WO2023186106A1 - Procédé de détection d'anticorps neutralisant - Google Patents

Procédé de détection d'anticorps neutralisant Download PDF

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WO2023186106A1
WO2023186106A1 PCT/CN2023/085530 CN2023085530W WO2023186106A1 WO 2023186106 A1 WO2023186106 A1 WO 2023186106A1 CN 2023085530 W CN2023085530 W CN 2023085530W WO 2023186106 A1 WO2023186106 A1 WO 2023186106A1
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cell surface
ligand
binding
car
surface receptor
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PCT/CN2023/085530
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English (en)
Chinese (zh)
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林志财
朱雷
孙艳
钱其军
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上海细胞治疗集团药物技术有限公司
上海细胞治疗集团有限公司
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Publication of WO2023186106A1 publication Critical patent/WO2023186106A1/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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • 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
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • 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
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to the detection of neutralizing anti-drug antibodies in peripheral blood of tumor-targeted cell therapy products, including CAR-T, CAR-NK, CAR-MC and other immune cell therapy products after clinical infusion therapy. Detection of neutralizing anti-drug antibodies.
  • Immunogenicity is a humoral or cell-mediated immune response to biological or biotechnological derivatives.
  • Most biological or biotechnologically derived proteins (biotherapeutics) induce an immune response that may be related to the patient, disease or product-related properties.
  • Clinical outcomes caused by immune responses to biologic products can range from no effect to benign reactions, loss of efficacy, or even severe life-threatening consequences. Therefore, immunogenicity testing is critical in any biotherapeutic drug development program.
  • ADA anti-drug antibodies
  • the production of ADA can affect the in vivo pharmacokinetics (PK) of antibody drugs, including the distribution of the drug. , metabolism and clearance.
  • the production of neutralizing antibodies will directly compete or block the combination of antibody drugs and targets, further affecting clinical efficacy.
  • CAR Chimeric antigen receptor
  • TCR T cell receptor
  • MHC major histocompatibility complex
  • scFv mouse monoclonal antibody
  • VHH antigen-recognizing variable region
  • scFv or VHH binds to a specific antigen target peptide and transmits an activation signal through a modular intracellular signaling domain.
  • Current CAR-T cells It is prepared from peripheral blood T cells in vitro. This process generally involves integrating CAR into the T cell genome using a vector lacking replication ability, and then expanding it to a considerable number through culture. After infusion back into the patient, these cells can recognize and eliminate tumor cells expressing the target antigen.
  • CAR-T cells have the risk of triggering humoral immunity and cellular immunity against themselves. These immune responses can be directed against the "non-self" components of the CAR structure, or against residual proteins on the gene transfer vector, which have natural immunogens. sex. This immune response may in turn limit the function of CAR-T cells and affect their clinical efficacy after infusion. Many CAR-T cells currently used in clinical applications are derived from mouse or other non-human monoclonal antibodies. Human or humanized scFv/VHH also contain “non-self” sequences because the variable binding region is derived from multiple gene rearrangements and somatic high-frequency mutations.
  • Antibodies that target a specific antibody sequence are called "anti-idiotype antibodies”.
  • proteins encoded by multiple genes are expressed on a single CAR peptide chain, and the fusion sequence at the junction generally does not exist in the human body.
  • the impact of CAR-specific immunogenicity on patient clinical outcomes after CAR-T infusion remains poorly understood and has not been well studied.
  • the detection of ADA produced by the human body after the reinfusion of various types of CAR gene-modified immune cells, especially the production of neutralizing antibodies, is crucial for the development of gene-modified cell drugs.
  • cell therapy drugs Due to their cutting-edge technological breakthroughs and more complex designs, cell therapy drugs currently have no mature regulations or guidelines to standardize the detection and management of immunogenicity issues.
  • ADA neutralizing antibody
  • CAR-T The detection of ADA after reinfusion of genetically modified cell therapy products represented by CAR-T also refers to the ADA method of protein drugs for detection. It mainly uses the gene expression product (CAR protein or ScFv) as the key reagent and passes the sandwich ELISA. The method is used for detection.
