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WO2021136571A1 - Essai d'activité biologique d'anticorps radiomarqués - Google Patents

Essai d'activité biologique d'anticorps radiomarqués Download PDF

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Publication number
WO2021136571A1
WO2021136571A1 PCT/DK2020/050405 DK2020050405W WO2021136571A1 WO 2021136571 A1 WO2021136571 A1 WO 2021136571A1 DK 2020050405 W DK2020050405 W DK 2020050405W WO 2021136571 A1 WO2021136571 A1 WO 2021136571A1
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Prior art keywords
antibody
antigen
minutes
binding fragment
antigen binding
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Ceased
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PCT/DK2020/050405
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English (en)
Inventor
Claus J. MØLLER SAN-PEDRO
Ahmed Mahiuddin
Sonia SEQUEIRA
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Y-mAbs Therapeutics Inc
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Y-mAbs Therapeutics Inc
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Publication of WO2021136571A1 publication Critical patent/WO2021136571A1/fr
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    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
    • A61K51/1096—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies radioimmunotoxins, i.e. conjugates being structurally as defined in A61K51/1093, and including a radioactive nucleus for use in radiotherapeutic applications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/36—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Actinomyces; from Streptomyces (G)

Definitions

  • the present invention relates to a radioimmunoreactivity or immune reactive fraction assay to evaluate the potency of radioimmunotherapies or radiodiagnostics (e.g. radiotheranostics), radiolabeled with alpha, beta or positron emitting radionuclides.
  • the invention further relates to a kit of parts adapted to carry out the inventive method and the production of the target antigens materials for the assay.
  • the present invention relates to a biotinylated antigen-streptavidin coated bead assay for the assessment of potency of B7FI3, GD2, CD33 or TIM-3 targeting radiolabeled antibodies for the diagnosis or treatment of cancer. Furthermore, a potency assay for 177 Lu-DTPA-8FI9 antibody (CAR 3), 131 I-8H9 antibody or 131 l-humanized 8H 9 antibody.
  • IRF is the measured fraction of an antibody or engineered derivative to its cognate antigen/target upon compared to a reference standard.
  • the IRF is used to evaluate the extent to which an antibody retains its binding to the target upon modifications such as bioconjugation and radiolabeling or as a measure of stability in defined conditions.
  • a rapid bead-based radioligand biding assay for the determination of target-binding fraction and quality control of radiopharmaceuticals, Nuclear Medicine and Biology 71, 2019) has reported a cell-free quantitative method of analysis to determine the IRF of radioligands that includes the incubation of magnetic beads functionalized with Ni-NTA (Nickel-Nitrilotriacetic acid; HisPurTM Ni-NTA magnetic beads; 88831: Thermo Scientific) or streptavidin (65601; DynaBeadsTM MyOneTM Streptavidin Tl; InvitrogenTM) with a histidine-tagged or biotinylated antigen of choice for 15 min, followed by the addition of the radioactive antigen-targeting agent for 30 min, washing of the beads and collection of supernatant, wash and beads for gamma counting.
  • Ni-NTA Nickel-Nitrilotriacetic acid
  • HisPurTM Ni-NTA magnetic beads 88831: Thermo Scientific
  • streptavidin
  • the assay included a positive and negative control, for specificity of radioligand binding to the cognate antigen in the presence of an excess of unlabeled ligand, and for non-specific binding (NSB) of the radioligand to beads lacking the target antigen, respectively.
  • the IRF of 1311-omburtamab was measured using a his-tagged B7-H3 antigen bound to streptavidin beads. IRF's of greater than 90% at end of synthesis (EOS) were consistently achieved and an assessment of stability over time indicated a reduction in potency at 76h plausibly due to 1311-induced radiolysis of the sample in the chosen storage conditions.
  • Immunoreactivity is an important quality control test to ensure that an immunodiagnostic (RID) or immunotherapy (RIT) retains the ability to bind to its target after radiolabeling.
  • the immunoreactivity influences the absolute uptake of an antibody by the tumor and the target-to-background ratio. Deterioration of immunoreactivity is usually caused by conjugation of chelating agents, labeling procedures, specific activity, and by radiolysis during storage. The FDA requires a potency test for the release of clinical products however there is no standard protocol.
