WO2024076704A1 - Élimination d'il -6 - Google Patents

Élimination d'il -6 Download PDF

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Publication number
WO2024076704A1
WO2024076704A1 PCT/US2023/034589 US2023034589W WO2024076704A1 WO 2024076704 A1 WO2024076704 A1 WO 2024076704A1 US 2023034589 W US2023034589 W US 2023034589W WO 2024076704 A1 WO2024076704 A1 WO 2024076704A1
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WIPO (PCT)
Prior art keywords
seq
amino acid
column
sequence
ligand
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PCT/US2023/034589
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English (en)
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Steven Francis Josephs
Michael MATHO
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Immunicom, Inc.
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Publication of WO2024076704A1 publication Critical patent/WO2024076704A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • A61M1/3472—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate
    • A61M1/3486—Biological, chemical treatment, e.g. chemical precipitation; treatment by absorbents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • A61M1/3496—Plasmapheresis; Leucopheresis; Lymphopheresis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3679—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by absorption
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2202/00—Special media to be introduced, removed or treated
    • A61M2202/04—Liquids
    • A61M2202/0413—Blood
    • A61M2202/0445—Proteins

Definitions

  • Apheresis typically includes the steps of extracting blood from a patient, separating the blood into blood components such as red blood cells and plasma, removing a particular element such as platelets from the plasma, and then recombining the blood components and returning them to the patient. Separation of the blood components and removal of the element are commonly performed using centrifugation or mechanical filtering.
  • Interleukin 6 is a cytokine that is involved with inflammatory processes including those caused by infections, autoimmune diseases such as rheumatoid arthritis, inflammatory bowel syndrome or Crohn’s disease, and the development and progression of cancer. Persistent elevation of IL-6 levels is deleterious and can lead to organ failure and death.
  • the IL-6 receptor complex consists of the IL-6 alpha receptor (IL-6Ra, gp80, CD126) and the signal transducing subunit glycoprotein 130 (gpl30, CD 130.) IL-6 first binds to IL- 6Ra with low affinity. The IL-6:IL-6Ra complex subsequently builds a high affinity complex with gp!30, as shown in Fig. 1, that is referred to herein as Protein Data Bank “1P9M.”
  • the present invention provides apparatus and methods for the selective removal of IL- 6 from the plasma component of the blood of a patient. Certain methods include use of an apheresis machine in conjunction with a column disclosed herein.
  • a column for removing IL-6 from a body fluid or a blood component of a patient is disclosed.
  • the column comprises a sequestering chamber configured to receive the body fluid or the blood component, and a substrate disposed within the sequestering chamber.
  • the substrate has a capture ligand immobilized thereon.
  • the capture ligand is configured to selectively bind to the IL-6 in the body fluid or the blood component.
  • the method comprises the steps of receiving a blood component from the patient, modifying the blood component by passing a portion of the blood component through a column containing a substrate having a capture ligand immobilized thereon, and returning the modified blood component to the patient.
  • the capture ligand is configured to bind to IL-6, wherein a portion of IL-6 that is in the blood component binds to the capture ligand and remains in the column.
  • a ligand adapted to capture IL-6 is also disclosed.
  • the ligand comprises a modified sequence comprising an amino acid substitution in a reference sequence that comprises a portion of a wild-type IL-6 alpha receptor (IL-6Ra) sequence.
  • Fig. 1 is a 3D depiction of the IL-6:IL-6Ra:gpl30 complex.
  • Fig. 2 is a 3D depiction of the IL-6:IL-6Ra complex.
  • Fig. 3 is a plot of the efficiency of capture of IL-6 by immobilized natural IL-6Ra ligands, according to certain aspects of the present disclosure.
  • Fig. 4 is a plot of the leaching of the natural IL-6Ra ligands, according to certain aspects of the present disclosure.
  • Fig. 5 is a plot of the efficiency of capture of IL-6 by different densities of the immobilized IL-6R ligands, according to certain aspects of the present disclosure.
  • Fig. 6 is a plot of the capture efficiency of natural and variants of IL-6, according to certain aspects of the present disclosure.
  • Fig. 7 depicts an exemplary apheresis column according to certain aspects of the present disclosure.
  • Fig. 8 depicts an enlarged view of an exemplary portion of the apheresis column of Fig. 7, according to certain aspects of the present disclosure.
