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WO2025040637A1 - Test immunologique pour le propofol - Google Patents

Test immunologique pour le propofol Download PDF

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Publication number
WO2025040637A1
WO2025040637A1 PCT/EP2024/073240 EP2024073240W WO2025040637A1 WO 2025040637 A1 WO2025040637 A1 WO 2025040637A1 EP 2024073240 W EP2024073240 W EP 2024073240W WO 2025040637 A1 WO2025040637 A1 WO 2025040637A1
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WO
WIPO (PCT)
Prior art keywords
seq
propofol
antibody
clone
buffer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/073240
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English (en)
Inventor
Mark O'connell
CHristopher BLENCOWE
Duncan PURVIS
Jennifer MURRAY
Kevin WOOLSTON
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Somnus Scientific Ltd
Original Assignee
Somnus Scientific Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Somnus Scientific Ltd filed Critical Somnus Scientific Ltd
Publication of WO2025040637A1 publication Critical patent/WO2025040637A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/948—Sedatives, e.g. cannabinoids, barbiturates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity

Definitions

  • the present invention relates generally to devices, methods, antigens and antibodies for conducting immunoassays and particularly, although not exclusively, to an immuno-biosensor for propofol.
  • Immuno-biosensors are a type of biosensor to detect the formation of an immunocomplex using an antibody or antigen as a bioreceptor.
  • Propofol (2,6-diisopropylphenol, formula I) is a hypnotic alkylphenol derivative and is a parenteral anaesthetic agent commonly used to produce sedation and general anaesthesia.
  • TIVA total intravenous anaesthesia
  • the optimisation of sedation using propofol is limited by the lack of suitable methods for the real-time monitoring of blood propofol concentration in at the point of care of patients receiving sedation.
  • any method must be capable of returning results within a sufficiently narrow window of time to provide information that is of practical use to anaesthetists or other healthcare professionals.
  • any sensor system needs to be capable of producing stable results over the duration of a surgical procedure, potentially 8 hours or longer. Furthermore, it has been shown that propofol will slowly redistribute between the plasma and blood cell membranes over time, meaning that the time between the collection and measurement of a sample needs to be tightly controlled. As such, any technique for real-time propofol monitoring should be suitable for automation, with minimal sample processing.
  • propofol will be co-administered with other drugs (e.g. lidocaine, opioid analgesics such as fentanyl or morphone, competitive neuromuscular blocking agents such as atracurium or rocuronium, antibiotics, and anti-inflammatory agents), and as such it is necessary that any propofol assay possess a sufficient degree of specificity.
  • other drugs e.g. lidocaine, opioid analgesics such as fentanyl or morphone, competitive neuromuscular blocking agents such as atracurium or rocuronium, antibiotics, and anti-inflammatory agents.
  • the antigen is injected into an animal and the resulting antibodies, generated as part of the immune response, are extracted.
  • Propofol is a small molecule: a small molecule is a low molecular weight ( ⁇ 1000 Da) organic compound.
  • hapten half antigen
  • Carrier proteins such as keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) are conjugated to multiple copies of the small molecule.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • Some of these antibodies are able to bind both the protein-conjugated and unconjugated small molecule, though the majority only bind to the conjugated form and so have to be screened. Once screened some of these antibodies exhibit good affinity for their small molecule target, with reported KD values in the nanomolar range; though this is often only achievable following extensive, stringent in vitro selection from immune libraries and affinity maturation.
  • An aspect of the present invention provides an antibody produced by immunisation of a nonhuman animal with propofol-4-carboxylic acid conjugated to a carrier protein.
  • An aspect of the present invention provides an antibody produced by immunisation of a nonhuman animal with an antigen consisting of propofol-4-carboxylic acid conjugated to a carrier protein.
  • a further aspect of the present invention provides an antibody produced by immunisation of a non-human animal with a propofol-4-carboxylic acid conjugated to a carrier protein.
  • a further aspect of the present invention provides an antibody produced by immunisation of a non-human animal with a propofol-4-carboxylic acid or an analogue or functional equivalent thereof conjugated to a carrier protein.
  • a further aspect of the present invention provides an antibody produced by immunisation of a non-human animal with a propofol-4-carboxylic acid, including salts and esters thereof, conjugated to a carrier protein.
  • a further aspect provides an antibody produced by immunisation of a non-human animal with HS357 conjugated to a carrier protein.
  • a further aspect provides an antibody which binds specifically to propofol, said antibody produced against an antigen comprising propofol-4-carboxylic acid conjugated to a carrier protein.
  • a further aspect provides an antibody which binds specifically to propofol, said antibody produced against an antigen comprising HS357 conjugated to a carrier protein.
