ADCC antibody-dependent cellular cytotoxicity
  • complement-mediated activity inflammatory activation or target cell lysis
  • an antibody (or a fragment thereof) specific for transferrin receptor can be used as the targeting element, in connection with effector elements suitable for the particular CNS disease.
  • Fc-based molecular constructs for the treatment of multiple sclerosis may use an scFv specific for integrin- ⁇ 4 as the effector element.
  • illustrative Fc-based molecular constructs can use an scFv specific for ⁇ -amyloid as the effector element.
  • the above-mentioned Fc-based molecular constructs for treating CNS diseases may take the configuration described in connection with any of FIGS. 1A to 1C , and FIG. 2 .
  • Fc-based molecular constructs for treating multiple sclerosis may also use INF- ⁇ 1a or INF- ⁇ 1b as the effector elements.
  • the Fc-based molecular constructs may take the configuration described in connection with FIG. 3A or 3B .
  • Fc-based molecular constructs for treating diseases/conditions associated with infection such as viral infections or bacterial infections
  • an antibody (or a fragment thereof) specific for a viral protein or bacterial protein as the targeting element.
  • an antibody (or a fragment thereof) specific for CD32 or CD16b can be used.
  • the essence of this invention is the rationalization and conception of the specific combination or pairing of the targeting and effector elements.
  • the adoption of Fc-fusion configuration in the molecular constructs is a preferred embodiment. It is conceivable for those skilled in the arts to link the pairs of targeting and effector elements of this invention employing other molecular platforms, such as peptides, proteins (e.g., albumin), polysaccharides, polyethylene glycol, and other types of polymers, which serve as a structural base for attaching multiple molecular elements.
  • the present disclosure also pertains to method for treating CNS diseases using the suitable Fc-based molecular construct.
  • the method comprises the step of administering to a subject in need of such treatment an effective amount of the Fc-based molecular construct according to embodiments of the present disclosure.
  • the present disclosure further pertains to method for treating infections using the suitable Fc-based molecular construct.
  • the method comprises the step of administering to a subject in need of such treatment an effective amount of the Fc-based molecular construct according to embodiments of the present disclosure.
  • the scFv1-CH2-CH3-scFv2 (human ⁇ 1) recombinant chain was configured by fusing two scFvs, in which the first one specific for Protein F of RSV fused to the N-terminal of CH2 domain of IgG1.Fc through a flexible hinge region, while the second one specific for ectodomain of CD32a was fused to the C-terminal of CH3 domain through a flexible linker, (GGGGS) 3 .
  • Both of the scFvs had an orientation of V L -linker-V H .
  • the V L and V H in each of the two scFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG.
  • the sequence of the recombinant chain in the IgG1.Fc fusion protein molecular construct is shown as SEQ ID NO: 1.
  • the gene-encoding sequence was placed in pcDNA3 expression cassette.
  • Expi293F cells were seeded at a density of 2.0 ⁇ 10 6 viable cells/ml in Expi293F expression medium and maintained for 18 to 24 hours prior to transfection to ensure that the cells were actively dividing at the time of transfection.
  • 7.5 ⁇ 10 8 cells in 255-ml medium in a 2-liter Erlenmeyer shaker flask were transfected by ExpiFectamineTM 293 transfection reagent. The transfected cells were incubated at 37° C.
  • FIG. 4C shows binding activity of the recombinant Fc-fusion protein to ectodomain of CD32a.
  • ELISA plates were coated with 5 ⁇ g/mL of recombinant ectodomain of CD32a.
  • Recombinant 2-chain (scFv ⁇ RSV)-hIgG1.Fc-(scFv ⁇ CD32a) fusion protein was detected by HRP-conjugated goat anti-human IgG1.Fc.
  • FIG. 4C shows that the recombinant 2-chain (scFv ⁇ RSV)-hIgG1.Fc-(scFv ⁇ CD32a) Fc-fusion protein has binding activity to recombinant ectodomain of CD32a.
  • Recombinant 2-chain (scFv ⁇ endotoxin)-IgG1.Fc protein was used as a control antibody.
  • the scFv1-CH2-CH3-scFv2 (human ⁇ 1) recombinant chain was configured by fusing two scFvs, in which the first one specific for endotoxin was fused to the N-terminal of CH2 domain of IgG1.Fc through a flexible hinge region, and the second one specific for ectodomain was fused to the C-terminal of CH3 domain through a flexible linker, (GGGGS) 3 .