  • Fig. 9 is a schematic of an exemplary salt bridge, according to certain aspects of the present disclosure.
  • Fig. 10 depicts a first exemplary salt bridge between a variant IL-6Ra and IL-6, according to certain aspects of the present disclosure.
  • Fig.11 depicts a second exemplary salt bridge between a variant IL-6Ra and IL-6, according to certain aspects of the present disclosure.
  • FIGs. 12 A and 12B illustrate an exemplary effect of a modification to the IL-6Ra, according to certain aspects of the present disclosure.
  • Fig. 13 depicts an exemplary synergistic modification of IL-6Ra, according to certain aspects of the present disclosure.
  • Reduction of IL-6 by a specific ligand as disclosed herein has significance in the context of a condition in which above-average levels of IL-6 are present in a blood component.
  • Many patients with underlying acute or chronic inflammatory conditions may present with an elevated level of IL-6, which may be measured in a biological fluid or in a blood component such as serum or plasma component.
  • an apheresis apparatus may comprise a commercially available apheresis machine, for example, a Terumo BUT Spectra Optia System, and may be used in conjunction with an adsorptive column for use in removing one or more disease-related components and/or treatment-related components from plasma.
  • the column has a compartment and a bead disposed within the compartment.
  • the bead comprises a substrate with an adsorbent coupled to the substrate, wherein the adsorbent comprises a capture ligand that exhibits specificity and selectivity for IL-6.
  • blood component or “body fluid” refers to a portion of blood from a human or animal and, in certain instances, may refer to unseparated whole blood.
  • target refers to a molecule, antigen, antibody, cellular element or a fragment thereof for which the ligand has specificity and/or selectivity.
  • molecule means any biologically active substance and includes cytokines, proteins, amino acids, nucleic acids, nucleotides, carbohydrates, and lipids.
  • adsorbent means a material that possesses an affinity to couple to a target.
  • An example coupling mechanism is binding of a site on the adsorbent to a portion of a target. Binding of a target to an adsorbent that is non-detachably coupled to a substrate captured within the column is intended to retain the target within the column.
  • An adsorptive column includes an adsorbent that selectively adsorbs a target molecule.
  • “selectively” indicates that sites on the adsorbent interact with sites on the target molecule in a manner similar to a “lock and key” mechanism, and that the target molecule binds to the adsorbent.
  • a selective adsorbent may comprise multiple different sites that will bind to multiple different target molecules.
  • tumor necrosis factor As used within this disclosure, the terms and phrases “tumor necrosis factor,” “TNF,” “tumor necrosis factor alpha,” “TNF-alpha,” “TNF-a,” “TNF-a,” and variations thereof are equivalent and interchangeable unless explicitly stated otherwise.
  • interleukin 6 interleukin 6
  • IL6 interleukin-6
  • IL-6 IL-6
  • interleukin 6 alpha receptor As used within this disclosure, the terms and phrases “interleukin 6 alpha receptor,” “interleukin-6 receptor,” “IL6R,” “IL-6R,” “IL-6Ra,” “IL-6Ra,” “IL-6RA,” and variations thereof and alternate names gp80 and CD126 are equivalent and interchangeable unless explicitly stated otherwise. [0036] As used within this disclosure, the terms “natural” and “native” and the phrase “wildtype” and variations thereof are interchangeable and refer to the form of a molecule, or portion thereof, created by natural processes within an organism.
  • the receptor IL-6Ra will bind to the cytokine IL-6 in the absence of gpl30.
  • Fig. 2 is a 3D depiction of the IL-6:IL-6Ra complex.
  • the IL-6Ra chain comprises extracellular domains D2-D3.
  • Extracellular domain D2 comprises VI 12-C211 of SEQ ID NO: 1 and D3 comprises G212-W315 of SEQ ID NO: 1.
  • IL-6Ra forms only low-affinity bonds to IL-6, with affinity of around 46 nanomolar (nM), while the affinity of IL-6 to the IL-6Ra:gpl30 complex is around lOOx greater. Given the low affinity that IL-6Ra has for IL-6, it was doubtful whether IL-6Ra would be successful in capturing significant quantities of IL-6. In human plasma, most of the soluble IL-6 remains free (i.e., unbound to IL-6Ra) despite a high concentration of IL-6Ra that are simultaneously present in the plasma. Furthermore, IL-6 is known to remain largely free in in vitro experiments where large molar excesses of IL-6 are incubated with IL-6Ra.