  • the most common radiopharmaceutical potency assay is the Lindmo cell-based assay (Lindmo, 1984) in which the fraction of radiolabeled antibody bound to antigen is extrapolated to conditions of "infinite antigen excess".
  • Cell based assays such as this are cell batch and experimental condition dependent, and results are provided post-release given the length of the procedure (several hours), not allowing for the determination of immunoreactivity before the radiopharmaceutical is given to the patient.
  • Cell based assays are also not commonly available to radiopharmacies.
  • the present invention relates to a robust, off the shelf assay that provides IRF results for radiolabeled antibodies in 60 minutes or under 30 minutes, reducing operator exposure time and reducing time from end-of-synthesis to patient administration. This is particularly important in the context of radiolabels with a short half-life.
  • the invention only requires disposable materials readily available in radiopharmacies and does not require specialized equipment.
  • the method may overcome inconsistencies and tediousness of cell- based IRF assays and provide a reproducible readout of stability and expiry for patient use.
  • a key part of the invention is the antigen pre-coated bead material, which obviates the need for an extra incubation step, reduces lot-to lot or operator variability, is stable for months and is ready for use.
  • the invention relates to off the shelf biotinylated 2lg and 4lg 4 B7-FI3, CD33 or GD2 coated beads, produced in stable yeast cell lines, followed by complete biotinylation according to the invention and binding to streptavidin.
  • the invention relates to the specific method that allows for testing IRF in 30-60 min, including amounts of protein, buffers, number and volume of washes, controls.
  • the invention relates to a potency assay for radioimmunotherapies or diagnostics labeled with a, b or g emitting radionuclides that include 177Lu-DTPA-omburtamab and 1311-humanized omburtamab.
  • this invention relates to an IRF or potency kit of parts, that include the pre-coated biotinylated antigen beads and incubation/wash buffer.
  • DynabeadsTM MyOneTM Streptavidin T1 magnetic beads are uniform, 1.0 pm superpara- magnetic beads with a streptavidin monolayer covalently coupled to the hydrophilic bead surface. This layer ensures negligible streptavidin leakage while the lack of excess adsorbed streptavidin ensures batch consistency and reproducibility of results.
  • a method for biotinylating a protein is described in US patent no.: 5,874,239. Biotinylation technology is also reported to be offered by the company Creative Biolabs using the trademark BtAP-Tag.
  • the invention concerns an in vitro method for determining the efficacy of a radiolabeled antibody for administration to an individual, comprising the steps of: i. Providing a biotinylated target antigen by attaching biotin to a target antigen, preferably wherein the biotinylation is single and site specific; ii. Conjugating said target antigen to a solid phase support; iii. Radiolabeling an antibody with a radioactive entity, preferably after step ii.; iv. Adding said conjugated antibody to said solid phase support; and v. Measuring the binding of said antibody to said target antigen, preferably using a gamma counter. Step i.
  • the invention concerns providing a target antigen attached to biotin. Conjugating a target antigen to a solid phase support may be performed before step iii. The solid phase support including the target antigen may be frozen for storage and thawed before use. Performing the subsequent step of conjugating the antibody with a radioactive entity after step ii. allows for the time for completion of the rest of the method to be minimized.
  • the invention concerns an in vitro method for determining the immune reactive fraction (IRF) of a radiolabeled antibody, or antigen binding fragment thereof, wherein said antibody or antigen binding fragment binds to a target antigen, comprising the steps: i. Conjugating the target antigen to a solid phase support; ii.
  • the beads are preferably pre-coated before use.
  • the target-binding fraction is the fraction of an antibody which retains its binding to the cognate target (TBF). This is also the case when the antibody has been undergoing modifications including bioconjugation and radiolabeling.
  • This parameter may be referred to as "immunoreactivity" or “immunoreactive fraction” (IRF)
  • the invention concerns a polypeptide conjugate comprising a self-assembly disassembly (SADA) polypeptide, and at least a first binding domain that binds to a first target and is covalently linked to the SADA polypeptide, wherein said polypeptide conjugate is for use in a method according to the invention.