  • Both of the scFvs had an orientation of V L -linker-V H .
  • the V L and V H in each of the two scFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG.
  • the sequence of the recombinant chain in the IgG1.Fc fusion protein molecular construct is shown as SEQ ID NO: 2.
  • the expression of the constructed gene in Expi293F cells and the purification of the expressed fusion protein were performed as in preceding Examples. Characterization of the new construct was performed with SDS-PAGE and ELISA. The SDA-PAGE results in FIG. 5A shows that the recombinant chain of the new construct has a size of about 85 kDa, consistent with the expected size.
  • FIG. 5B shows ELISA results of the recombinant 2-chain (scFv ⁇ endotoxin)-(scFv ⁇ CD32a)-hIgG1.Fc binding to E. coli LPS 0111:B4 (Sigma Aldrich).
  • ELISA plates were coated with 50 ⁇ g/ml poly-L-lysine. Subsequently, the poly-L-lysine-coated plates were further coated with 10 ⁇ g/ml E. coli LPS 0111:B4.
  • the recombinant fusion protein was detected by HRP-conjugated goat anti-human IgG.Fc.
  • the ELISA results show that the present recombinant Fc-fusion protein has binding activity to E. coli LPS 0111:B4 (Sigma Aldrich);
  • FIG. 5C shows that the recombinant Fc-fusion protein has binding activity to ectodomain of CD32a.
  • the 2-chain IgG.Fc fusion protein was prepared by configuring (interferon- ⁇ -1a)-CH2-CH3-(scFv ⁇ TfR1) (human ⁇ 4) in a recombinant chain.
  • the C-terminal of the interferon- ⁇ -1a was fused to the N-terminal of CH2 via a linker, GGGGSGGGASGGS.
  • the scFv specific for ectodomain of TfR1 was fused to the C-terminal of CH3 domain through a flexible linker, (GGGGS) 3 .
  • the scFv (specific for ectodomain of TfR1) had an orientation of V L -linker-V H .
  • the V L and V H in the scFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG.
  • the sequence of the recombinant chain in the IgG4.Fc fusion protein molecular construct is shown as SEQ ID NO: 3.
  • the gene-encoding sequence was placed in pcDNA3 expression cassette.
  • Expi293F cells were seeded at a density of 2.0 ⁇ 10 6 viable cells/ml in Expi293F expression medium and maintained for 18 to 24 hours prior to transfection to ensure that the cells were actively dividing at the time of transfection.
  • 7.5 ⁇ 10 8 cells in 255-ml medium in a 2-liter Erlenmeyer shaker flask were transfected by ExpiFectamineTM 293 transfection reagent. The transfected cells were incubated at 37° C.
  • Binding activity of recombinant (Interferon- ⁇ -1a)-hIgG1.Fc-(scFv ⁇ TfR1) was assayed by ELISA using a 96-well plate coated with recombinant (Interferon- ⁇ -1a)-hIgG1.Fc-(scFv ⁇ TfR1) protein in 5 ⁇ g/ml concentration, 100 ⁇ l per well.
  • the scFv specific for ectodomain of TfR1 is as a negative control.
  • Recombinant 2-chain (Interferon- ⁇ -1a)-hIgG1.Fc-(scFv ⁇ TfR1) was detected by HRP-conjugated rabbit anti-human interferon- ⁇ polyclonal antibody (Santa Cruz Biotechnology, Dallas, USA).
  • HRP-conjugated rabbit anti-human interferon- ⁇ polyclonal antibody Santa Cruz Biotechnology, Dallas, USA.
  • 50 ⁇ l of TMB substrate was added for color development.
  • the reaction was stopped by 50 ⁇ l of 1M HCl. Absorbance at 450 nm was measured with a plate reader, Each bar represents the mean OD450 value of duplicate samples.
  • FIG. 6B shows ELISA analysis of the present the molecular construct.
  • the ELISA results show that (Interferon- ⁇ -1a)-hIgG1.Fc-(scFv ⁇ TfR1) fusion protein bound specifically to recombinant ectodomain of TfR1 protein.
  • the V L and V H of the scFv specific for integrin ⁇ 4 were from monoclonal antibody natalizumab.
  • the 2-chain IgG.Fc fusion protein was prepared by configuring (scFv ⁇ integrin ⁇ 4)-CH2-CH3-(scFv ⁇ TfR1) (human ⁇ 4) in a recombinant chain.
  • the C-terminal of the scFv specific for integrin ⁇ 4 was fused to the N-terminal of CH2 via a linker, GGGGSGGGASGGS.