  • SEQ ID NO: 1 Human IL-6Ra
  • SEQ ID NO: 2 natural IL-6R Peptide Sequence
  • IL-6Ra As a capture ligand, purified human IL-6Ra (Peprotech, Cranbury, NJ) was coupled to Sepharose Fast Flow 6 beads (Cytiva, Marlborough MA) that had been activated by treatment with sodium metaperiodate. 1 ml of IL-6Ra was coupled to 1 ml of the activated beads by Schiff base formation followed by treatment with sodium cyanoborohydride thereby producing a secondary amine bond between the IL-6Ra and the bead. The IL-6 coupled beads as well as control beads (background) that were not previously coupled to IL-6Ra were then exposed to ethanolamine, which quenches any unbound methyl groups on the bead surface.
  • Test solutions were prepared of (a) PBS spiked with 1 ng/ml of human IL-6 and 0.025% BSA and (b) human plasma and passed through the columns sequentially at the indicated flow rates. The outflow of the column was collected for 1 minute at each flow rate.
  • the IL-6 concentration of the test solutions and each sample was measured using a human IL-6 MSD assay (Mesoscale Discovery, Rockville MD). The percentage of IL-6 capture was calculated using the formula (1-Co/Ci)*100 where Ci is the IL-6 concentration of the test solution and Co is the IL-6 concentration of the sample.
  • Fig. 3 is a plot 300 of the efficiency of capture of IL-6 by immobilized natural IL-6R ligands, according to certain aspects of the present disclosure.
  • the column was tested with both human plasma and phosphate buffered saline (PBS), each spiked with human IL-6.
  • the extent of IL-6 removal (capture efficiency) was determined based on the formula (1- Co/Ci)*100 where Ci is the concentration of IL-6 in the inflow solution and Co is the concentration of IL-6 in the outflow solution. Efficient capture of IL-6 was observed in both PBS and plasma.
  • the capture efficiency of a column comprising natural IL 6R ligands is greater than 70%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, and greater than 97%.
  • Fig. 4 is a plot 400 of the leaching of the IL-6Ra ligands from an experimental column, according to certain aspects of the present disclosure.
  • Leaching is a concern as an adsorbent, e.g., natural TNF, that leaches from the column into the blood component being returned to the patient may be toxic to the patient.
  • IL-6Ra is naturally present in the blood of healthy humans.
  • Adsorbent columns are routinely flushed, for example with saline or PBS, to remove any residual unbound ligand before clinical use with a patient.
  • the 1 ml test column was not flushed prior to the testing.
  • the initial outflow showed a higher level of IL-6Ra compared to subsequent samples in the sequence of samples.
  • the level of IL-6Ra was measured in each of the samples collected as described above from an experimental column.
  • the amount of IL-6Ra in the samples was measured using an MSD assay for human IL-6Ra.
  • the initial leaching rate of 6.8 ng/min at a flow rate of 5.12 ml/min is equivalent to a density of 1.2 ng/ml. Scaling this up to a commercial 15 ml column would indicate the initial leaching might be as high as 18 ng/ml.
  • the leaching rates of samples 2-5 are more representative of clinical performance and scale up to levels of 4.4-12.5 ng/ml. In all cases, the leaching rate compares favorably with the normal healthy level of IL-6Ra in the blood of 50-75 ng/ml and suggests that leaching is not a concern with this capture ligand.
  • Fig. 5 is a plot 500 of the efficiency of capture of IL-6 by different densities of the immobilized IL-6R ligands, according to certain aspects of the present disclosure. This test was conducted using 1-3 mg of IL-6Ra on 1 ml of beads (1 mg/ml to 3 mg/ml) and comparatively tested. Purified IL-6Ra was coupled to beads at 0, 1 mg and 3 mg of protein per mL of beads followed by coupling to ethanolamine as described in Fig. 1. The capture efficiency was determined using a test solution of PBS, 0.25% BSA spiked with 20 ng/mL of IL-6. Samples obtained at the flow rates described and likewise assayed for capture efficiency. Fig. 5 presents an average capture efficiency calculated from all the flow rates for each column.
  • polypeptide of SEQ ID NO: 2 was generated with a 10 amino acid Histidine Tag juxtaposed to a C terminal Flag Tag, thus creating SEQ ID NO: 3 that was used for testing.