  • SADA self-assembly disassembly
  • the invention concerns a reagent for use in a method according to the invention, comprising a solid phase support, a primary coating of antigen on the solid phase support, adapted to support a secondary coating of an antibody or antigen binding fragment thereof over said primary coating.
  • the invention concerns a vial comprising the reagent according to the invention.
  • the invention concerns a method of preparing the antigen according to the invention, wherein said method comprises the steps: i. Reconstitution of said antigen in sterile deionized water. ii. Storing at 2 - 8 °C and/or -70 °C.
  • the invention concerns a method of preparing a solid phase support according to the invention, wherein said method comprises the steps: i. Providing a magnetic bead ii. Incubation with the antigen according to the invention iii. Separation of antibody-coated beads with a magnet iv. Transfer of supernatant to a first vial v. Measurement of UV absorbance, preferably by nanodrop vi. Wash of said antigen-coated beads vii. Collecting the washes of step vi. in a second vial viii. Measurement of the UV absorbance in the second vial, preferably by nanodrop ix. Wash of said antigen coated beads x. Separation of the antigen-coated beads with a magnet xi. Resuspending the antigen-coated beads in PBS xii. Storing at - 80 °C.
  • said magnetic beads are conjugated to streptavidin, and wherein said antigen is biotinylated, and wherein said magnetic beads are conjugated to said antigen through binding of streptavidin to biotin.
  • PBS may be referred to as phosphate-buffered saline.
  • the invention concerns a method of treating cancer in an individual, wherein the method comprises use of an radiolabeled antibody or antigen binding fragment thereof, and wherein the potency and/or efficacy of said antibody is tested in a method according to the invention before use in the method of treatment. Potency may be defined as a measure of drug activity expressed in terms of the amount required to produce an effect of a given intensity.
  • the invention concerns a kit of parts, wherein said kit comprises a vial, and wherein said vial comprises a solid phase support according to the invention, and wherein said kit further comprises an antigen according to the invention, and wherein said kit further comprises a wash buffer and/or a wash formulation buffer, and wherein said kit is adapted to be used in a method according to the invention.
  • the invention concerns a kit of parts, wherein said kit comprises a magnetic bead surface coated with streptavidin conjugated to biotinylated antigen, and wherein said kit further comprises a wash buffer and/or a wash formulation buffer, and wherein said kit is adapted to be used in a method according to the invention.
  • affinity is a measure of the tightness with a particular ligand (e.g., an antibody) binds to its partner (e.g., an epitope). Affinities can be measured in difference ways.
  • Antibody is art-recognized terminology and is intended to include molecules or active fragments of molecules that bind to known antigens. Examples of active fragments of molecules that bind to known antigens include Fab and F(ab')2fragments. These active fragments can be derived from an antibody of the present invention by a number of techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and subjected to HPLC gel filtration. The appropriate fraction containing Fab fragments can then be collected and concentrated by membrane filtration and the like.
  • the term “antibody” also includes bispecific and chimeric antibodies and other available formats.
  • Antibody fragment is a portion of an antibody such as F(ab')2, F(ab)2, Fab', Fab, Fv, sFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. For example, an 3F8 monoclonal antibody fragment binds with an epitope recognized by 3F8.
  • antibody fragment also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.
  • antibody fragments include isolated fragments consisting of the variable regions, such as the "Fv” fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker ("scFv proteins”), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.
  • variable regions such as the "Fv” fragments consisting of the variable regions of the heavy and light chains
  • scFv proteins recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker
  • minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.
  • Bispecific antibody is an antibody that can bind simultaneously to two targets which are of different structure.
  • Bispecific antibodies (bsAb) and bispecific antibody fragments (bsFab) have at least one arm that specifically binds to an antigen, for example, GD2 and at least one other arm that specifically binds to another antigen, for example a targetable conjugate that bears a therapeutic or diagnostic agent.
  • bsAb bispecific antibodies
  • bsFab bispecific antibody fragments
  • a variety of bispecific fusion proteins can be produced using molecular engineering.
  • the bispecific fusion protein is divalent, consisting of, for example, a scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen.
  • the bispecific fusion protein is tetravalent, consisting of, for example, an IgG with two binding sites for one antigen and two identical scFv for a second antigen.