  • the scFv specific for ectodomain of TfR1 was fused to the C-terminal of CH3 domain through a flexible linker, (GGGGS) 3 .
  • the result of 8% SDA-PAGE in FIG. 7A shows that the recombinant chain of the new construct has a size of about 85 kDa (indicated by arrow), consistent with the expected size.
  • the two scFv had the orientation of V L -linker-V H .
  • the V L and V H in each of the two scFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG.
  • the sequence of the recombinant chain in the IgG4.Fc fusion protein molecular construct is shown as SEQ ID NO: 4.
  • Illustrated herein is the configuration of the prepared 2-chain (scFv ⁇ integrin ⁇ 4)-IgG4.Fc-(scFv ⁇ TfR1) molecular construct.
  • Jurkat T cells 1 ⁇ 10 6 Jurkat T cells was maintained in the RPMI1640 medium supplemented with 10% FBS at a density of 1 ⁇ 10 5 . The cells were kept in 37° C. with 5% CO 2 in a humidified chamber. 1 ⁇ 10 6 Jurkat T cells were washed with the binding buffer (phosphate-buffered saline with 0.1% FBS, 2 mM EDTA and 20 ng/ml NaN 3 ) twice. 10 ⁇ g/ml of Human BD Fc BlockTM (BD Biosciences, San Jose, US) was added to the washed Jurkat T cells to block Fc receptor mediated.
  • the binding buffer phosphate-buffered saline with 0.1% FBS, 2 mM EDTA and 20 ng/ml NaN 3
  • FIG. 7B shows results of the cell staining analysis of recombinant 2-chain (scFv ⁇ integrin ⁇ 4)-IgG4.Fc-(scFv ⁇ TfR1) protein on integrin ⁇ 4-expressing Jurkat T cells.
  • ELISA was to determine the amount of secreted TNF- ⁇ in the supernatant by macrophage-like U937 cells.
  • U937 cells were maintained in RPMI1640 supplemented with 10% fetal bovine serum (Gibco) and 100 U/ml penicillin-streptomycin (Gibco), at the density between 3 ⁇ 10 5 and 2 ⁇ 10 6 cells/ml. The cells were kept in 37° C. with 5% CO 2 in a humidified chamber. To differentiate U937 into macrophage-like cells, 1 ⁇ 10 6 cells/ml of U937 were incubated with 10 ng/ml of phorbol 12-myristate ⁇ -acetate (PMA, Sigma Aldrich). After 48 hours, non-adherent cells were removed, and adherent cells were washed and seeded into 96-well plates.
  • PMA phorbol 12-myristate ⁇ -acetate
  • TNF- ⁇ levels in U937 supernatant were measured using an ELISA kit from R&D Systems.
  • the wells of ELISA plates (Greiner Bio-One) were coated with 4 ⁇ g/mL of capture antibody in PBS at 4° C. overnight. Wells were subsequently blocked by 0.5% in PBS for 1 hour and incubated with diluted culture supernatant for 2 hours. 400 ng/mL of biotin-labeled detection antibody was used followed by Streptavidin-HRP to detect bound TNF- ⁇ . Chromogenic reaction was carried out using TMB substrate (Clinical Science Products), and stopped by adding 1N HCl. Plates were read at 450 nm absorbance. Concentrations of TNF- ⁇ were determined by extrapolation from four-parameter logistic fit standard curves generated from dilutions of standard protein supplied by the manufacturer.
  • FIG. 8 shows that recombinant 2-chain (scFv ⁇ endotoxin)-hIgG1 Fc-(scFv ⁇ CD32a) significantly reduced TNF- ⁇ secretion stimulated by E. coli LPS 0111:B4, compared to control antibodies 2-chain (scFv ⁇ endotoxin)-hIgG1 Fc protein and anti-CD32a scFv or medium alone.

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RU2017144294A3 (ru) 2019-06-20
US9975951B2 (en) 2018-05-22
US9988454B2 (en) 2018-06-05
US20160339110A1 (en) 2016-11-24
US20160339116A1 (en) 2016-11-24
US10202455B2 (en) 2019-02-12
TWI614030B (zh) 2018-02-11
IL255785B (en) 2021-09-30
TW201706312A (zh) 2017-02-16
TWI614264B (zh) 2018-02-11
IL255785A (en) 2018-01-31
CA2986486C (en) 2023-03-07
RU2703952C2 (ru) 2019-10-22

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