  • SEQ ID NO: 3 IL-6Ra Peptide with tag
  • the fluid is blood or a portion thereof, for example plasma, and the target component to be removed is IL-6.
  • a portion of the IL-6R is produced as an IL-6 adsorbent, which is then non-detachably bound to a substrate.
  • the portion of the IL-6R comprises a primary amine group.
  • the portion of IL-6R is bound to the substrate with a secondary amine bond.
  • the capture ligand comprises the binding sites of IL-6R.
  • a truncated capture ligand comprises a portion of the amino acids of IL-6R as shown in SEQ ID NO: 4. The amino acid 1 -letter codes are listed in Table 1.
  • human IL-6Ra with a native-sequence is used as a ligand for binding to IL-6 and for removal of IL-6 from a blood component.
  • an apheresis column comprising native-sequence human IL-6Ra is used for removal of IL-6 from a blood component.
  • a ligand for removal of IL-6 from a blood component comprises SEQ ID NO: 1.
  • the ligand comprises V112-C211 of SEQ ID NO: 1.
  • a ligand comprises G212-W315 of SEQ ID NO: 1
  • a capture ligand for use in an adsorptive column comprises L-6Ra molecules with conservative amino acid substitutions at one or more sites. In certain embodiments, only lysine (K) sites are substituted.
  • the natural IL6-Ra peptide sequence of SEQ ID NO: 2 contains multiple lysines. Certain lysines (e.g, K124, K152, K173) contribute to the structural integrity and folded configuration of the IL-6Ra molecule and substitutions for these lysines adversely affect the structure. Amino acid substitutions for other lysines (e.g, K201, K204, K263, K271) do not compromise the structure of IL-6Ra. Protein coupling to the support structure occurs by amine coupling at exposed lysines.
  • lysines e.g, K124, K152, K173
  • Amino acid substitutions for other lysines e.g, K201, K204, K263, K271
  • Protein coupling to the support structure occurs by amine coupling at exposed lysines.
  • K271 is modified to G271 in SEQ ID NO: 6
  • K271 is modified to R271 in SEQ ID NO: 7.
  • Table 2 shows groupings of amino acids that are similar to each other.
  • any amino acid, other than lysine can be substituted for a lysine in IL-6R.
  • the amino acid being substituted does not comprise an amine group.
  • the amino acid being substituted for a lysine is selected from Table 2.
  • the amino acid being substituted for a lysine is selected from the group of basic amino acids of Table 2.
  • the amino acid being substituted for a lysine is selected from the group of aliphatic amino acids of Table 2.
  • any amino acid may be substituted for a non-lysine in IL-6R in order to modify the IL-6R structure so as to improve the availability 7 of an IL-6 binding site.
  • an amino acid is substituted for a non-lysine that is adjacent to a lysine that is also being substituted.
  • the amino acid being substituted for a non-lysine is selected so as to form a salt bridge with a proximate portion of an IL-6 molecule.
  • the amino acid being substituted for a non-lysine is selected from Table 2.
  • the amino acid being substituted for a non- lysine is selected from the group of basic amino acids of Table 2.
  • the amino acid being substituted for a non-lysine is selected from the group of aliphatic amino acids of Table 2.
  • the natural IL-6Ra of SEQ ID NO: 2 is modified by various amino acid substitutions to produce the sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
  • the protease cleavage site of IL-6Ra is modified.
  • SEQ ID NO: 5 has “PVQD” at the protease cleavage site replaced with “GGS.”
  • SEQ ID NO: 6 has this same ; ‘GGS” substitution as well as “KD” at sites 271-272 replaced with “GR ”
  • SEQ ID NO: 7 has the same “GGS” substitution as well as KD " replaced with “GD.” Modification of the protease cleavage site prevents unwanted protease activity resulting in expression of the full-length intact protein. For sequences with an intact protease cleavage site, the His Tag can be cleaved off obviating IMAC (Nickel Column) purification.
  • IMAC Nickel Column
  • SEQ ID NO: 6 IL-6Ra Variant 1
  • SEQ ID NO: 7 IL-6Ra Variant 2
  • the natural IL-6Ra of SEQ ID NO: 2 is a reference sequence that serves as a basis for creation of a modified sequence by addition, substitution, or substitution of an amino acid in the reference sequence.
  • the reference sequence is a portion of SEQ ID NO: 1.