  • a chimeric antibody is a recombinant protein that contains the variable domains including the complementarity-determining regions (CDRs) of an antibody derived from one species, for example a rodent antibody, while the constant domains of the antibody molecule is derived from those of a human antibody.
  • the constant domains of the chimeric antibody may also be derived from that of other species, such as a cat or dog.
  • Effective amount refers to an amount of a given compound, conjugate or composition that is necessary or sufficient to realize a desired biologic effect.
  • An effective amount of a given compound, conjugate or composition in accordance with the methods of the present invention would be the amount that achieves this selected result, and such an amount can be determined as a matter of routine by a person skilled in the art, without the need for undue experimentation.
  • Humanized antibody is a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent antibody, is transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains.
  • the constant domain of the antibody molecule is derived from those of a human antibody.
  • a human antibody may be an antibody obtained from transgenic mice that have been "engineered” to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci.
  • the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas.
  • Prevent refers to the prevention of the recurrence or onset of one or more symptoms of a disorder in a subject as result of the administration of a prophylactic or therapeutic agent.
  • Radioactive isotope examples include, but are not limited to, 211 At, 14 C, 51 Cr, 57 Co, 58 Co, 67 Cu, 152 Eu, 67 Ga, 3 H, m ln, 59 Fe, 212 Pb, 177 Lu, 32 P, 223 Ra, 224 Ra, 186 Re, 188 Re, 75 Se, 35 S, 99m Tc, 227 Th, 89 Zr, 90 Y, 123 l, 124 l, 125 l, 131 l, 94m Tc, 64 Cu, 68 Ga, 66 Ga, 76 Br, 86 Y, 82 Rb, 110m ln, 13 N, n C, 18 F and alpha-emitting particles.
  • alpha-emitting particles include
  • Subject By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans and other primates, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like.
  • treatment refers to prophylaxis and/or therapy, particularly wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of multiple sclerosis.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • the invention concerns an in vitro method for determining the efficacy of a radiolabeled antibody for administration to an individual, comprising the steps of: i. Providing a biotinylated target antigen by attaching biotin to a target antigen, preferably wherein the biotinylation is single and site specific; ii. Conjugating said target antigen to a solid phase support; iii. Radiolabeling an antibody with a radioactive entity, preferably after step ii.; iv. Adding said conjugated antibody to said solid phase support; and v. Measuring the binding of said antibody to said target antigen, preferably using a gamma counter.
  • the invention concerns an in vitro method for determining the immune reactive fraction (IRF) of a radiolabeled antibody, or antigen binding fragment thereof, wherein said antibody or antigen binding fragment binds to a target antigen, comprising the steps: i. Conjugating the target antigen to a solid phase support; ii. Radiolabeling an antibody with a radioactive entity after step i.; iii. Adding said antibody or antigen binding fragment to said solid phase support; and iv. Measuring the binding of said antibody or antigen binding fragment to said target antigen, preferably using a Geiger gamma counter.
  • IRF immune reactive fraction
  • the invention concerns the method, wherein said steps are performed in said order.
  • the invention concerns the method, wherein a plurality of solid phase supports are prepared in step i. for subsequent use.
  • the invention concerns the method, wherein one or more solid phase supports are frozen after step i. and thawed before step ii.
  • the invention concerns the method, wherein said solid phase comprises a magnetic bead.
  • the invention concerns the method, wherein step ii. thorough iv. and/or step ii. through iv. is adapted to be performed in less than 120 minutes, 115 minutes, 110 minutes, 105 minutes, 100 minutes, 95 minutes, 90 minutes, 85 minutes, 80 minutes, 75 minutes, 70 minutes, 65 minutes, 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 29 minutes, 28 minutes, 27 minutes, 26 minutes, 25 minutes, 24 minutes, 23 minutes, 22 minutes, 21 minutes, 20 minutes, 19 minutes, 18 minutes, 17 minutes, 16 minutes, 15 minutes, 14 minutes, 13 minutes, 12 minutes, 11 minutes or less than 10 minutes.