  • the quantification of this method must use the gene expression product (CAR protein or ScFv) to immunize heterogeneous animals (rabbits, mice, etc.), and the polyclonal obtained A standard curve is established for the antibody for relative quantification (for example, see CN112394179A).
  • CAR protein or ScFv gene expression product
  • this method has the following problems: the preparation of heterogeneous polyclonal antibodies for immunization requires a rather long development cycle. There are certain differences between the CAR protein or ScFv expressed by cells and the proteins expressed by genes in other cell lines, and cannot fully represent the expression of immune cell genes after gene modification. The corresponding affinities and binding sites may be different, and there are certain differences in the binding properties of the polyclonal antibodies produced by immunization.
  • the existing immunogenicity detection scheme refers to the ADA detection method of protein drugs. Based on the ability of bivalent and above-bivalent antibodies to bind to target protein drugs, the protein drugs are marked with color signals to display or quantify the production of anti-drug antibodies.
  • the routine process of ADA detection includes: ADA characterization and qualitative analysis, including competitive ligand binding analysis, isoform analysis, binding stability analysis, epitope specificity analysis, binding stability, and neutralization ability analysis; ADA antibody quantification, including Based on the standard curve of the positive control antibody, a relative titer is established for quantification; neutralization detection includes neutralizing antibody detection and neutralizing ability analysis.
  • the commonly used method is competitive ligand binding (CBL). CBL is neutralizing antibody. and preferred method of detection.
  • ADA and neutralizing antibodies are different from those of chimeric antibodies. Furthermore, the detection and quantification of neutralizing antibodies is mainly based on competitive ligand binding methods. The development of detection methods requires the development and preparation of neutralizing antibodies with competitive binding, which can be used as positive control antibodies for detection and quantification, and the establishment of a quantitative standard curve. . Positive control antibodies for neutralizing antibody detection need to be screened and established to establish monoclonal cell lines to ensure the continuous preparation of neutralizing antibody positive control antibodies. The cycle of positive control antibody preparation, method development, and validation is long. Also, the results of neutralizing antibody detection using modified gene expression products cannot fully represent the neutralizing effect of chimeric antibodies of genetically modified immune cell therapy products.
  • ADA and neutralizing antibodies In the early stages of the development of genetically modified immune cell drugs, we face a variety of different sequences. The production of ADA and neutralizing antibodies depends on the development cycle of key reagents and positive control antibodies for the detection method. Detection and identification cannot be performed early in development studies.
  • CAR-T products targeting hematological tumor targets such as CD19 and BCMA have achieved breakthroughs in the treatment of late relapsed lymphoma and multiple myeloma.
  • the therapeutic effect of two special targets can achieve excellent clinical efficacy, which is related to target specificity and tumor characteristics of hematoma. It is also related to the elimination of B cells and plasma cells that can produce anti-drug antibodies by CAR-T products. There is a certain correlation.
  • Solid tumor immune cell therapy drugs are potentially immunogenic to cells. has a significant impact on its in vivo retention and clinical efficacy.
  • the detection of neutralizing antibodies potentially produced by the infusion of genetically modified immune cell products targeting different targets of solid tumors into the human body is important for whether the product can ultimately achieve excellent clinical efficacy and whether the genetically modified immune cells can persist in the patient's body. indicating meaning.
  • a first aspect of the present invention provides a non-diagnostic or therapeutic method for detecting neutralizing antibodies against cell surface receptors in a sample, including the steps:
  • step (2) includes detecting the binding complex of the ligand to cells containing the cell surface receptor and comparing its level to the total level of cells containing the cell surface receptor. , thereby detecting neutralizing antibodies against the cell surface receptors in the sample.
  • the cell surface receptor comprises an extracellular portion that is an extracellular ligand binding domain, such as an extracellular antigen binding domain, and a membrane anchoring region.
  • the extracellular antigen-binding region is an antibody or an antigen-binding fragment thereof, preferably a Nanobody.