  • Table 3 shows that variant SEQ ID NO: 6 has 99.4% identity with natural SEQ ID NO: 2 in the overlap of 335 residues.
  • each residue has an asterisk (*) where the residues are identical and an underscore (_) where the amino acids are different.
  • SEQ ID NO: 2 171 SQKFSCQLAVPEGDSSFYIVSMCVASSVGSKFSKTQTFQGCGILQPDPPA
  • SEQ ID NO: 7 171 SQKFSCQLAVPEGDSSFYIVSMCVASSVGSKFSKTQTFQGCGILQPDPPA
  • Table 4 shows that SEQ ID NO: 7 has 99.7% identify with natural SEQ ID NO: 2 in the overlap of 335 amino acid residues.
  • each residue has an asterisk (*) where the residues are identical and an underscore (_) where the amino acids are different.
  • the modified sequence has at least 60%, 70%, 75%, 80% , 85%, 90% , 95% , 96% , 9% , 98 % , 99% or more identify to the reference sequence. In certain embodiments, the modified sequence has less than 99.9% identity to the reference sequence. In certain embodiments, the modified sequence has less than 99% identify to the reference sequence. In certain embodiments, the modified sequence has less than 98% identify to the reference sequence. In certain embodiments, the modified sequence has less than 97%, 96%, 95%, 90%, 85% or less identify to the reference sequence.
  • the adsorbent of a column comprises a complex of a modified IL-6R and a gpl30 molecule (IL-6R:gpl30) that binds to IL-6 in a blood component passing through the column.
  • one or more residues of the IL-6R are modified so as to modify the 3D structure and/or the orientation of a IL-6R:gpl30 complex that is attached to a substrate so as to increase one or more of the binding affinity of the IL-6R:gpl30 complex for IL-6, improve the exposure of certain residues to the blood component, and increase the capacity of the adsorbent to bind to IL-6.
  • Fig. 6 is a plot of the capture efficiency of natural and variants of IL-6, according to certain aspects of the present disclosure.
  • Experimental columns were prepared, respectively using beads prepared with ligands of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 as well as a control column that had only activated beads quenched in ethanolamine.
  • a test solution of PBS spiked with 1 ng/ml of IL-6 and 0.025% BSA was passed through the columns at the indicated flow rates of 1, 2, and 3 ml/min. The measured capture efficiencies are shown in the bottom row of Fig. 6.
  • Table 5 shows the measured affinities of the tested capture ligands.
  • the on-rate (kon) and off-rate (koff) are parameters that reflect the rate at which molecules associate and dissociate in a solution.
  • Both SEQ ID NOS. 6-7 show almost a 2-fold increase in binding affinity for IL-6.
  • Fig. 7 depicts an exemplary apheresis column 700 according to certain aspects of the present disclosure.
  • the column 600 comprises a body 710 that comprises a compartment 720 having an inlet 730 and an outlet 734.
  • the compartment 720 is generally a right cylinder wherein the inlet 730 and outlet 734 are both planar circular disks.
  • the cross-sectional shape of the compartment 720 may be oval, rectangular, or other regular or irregular or nonplanar geometnc shape.
  • the size and shape of one or both of the inlet 730 and outlet 734 may be different from the size and shape of the nominal cross-section of the compartment 720.
  • fluid enters an entrance port 732 and is conveyed to the inlet 730.
  • fluid coming out of the outlet 734 is conveyed to an exit port 736.
  • the column may be oriented in any direction, including upside down, such that the direction of gravity in Fig. 7 may be in any direction.
  • one or both of the inlet 730 and outlet 734 comprise a porous wafer, commonly referred to as a “frit,” that is fabricated by melting polyethylene beads together.
  • the diameter of the beads and the degree of compression are chosen to produce an average pore size.
  • the average pore size is 20 microns.
  • the frit is formed by sintering beads comprising a metal or a ceramic, with the same effect. It is generally desirable to select an average pore size for the frit that allows the largest elements present in the incoming fluid to pass through the inlet 730 and outlet 734, thereby avoiding clogging of the column 700. It is further desirable to select the average pore size to retain the substrates, such as the beads 750 of Fig. 8, within the compartment 720.
  • Fig. 8 depicts an enlarged view of an exemplary portion “A” of the apheresis column 600 of Fig. 7, according to certain aspects of the present disclosure.