  • the invention concerns the method, wherein said method measure the immunoreactive fraction/target binding fraction of said antibody or antigen binding fragment thereof, and wherein said immunoreactive fraction/target binding fraction is above 60 %, 65 %, 70%, 75 %, 80 %, 85 %, 87 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or above 99 %.
  • the invention concerns the method, wherein said measuring of the binding of said antibody or antigen binding fragment to the target antigen, is measured by UV absorbance.
  • the invention concerns the method, wherein said method comprises at least one step of incubation. According to an embodiment, the invention concerns the method, wherein said method comprises at least one step of isolating an antibody-coated bead using a magnet.
  • the invention concerns the method, wherein said antibody is for use in a method of treatment of a disease and/or for use in a prophylaxis of a disease.
  • the invention concerns the method, wherein said antibody is use in the diagnostics of a disease.
  • the invention concerns the method, wherein said disease is a cancer, tumor, metastasis or a neurodegenerative disease.
  • the invention concerns the method, wherein said cancer, said tumor and/or said metastasis is prostate cancer, a desmoplastic small round cell tumor, ovarian cancer, gastric cancer, pancreatic cancer, liver cancer, renal cancer, breast cancer, non-small cell lung cancer, melanoma, alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma, Ewing sarcoma, Wilms tumor, neuroblastoma, ganglioneuroblastoma, ganglioneuroma, medulloblastoma, high-grade glioma, diffuse intrinsic pontine glioma, embryonal tumors with multilayered rosettes, or a cancer expressing B7H3, GD2, CD33, TIM- 3, CD3 and/or CD22.
  • the invention concerns the method, wherein said target binding fraction or immune reactive fraction is determined prior to administration of said antibody.
  • the present invention relates to a robust, off the shelf assay that provides IRF results for radiolabeled antibodies in 60 minutes or under 30 minutes, reducing operator exposure time and reducing time from end-of-synthesis to patient administration. This is particularly important in the context of radiolabels with a short half-life.
  • the invention concerns the method, wherein said magnetic beads are coated with streptavidin.
  • the invention concerns the method, wherein said antigen is selected among B7H3, GD2, CD33, TIM-3 and CD22.
  • the invention concerns the method, wherein said antigen is produced in yeast.
  • the invention concerns the method, wherein said antigen comprises the sequence according to SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 15 or SEQ ID No. 16. According to an embodiment, the invention concerns the method, wherein said antigen comprises the sequence according to SEQ ID No. 13 or SEQ ID No.14.
  • the invention concerns the method, wherein a single lysine residue in said antigen is labeled with biotin. According to an embodiment, the invention concerns the method, wherein a single lysine residue in sequence ID No. SEQ ID No. 13, 15 or 16 is labeled with biotin.
  • the invention concerns the method, wherein said antigen is biotinylated.
  • Biotinylation is the process of attaching biotin to proteins and other molecules. Biotinylation may not disturb the natural function of the molecule.
  • the invention concerns the method, wherein said biotinylation is above 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or about 100 %.
  • the invention concerns the method, wherein said antigen binding fragment is a single chain variable fragment (scFv).
  • scFv single chain variable fragment
  • the invention concerns the method, wherein said antibody or antigen binding fragment is a murine antibody or an antigen binding fragment thereof.
  • the invention concerns the method, wherein said antibody or antigen binding fragment is a chimeric antibody or an antigen binding fragment thereof. According to an embodiment, the invention concerns the method, wherein said antibody or antigen binding fragment is a humanized antibody or an antigen binding fragment thereof.
  • the invention concerns the method, wherein said radioactive isotope is selected among a PET label and a SPECT label.
  • the invention concerns the method, wherein said PET label is selected among 1241, 68Ga and 89Zr.
  • the invention concerns the method, wherein said SPECT label is selected among 1311, 177Lu, 99mTc, 64Cu and 89Zr.
  • the invention concerns the method, wherein said antibody or antigen binding fragment thereof is conjugated to a chelator compound.
  • the invention concerns the method, wherein said chelator compound is bound to a radioactive isotope.
  • the invention concerns the method, wherein said radioactive isotope is selected among 1241, 1311, 68Ga and 177Lu or 99mTc, 64Cu and 89Zr.
  • the invention concerns the method, wherein said chelator compound is selected among DOTA, DTPA, NOTA and DFO and their derivatives.