  • the carrier is KLH.
  • the carrier is BSA.
  • Antibodies formed in accordance with the present invention may be monoclonal or polyclonal. In some aspects and embodiments the antibody is selected to be specific to the hapten.
  • the present invention also provides an antibody binding to propofol, which comprises HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2 and LCDR3 sequences of SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56.
  • the present invention also provides an antibody binding to propofol, which comprises HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2 and LCDR3 sequences of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64.
  • the present invention also provides an antibody binding to propofol, which comprises HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2 and LCDR3 sequences of SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71 , SEQ ID NO: 72.
  • the present invention also provides an antibody binding to propofol, which comprises HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2 and LCDR3 sequences of SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80.
  • the present invention also provides a monoclonal antibody for binding to propofol, the antibody comprising one or more of SEQ ID NO: 1 to SEQ ID NO: 80.
  • a further aspect provides an immunoassay for measuring propofol in a sample, comprising an antibody as defined herein.
  • the immunoassay may be provided in a plate-based ELISA format.
  • a further aspect provides a rapid, point-of-care immune-biosensor for propofol in blood plasma based on an antibody as defined herein.
  • Propofol will be conjugated to both KLH and BSA carrier proteins for immunisation and screening.
  • Propofol - purchased from Sigma Aldrich P1643-1 ML lot LRAC4221 Pharmaceutical secondary standard reference material.
  • Buffer concentration - Further conjugates were prepared using conventional Fraction V BSA, varying the MES/NaCI buffer concentration, amount of ethanol added and the relative concentration of formaldehyde. All variations showed precipitation, but the standard method as already tried was the least bad.
  • KLH tubes 200ul/tube at 10mgml + 50ul formaldehyde + 25ul of ethanol containing propofol at 80mgml.
  • cBSA tubes 200ul/tube at 10mgml + 200ul CP buffer + 50ul formaldehyde + 75ul ethanol containing propofol at 80mgml.
  • Protein-propofol material collected by centrifugation at 17,000g for 10min. Pellets resuspended in DPBS and stored frozen.
  • mice from a similar cohort showed a strong response as expected, therefore we do not believe there were any issues with the animals themselves or the immunisation process.
  • the small propofol molecule is either not very immunogenic; or that the antibodies raised can only weakly bind the small target.
  • HS245 includes a hydroxyamide moiety
  • HS357 incorporates a six-carbon linker between the phenyl and hydroxyamide moieties.
  • propofol-BSA for screening may be problematic since there are two pocket binding sites for propofol in serum albumin. Will this be beneficial and produce two sets of antibodies specific for free propofol and bound propofol? Propofol-BSA cannot really be used in assay development for the same reason; it will non- specifically bind propofol in the sample and give a bias in measurement of low concentrations of propofol.
  • Proposal propofol analogue e.g., 2-(3-ethyl-4-hydroxy-5-isopropyl-phenyl)-3,3,3-trifluoro-2- hydroxypropionamide.
  • Propofol-4-carboxylic acid 2 (TRC, P829760-1G) will be conjugated to KLH (Sigma, H7017) and BSA (Sigma, A7030) using EDC/NHS coupling chemistry, to generate the propofol immunogen and screening conjugate, respectively.
  • the products will be purified by gel filtration (e.g. Sephadex G25) eluting with PBS pH 6.7, to remove unreacted hapten and coupling reagents. Protein concentrations will be ascertained by UV-vis spectrophotometry. A qualitative assessment of hapten incorporation will be made by TNBS assay and UV-vis spectrophotometry.
  • the products will be normalised at >1 mg/ml and terminally filtered to 0.2 pm.
  • An appropriate quantity of immunogen and screening conjugate is provided from Stage 1 , and an immunisation program is carried out to generate monoclonal antibody against propofol.
  • Monoclonal antibody development programme is outlined below:
  • Phase 1 Immunisation and test bleed analysis - 4 * mice.
  • Phase 2 Fusion of 2 * mice spleen and screening.
  • Phase 3 Limiting dilution cloning, per round, per cell line.
  • Phase 4 Final production per cell line, up to 5mg approx. Hybridomas - Pilot scale up and purification.
  • Stage 3 Evaluate polyclonal antisera produced using the immunogen produced in Stage 2 and carry out proof of concept development in an immunoassay format.
  • the aim of this phase was to evaluate the polyclonal antisera produced using the immunogen produced and carry out proof of concept development in an immunoassay format.
  • the polyclonal antisera has been raised to be sensitive to Propofol, also known as Diprivan, a commonly used anaesthetic involved in the starting and maintenance of general anaesthesia as well as other medical applications.
  • Propofol also known as Diprivan
  • the polyclonal antisera was tested before assay development was undertaken using the Propofol-BSA conjugate showing comparable results for both hosts. These were then titrated at FBL at a range of concentrations (Propofol-BSA: 20- 0.156pg/ml, Antisera: 1/250 - 1/512000) using an Anti-Rabbit IgG-HRP detection conjugate.