  • the compartment 720 is at least partially filled with a substrate, for example a plurality of beads 750 as shown in Fig. 8.
  • the beads 750 are spherical with a diameter in a range of 10-10000 microns, 20-1000 microns, 30-500 microns, 40-250 microns, 45-165 microns, 75-125 microns, or other ranges of diameters.
  • the beads 750 have a common nominal diameter of 25, 50, 75, 100, 125, or 150 microns or other nominal diameter.
  • the beads 750 are comprise a plurality of nominal diameters.
  • the term “substrate” means an object that provides structure while not necessarily interacting with material proximate to the substrate.
  • a substrate may comprise one or more organic materials, such as a polysaccharide polymer, and also may comprise one or more inorganic materials, such as metal, ceramic, and water.
  • a substrate may comprise a portion that has been converted to a different form, for example an oxide, by exposure to a substance, treatment, and/or environment.
  • a substrate may comprise one or more layers, for example a coating or plating.
  • a substrate may also be referred to as a “support.”
  • a bead is used to describe an exemplary structural embodiment without excluding other geometric shapes or structures.
  • a bead may provide a substrate as a solid form, such as a solid sphere, or have structure, such as a hollow element or an open-cell foam.
  • a bead may comprise a simple geometric form, for example a sphere or rod, or a more complex form such as a “multi-arm star,” e.g. a child’s toy jack.
  • a bead may comprise other materials, such as a coating of an adsorbant or a catalyst, intended to interact with material proximate to the bead.
  • the compartment 720 may contain a substrate comprising an open-cell foam.
  • a single instance of the substrate may fill the compartment 720 or an entire cross-sectional area and a portion of the length of the compartment 720.
  • the “diameter” of the substrate may be the average width of passages through the foam.
  • Fig. 9 is a schematic of an exemplary salt bridge between a lysine (K) and a glutamic acid (E), according to certain aspects of the present disclosure.
  • Salt bridges are among the highest energy interactions that may be elicited in inter-molecular interfaces.
  • a salt bridge is created between oppositely charged residues, for example between the anionic carboxylate (RCOO-) of aspartic acid (D) and the cationic ammonium (RNH3+) from lysine (K) or the guanidinium (RNHC(NH2)2+) of arginine (R).
  • a salt bridge is formed with other residues having an ionizable side chain, e.g., histidine, tyrosine, and serine.
  • the distance between the residues participating in the salt bridge is important and generally required to be less than 4 angstroms (A).
  • Substitution of an amino acid that increases the ability of the IL-6Ra to form a salt bridge with an IL-6 molecule increases the affinity of the IL-6Ra for IL-6 and thus improves the effectiveness of the IL-6Ra as an adsorbent.
  • Fig. 10 depicts a first exemplary salt bridge between a variant IL-6Ra and IL-6, according to certain aspects of the present disclosure.
  • Evaluation of the 3D computer model of the IL-6Ra:IL-6 binding indicated that residue K271 of the IL-6Ra is ⁇ 1A short of eliciting a strong salt bridge with K27 of IL-6 (using a 1P9M model.
  • Fig. 11 depicts a second exemplary salt bridge between a variant IL-6Ra and IL-6, according to certain aspects of the present disclosure.
  • an aspartic acid (D) is substituted for the cysteine (C) of residue 211 (C21 ID) of IL-6Ra to generate a salt bridge with IL-6 K54.
  • a lysine (K) is substituted for the C at residue 211 (C21 IK) to create a more attractive 2.7 A salt bridge with IL-6 residue E172.
  • Figs. 12A and 12B illustrate an exemplary effect of a modification to the IL-6Ra, according to certain aspects of the present disclosure.
  • Evaluation of the 3D computer model of the IL-6Ra:IL-6 binding indicated that the distance between C211 and E172 of the IL-6 is 5.5 A, which is slightly too large to enable a salt bridge to form.
  • Substitution of K for the C (C21 IK) reorients the side chain, as can be seen in a comparison of C211 of Fig. 11A with C21 IK of Fig. 1 IB.
  • the change in structural configuration reduces the distance between the binding sites to 4.5 A, which enables the formation of a salt bridge that will increase the binding affinity of the variant IL-6Ra for IL-6.
  • a change in the 3D structure and shape that is induced by a substitution of an amino acid will increase the expose of capture sites on a variant IL-6Ra ligand that is immobilized on a substrate, thereby increasing one or both of the binding affinity and the binding capacity of the ligand for IL-6.