  • the invention concerns the method, wherein said DOTA is a variant of DOTA, such as Benzyl-DOTA.
  • the invention concerns the method, wherein said DTPA is a variant of DTPA, such as CHX-A"-DTPA.
  • the invention concerns the method, wherein said antibody or antigen binding fragment is conjugated to a DTPA, and wherein said DTPA is bound to 177Lu.
  • the invention concerns the method, wherein said antibody or antigen binding fragment is bound to 1311.
  • the invention concerns the method, wherein said antibody or antigen binding fragment is bound to 225Ac or 211At, and wherein said antigen is CD33.
  • the invention concerns the method, wherein said antibody or antigen binding fragment is bound to 225Ac or 211At, and wherein said antigen is TIM-3.
  • the invention concerns the method, wherein said antibody or antigen binding fragment is bound to 225Ac or 211At, and wherein said antigen is GD2.
  • the invention concerns the method, wherein said antibody or antigen binding fragment comprises at least one sequence selected among a heavy chain variable region CDR1 according to SEQ ID No. 3, a heavy chain variable region CD2 according to SEQ ID No. 4, a heavy chain variable region CDR3 according to SEQ ID No. 5 a light chain variable region CDR1 according to SEQ ID No. 6, a light chain variable region CDR2 according to SEQ ID No. 7 and a light chain variable region CDR3 according to SEQ ID No. 8.
  • the invention concerns the method, wherein said antibody or antigen binding fragment comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2. According to an embodiment, the invention concerns the method, wherein said antibody or antigen binding fragment thereof is humanized.
  • the invention concerns the method, wherein said antibody or antigen binding fragment comprises a heavy chain sequence according to SEQ ID No. 11 and a light chain sequence according to SEQ ID No. 12.
  • the invention concerns the method, wherein said radioactive isotope is an alpha, beta or positron emitting radionuclide.
  • the invention concerns the method, wherein said antibody or antigen binding fragment thereof is a bispecific and/or trispecific binding antibody.
  • the invention concerns the method, wherein said bispecific and/or trispecific binding antibody comprises a first antibody or antigen binding fragment thereof according to the invention, and a second antibody or antigen binding fragment for binding to a second antigen, and wherein said antibody is adapted for use in a method according to the invention.
  • the invention concerns the method, wherein said second antibody or antigen binding fragment thereof binds to derivatives of DOTA.
  • the invention concerns the method, wherein said antibody is linked to a self-assembly disassembly (SADA) polypeptide.
  • SADA self-assembly disassembly
  • the antibody of antigen binding fragment thereof is linked to a self- assembly disassembly (SADA) polypeptide disclosed in International Patent Application Publication No. WO2018204873, all of which is incorporated by reference in its entirety.
  • SADA self- assembly disassembly
  • the invention concerns the method, wherein said self- assembly disassembly (SADA) polypeptide has an amino acid sequence that shows at least 75% identity with that of a human homo-multimerizing polypeptide and being characterized by one or more multimerization dissociation constants (KD).
  • SADA self- assembly disassembly
  • the invention concerns the method, wherein said method comprises use of a polypeptide conjugate, wherein said conjugate comprising the self- assembly disassembly (SADA) polypeptide according to the invention, and wherein said conjugate further comprises the bispecific antibody according to the invention, wherein said first antigen is B7H3 and wherein said second antigen is DOTA.
  • SADA self- assembly disassembly
  • the invention concerns the method, wherein said polypeptide conjugate comprises the self-assembly disassembly (SADA) polypeptide according to the invention, and wherein said conjugate further comprises the bispecific antibody according to the invention, wherein said first antigen is B7H3 and wherein said second antigen is DOTA.
  • the invention concerns a polypeptide conjugate comprising a self-assembly disassembly (SADA) polypeptide, and at least a first binding domain that binds to a first target and is covalently linked to the SADA polypeptide, wherein said polypeptide conjugate is for use in a method according to the invention.