  • the assay conditions which were optimised in Objective 1 were used to test serial dilutions of each of the interferents identified. These were tested in duplicate with a control on each plate. All interferents were made up at the same concentration as the control series to calculate relative displacement (DV337/17.24). This was only necessary for two of the interferents, Propofol-O-Glucuronide and Propofol-O-Sulphate. All other potential interferents tested produced no antibody displacement over the assessed concentration range.
  • Anti-Propofol Antibody Anti-Propofol serum (Harvest Bleed, Rabbit 1 ) sourced the antibody generation program (lot number: 22:05/1551-3).
  • Anti-Rabbit HRP Anti-rabbit HRP sourced from Merck, (catalogue code: lot number: 3920753).
  • Propofol- BSA solution Propofol-BSA conjugate prepared by FBL and provided by SOM/APS (lot number: SOM1/2), stock concentration 1.7mg/ml.
  • Buffer 21 See appendix for formulation.
  • Buffer 111 See appendix for formulation.
  • ⁇ Buffer 58 See appendix for formulation.
  • Propofol-BSA preparation Optimal assay concentration was found to be 20pg/ml for Propofol-BSA coating.
  • Optimal assay concentration was found to be a 1/500 dilution.
  • Optimal assay concentration was found to be a 1/5000 dilution.
  • Standard series started from a concentration of 4pg/ml.
  • a serial dilution of 1 in 4 was performed to produce 7 standards in the range 4pg/ml to 0.98ng/ml.
  • Table 3 Table showing relative displacement of interferents (DV337/24)
  • Table 6 Back calculated values for undiluted QC samples based on 4PL fit of standard curve for the respective day of testing (DV337/32,34,37).
  • the assay developed demonstrated an effective range of between 0.02-80pg/ml with a serum dilution of 1/20 applied which meets the target range of 0.1-20pg/ml.
  • the assay appears to be tolerant of up to 20% serum, but calibrators should be prepared in diluted matrix pool. If maximising sensitivity is required, then using a 1/5 sample dilution gives an effective assay range of 0.005-20pg/mL.
  • the antiserum Whilst there is a relatively low level of cross-reactivity with the metabolites, particularly given that the glucuronide is the primary metabolite, the antiserum is not 100% specific for propofol only. Whilst it had been hoped that the design of the immunogen would give something that was reactive with propofol only, the low level of cross-reactivity, particularly for the glucuronide metabolite, does give options. It should be remembered that this is crude polyclonal antiserum and so contains antibodies with a range of different specificities. For a lateral flow type test to be manufactured, a monoclonal antibody would be the preferred embodiment. In which case, this data gives guidance for the clone screening approach to be used.
  • the same immunogen can be used for the immunisation, but for screening hits and clones a tiered approach would need to be taken.
  • the initial screen would be for binding to the screening conjugate, after which subsequent testing would be done to exclude those clones which displace with the metabolites. In this way the clones for investigation can be selected on the basis of minimal cross-reactivity.
  • the antisera had shown a degree of reactivity to the sulphate and, to a lesser extent, glucuronide metabolites of propofol.
  • the aim of this phase of the project is to attempt to deplete the polyclonal antiserum of the unwanted metabolite reactivity whilst maintaining reactivity with the parent compound.
  • Propofol-BSA hapten conjugate will be produced using the method established in project S0M1.
  • the resulting conjugate will then be immobilised on Cytiva HiTrap NHS-activated HP 1 mL column.
  • Such columns can be readily scaled if required in the future for process scale up.
  • 1 mL of crude antiserum will be passed down the column and allowed to bind.
  • Captured antibodies will be eluted by acidification. This will demonstrate utility of the column and provide a baseline for the proportion of the antiserum which is propofol specific.
  • the concentration of eluted antibodies will be determined by UV absorbance.
  • the eluate products from objectives 1 and 2 will be assessed for reactivity using the proof of concept assay developed under Phase 3.
  • the products and crude antiserum will be run in the assay against standard curves of propofol, and the sulphate and glucuronide metabolites.
  • Working strengths for the eluate products will be estimated based on the known working strength of the crude antiserum and the product concentrations determined under objectives 1 and 2.
  • the products will be assessed relative to the crude antiserum and cross reactivity with the metabolites estimated based on the displacement seen relative to propofol itself.
  • Monoclonal antibodies have been generated using a hybridoma technology approach. It involves fusing B cells, which produce antibodies, with myeloma cells, which are cancerous cells that grow indefinitely. The resulting hybrid cells, known as hybridomas, can produce a large quantity of identical mAbs that target a specific antigen.
  • the general process of creating hybridomas involves several steps, including immunization of an animal with the antigen of interest, harvesting B cells from the spleen, fusing the B cells with myeloma cells using a chemical or electrical method, selecting the hybridomas that produce the desired antibody, and finally, growing and maintaining the selected hybridomas in culture.
  • cAb11051 aka CE8
  • cAb11052 aka CE7
  • cAb11053 aka FH10
  • cAb11131 aka GH5