  • a change in the 3D structure and shape that is induced by a substitution of an amino acid will promote the formation of a salt bridge between the ligand and a captured IL-6.
  • Fig. 13 depicts an exemplary synergistic modification of IL-6Ra, according to certain aspects of the present disclosure.
  • the formation of a salt bridge between C211D and El 72 at a distance of 4.5 A has been discussed with respect to Figs. 12A-12B.
  • An additional modification Q209E reduces the distance of residue 209 to K64 of the IL-6 to 4.5 A, thereby enabling formation of a salt bridge between these two sites, and also further reduces the distance between C211K to E172 from 4.5 to 4.1 A.
  • the combination of Q209E and C211K thus synergistically promote an stronger C211K-E172 salt bridge that will increase the affinity of IL-6Ra to IL-6 greater than either of the modifications by themselves.
  • Exemplary SEQ ID NO: 8 incorporates Q209E, C21 IK, K271G, and D272E.
  • SEQ ID NO: 8 IL-6Ra Variant 3
  • Headings and subheadings, if any, are used for convenience only and do not limit the invention.
  • top should be understood as referring to an arbitrary frame of reference, rather than to the ordinary gravitational frame of reference.
  • a top surface, a bottom surface, a front surface, and a rear surface may extend upwardly, downwardly, diagonally, or horizontally in a gravitational frame of reference.
  • a phrase such as an “aspect” does not imply that such aspect is essential to the subject technology or that such aspect applies to all configurations of the subject technology.
  • a disclosure relating to an aspect may apply to all configurations, or one or more configurations.
  • a phrase such as an aspect may refer to one or more aspects and vice versa
  • a phrase such as an “embodiment” does not imply that such embodiment is essential to the subject technology or that such embodiment applies to all configurations of the subject technology.
  • a disclosure relating to an embodiment may apply to all embodiments, or one or more embodiments.
  • a phrase such as an embodiment may refer to one or more embodiments and vice versa.
  • the word “exemplary” is used herein to mean “serving as an example or illustration.” Any aspect or design described herein as “exemplary ” is not necessarily to be construed as preferred or advantageous over other aspects or designs.

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Abstract

La présente invention concerne une colonne pour éliminer l'IL-6 d'un composant sanguin d'un patient et des procédés de traitement d'un patient à l'aide d'une telle colonne. La colonne a une chambre de séquestration conçue pour recevoir le fluide corporel et un substrat disposé à l'intérieur de la chambre de séquestration. Le substrat comporte un ligand de capture immobilisé sur celui-ci. Le ligand de capture est conçu pour se lier sélectivement à l'IL-6 dans le fluide corporel.
PCT/US2023/034589 2022-10-05 2023-10-05 Élimination d'il -6 WO2024076704A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6217858B1 (en) * 1997-02-11 2001-04-17 Hadasit & Medical Research Services & Development Company, Ltd. Pharmaceutical composition for treating hepatitis B virus (HBV) infection
WO2008065378A2 (fr) * 2006-11-30 2008-06-05 Astrazeneca Ab Composés
US7531644B2 (en) * 1998-07-06 2009-05-12 Tosoh Corporation IL-6 receptor•IL-6 direct fusion protein
US20200171232A1 (en) * 2012-05-14 2020-06-04 Children's Medical Center Corporation Systems and methods for extracorporeal blood modification
US10905818B2 (en) * 2014-03-31 2021-02-02 Zoll Medical Corporation Blood filtering of inflammatory biomarkers to treat post-resuscitation syndrome

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6217858B1 (en) * 1997-02-11 2001-04-17 Hadasit & Medical Research Services & Development Company, Ltd. Pharmaceutical composition for treating hepatitis B virus (HBV) infection
US7531644B2 (en) * 1998-07-06 2009-05-12 Tosoh Corporation IL-6 receptor•IL-6 direct fusion protein
WO2008065378A2 (fr) * 2006-11-30 2008-06-05 Astrazeneca Ab Composés
US20200171232A1 (en) * 2012-05-14 2020-06-04 Children's Medical Center Corporation Systems and methods for extracorporeal blood modification
US10905818B2 (en) * 2014-03-31 2021-02-02 Zoll Medical Corporation Blood filtering of inflammatory biomarkers to treat post-resuscitation syndrome

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