  • the invention concerns the polypeptide conjugate according to the invention, wherein said self-assembly disassembly (SADA) polypeptide has an amino acid sequence that shows at least 75% identity with that of a human homo-multimerizing polypeptide and being characterized by one or more multimerization dissociation constants (KD); and wherein said conjugate is being constructed and arranged so that it adopts a first multimerization state and one or more higher-order multimerization states, wherein: the first multimerization state is less than about -70 kDa in size, at least one of the higher-order multimerization states is a homo-tetramer or higher-order homo multimer greater than 150 kDa in size, wherein the higher-order homo-multimerized conjugate is stable in aqueous solution when the conjugate is present at a concentration above the SADA polypeptide KD, and the conjugate transitions from the higher-order multimerization state(s) to the first multimerization state under physiological conditions when
  • the invention concerns a reagent for use in a method according to the invention, comprising a solid phase support, a primary coating of antigen on the solid phase support, adapted to support a secondary coating of an antibody or antigen binding fragment thereof over said primary coating.
  • the invention concerns the reagent, wherein said solid phase support comprises a magnetic bead.
  • the invention concerns the reagent, wherein said magnetic bead is conjugated to streptavidin.
  • the invention concerns the reagent, wherein said antigen is B7H3.
  • the invention concerns the reagent, wherein said antigen is biotinylated.
  • the invention concerns the reagent, wherein said antibody or antigen binding fragment is an antibody or antigen binding fragment according to the invention.
  • the invention concerns the reagent, wherein said reagent is adapted to withstand freezing.
  • the invention concerns a vial comprising the reagent according to the invention.
  • the invention concerns the vial, wherein said vial is adapted to be frozen.
  • the invention concerns a method of preparing the antigen according to the invention, wherein said method comprises the steps: i. Reconstitution of said antigen in sterile deionized water. ii. Storing at 2 - 8 °C and/or -70 °C.
  • the invention concerns a method of preparing a solid phase support, wherein said method comprises the steps: i. Providing a magnetic bead ii. Incubation with the antigen according to the invention iii. Separation of antibody-coated beads with a magnet iv. Transfer of supernatant to a first vial v. Measurement of UV absorbance, preferably by nanodrop vi. Wash of said antigen-coated beads vii. Collecting the washes of step vi. in a second vial viii. Measurement of the UV absorbance in the second vial, preferably by nanodrop ix. Wash of said antigen coated beads x. Separation of the antigen-coated beads with a magnet xi. Resuspending the antigen-coated beads in PBS xii. Storing at - 80 °C.
  • said magnetic beads are conjugated to streptavidin, and wherein said antigen is biotinylated, and wherein said magnetic beads are conjugated to said antigen through binding of streptavidin to biotin.
  • PBS may be referred to as phosphate-buffered saline.
  • the invention concerns the method, wherein said method detects the clinical benefit of a radiolabeled antibody or antigen-binding fragment thereof in the treatment of cancer in an individual.
  • the invention concerns a method of treating cancer in an individual, wherein the method comprises use of an radiolabeled antibody or antigen binding fragment thereof, and wherein the potency and/or efficacy of said antibody is tested in a method according to the invention before use in the method of treatment.
  • the invention concerns the method, wherein the dosage to be used in the method of treatment is determined by said testing of said antibody or antigen binding fragment.
  • the invention concerns a kit of parts, wherein said kit comprises a vial, and wherein said vial comprises a solid phase support according to the invention, and wherein said kit further comprises an antigen according to the invention, and wherein said kit further comprises a wash buffer and/or a wash formulation buffer, and wherein said kit is adapted to be used in a method according to the invention.
  • the invention concerns a kit of parts, wherein said kit comprises a magnetic bead surface coated with streptavidin conjugated to biotinylated antigen, and wherein said kit further comprises a wash buffer and/or a wash formulation buffer, and wherein said kit is adapted to be used in a method according to the invention.
  • the invention concerns the kit, wherein said kit further comprises an antibody or antigen binding fragment according to the invention.
  • the invention concerns the kit, wherein said kit further comprises a magnet.
  • the invention concerns the kit, wherein said antigen is B7H3, TIM-3, CD33 and/or GD2.
  • the invention concerns the kit, wherein said antigen is 70- 100 % biotinylated.
  • the invention concerns the kit, wherein said wash buffer and/or a wash formulation buffer is 1% FBS in PBS.
  • FBS may be referred to as Fetal Bovine Serum.