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  • Health & Medical Sciences (AREA)
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  • Physics & Mathematics (AREA)
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  • Pharmacology & Pharmacy (AREA)
  • General Physics & Mathematics (AREA)
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  • Food Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Anesthesiology (AREA)
  • Epidemiology (AREA)
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Abstract

L'invention concerne un anticorps destiné à se lier au propofol. L'invention concerne également un test immunologique pour le dosage du propofol et un biocapteur immunologique rapide hors laboratoire pour le propofol.
PCT/EP2024/073240 2023-08-18 2024-08-19 Test immunologique pour le propofol Pending WO2025040637A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB2312657.6 2023-08-18
GBGB2312657.6A GB202312657D0 (en) 2023-08-18 2023-08-18 Immunoassay

Publications (1)

Publication Number Publication Date
WO2025040637A1 true WO2025040637A1 (fr) 2025-02-27

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PCT/EP2024/073240 Pending WO2025040637A1 (fr) 2023-08-18 2024-08-19 Test immunologique pour le propofol

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000059945A1 (fr) * 1999-04-06 2000-10-12 The Horticulture And Food Research Institute Of New Zealand Limited Ameliorations apportees a des dosages immunologiques d'anesthesiques
WO2021239495A1 (fr) * 2020-05-29 2021-12-02 Somnus Scientific Ltd Capteur de propofol

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000059945A1 (fr) * 1999-04-06 2000-10-12 The Horticulture And Food Research Institute Of New Zealand Limited Ameliorations apportees a des dosages immunologiques d'anesthesiques
WO2021239495A1 (fr) * 2020-05-29 2021-12-02 Somnus Scientific Ltd Capteur de propofol

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GB202312657D0 (en) 2023-10-04

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