  • Biotin Protein ratio is 0.7 (only 70% of the B7H3 is biotinylated).
  • Magnetic beads with a streptavidin monolayer covalently coupled to the hydrophilic bead surface Magnetic beads with a streptavidin monolayer covalently coupled to the hydrophilic bead surface.
  • iii Collect the supernatant from the washes in a separate vial for UV absorbance. f) Wash the antigen coated beads an additional 4 times as above. g) After the last wash, separate the antibody-coated beads with a magnet for 5 min, remove supernatant. h) Resuspend the beads in a total of 280 ⁇ L of PBS. i) Store beads at -80°C until use. j) Run the samples collected for UV absorbance in order of low protein concentration to high protein concentration (wash-supernatant, incubation- supernatant, B7H3).
  • Example 2 Specific bead production batches Immunoreactivity Methods Antigen (B7H3) conjugated streptavidin beads were produced as described above. Specific bead production batches are described in Table 1. Table 1: B7H3-bead production summaries
  • Example 3 Immunoreactivity assay performed on the 177Lu-8H9 antibody derivatives Objective: To assess the immunoreactivity of Lutetium-177 labeled 8H9 antibody. Techniques: Radiochemistry, bead IRF assay.
  • IRF assay Perform once per Lutetium-mAb.
  • SEQ ID NO: 1 Murine 8H9 Heavy Chain
  • SEQ ID NO: 2 Murine 8H9 Light Chain
  • SEQ ID NO: 3 8H9 Heavy Chain CDR-1

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Abstract

La présente invention concerne un dosage de radioimmunoréactivité ou de fraction réactive immunitaire pour évaluer la puissance de radioimmunothérapies ou de radiodiagnostics (par exemple, la radiothéranostique), radiomarqués avec des radionucléides émetteurs alpha, bêta ou émetteurs de positrons. L'invention concerne également une trousse de pièces adaptée à la mise en œuvre du procédé de l'invention et à la production des matériaux d'antigènes cibles pour le dosage. Plus particulièrement, la présente invention concerne un dosage par billes revêtues d'antigène biotinylé et de streptavidine pour l'évaluation de la puissance des anticorps radiomarqués ciblant le B7H3, le GD2, le CD33 ou le TIM-3 pour le diagnostic ou le traitement du cancer. En outre, l'invention concerne un essai d'activité biologique pour l'anticorps 177Lu-DTPA-8H9 (CAR 3), l'anticorps 131I-8H9 ou l'anticorps 8H9 131I-humanisé.
PCT/DK2020/050405 2019-12-30 2020-12-22 Essai d'activité biologique d'anticorps radiomarqués Ceased WO2021136571A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021213601A1 (fr) * 2020-04-24 2021-10-28 Y-Mabs Therapeutics, Inc. Anticorps b7h3 avec chélateurs

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US5874239A (en) 1993-07-30 1999-02-23 Affymax Technologies N.V. Biotinylation of proteins
WO2018204873A1 (fr) 2017-05-05 2018-11-08 Memorial Sloan Kettering Cancer Center Technologies d'auto-assemblage/désassemblage modulaire (sada)
WO2019092742A1 (fr) * 2017-11-09 2019-05-16 University Of Delhi South Campus Procédé d'immobilisation de polypeptides

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WO2018204873A1 (fr) 2017-05-05 2018-11-08 Memorial Sloan Kettering Cancer Center Technologies d'auto-assemblage/désassemblage modulaire (sada)
WO2019092742A1 (fr) * 2017-11-09 2019-05-16 University Of Delhi South Campus Procédé d'immobilisation de polypeptides

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CHATTOPADHAYA SOUVIK ET AL: "Use of intein-mediated protein ligation strategies for the fabrication of functional protein arrays", METHODS IN ENZYMOLOGY, vol. 462, 24 July 2009 (2009-07-24), pages 195 - 223, XP009141424, ISSN: 1557-7988, [retrieved on 20090724], DOI: 10.1016/S0076-6879(09)62010-3 *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021213601A1 (fr) * 2020-04-24 2021-10-28 Y-Mabs Therapeutics, Inc. Anticorps b7h3 avec chélateurs

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