  • the membrane anchoring region is a transmembrane region.
  • the cell surface receptor is a genetically modified somatic cell surface expressed protein receptor of interest.
  • the cell surface receptor is a CAR.
  • the CAR comprises an extracellular antigen-binding region, a hinge region, a transmembrane region, and an intracellular region.
  • the cells are somatic cells, such as immune cells, red blood cells, stem cells, etc., preferably immune cells, and further preferably T cells, NK cells, MC cells (macrophages).
  • the cell is a CAR-T cell expressing the CAR.
  • the ligand is an antigen specifically bound by the extracellular antigen-binding region of the CAR or a fragment of this antigen capable of binding to the CAR.
  • the antigen is a tumor associated antigen, such as a tumor surface antigen.
  • the sample is a sample derived from blood, such as plasma, serum or whole blood; preferably serum.
  • the sample is from a subject.
  • the subject has been exposed to (eg, has or has been administered) the cell surface receptor or a cell containing the cell surface receptor.
  • the method further includes: prior to contacting the subject with the cell surface receptor or cells containing the cell surface receptor, subjecting a sample from the subject containing the cell surface receptor to The cells of the body are mixed and incubated with a ligand that specifically recognizes the extracellular portion of the cell surface receptor, and the binding complex between the ligand and the cell containing the cell surface receptor is detected.
  • the ligand is coupled to a detectable label.
  • the detectable label is biotin or a fluorophore.
  • the method includes: (1) incubating a mixture of samples derived from plasma or serum, CAR-T cells, biotin or fluorescently labeled ligands, the plasma or serum being contacted with The plasma or serum of a subject who has passed the CAR-T cells, the extracellular portion of the CAR has an antibody against the ligand, (2) identify the level of labeled CAR-T cells, and the level of total CAR-T cells Compare, thereby detecting neutralizing antibodies against the CAR in the sample.
  • step (2) includes determining the neutralizing antibody neutralizing efficacy by Rate:
  • Neutralizing antibody neutralization efficiency (Day0 positive rate - DayN positive rate)/Day0 positive rate
  • the Day0 positivity rate represents the level of labeled cells detected before the subject comes into contact with cells containing cell surface receptors
  • the DayN positivity rate represents the level of labeled cells detected after the subject comes into contact with cells containing cell surface receptors
  • N is the number of days.
  • the present invention also provides the use of cells containing cell surface receptors and ligands that specifically recognize the extracellular part of the cell surface receptors in the preparation of kits for evaluating cell surface receptors or cells containing them. In vivo effects of neutralizing antibodies on cells that compete with the ligand for binding to the cell surface receptor.
  • the assessment includes detecting neutralizing antibodies directed against a cell surface receptor in a sample of a subject administered the cell surface receptor or cells containing the cell surface receptor. In one or more embodiments, the assessment further includes detecting neutralizing antibodies against the cell surface receptor or cells containing the cell surface receptor in a sample from the subject prior to administering the cell surface receptor or cells containing the cell surface receptor to the subject and Compare with results after application.
  • the detection includes the steps of mixing the sample to be tested, the cells and the ligand, and detecting binding of a cell surface receptor to the ligand.
  • the cell surface receptor comprises an extracellular portion that is an extracellular ligand binding domain, such as an extracellular antigen binding domain, and a membrane anchoring region.
  • the extracellular antigen-binding region is an antibody or an antigen-binding fragment thereof, preferably a Nanobody.
  • the membrane anchoring region is a transmembrane region.
  • the cell surface receptor is a CAR.
  • the CAR comprises an extracellular antigen-binding region, a hinge region, a transmembrane region, and an intracellular region.
  • the cells are immune cells, preferably T cells, NK cells, MC cells.
  • the cell is a CAR-T cell expressing the CAR.
  • the ligand is an antigen specifically bound by the extracellular antigen-binding region of the CAR or a fragment of this antigen capable of binding to the CAR.
  • the antigen is a tumor associated antigen, such as a tumor surface antigen.
  • the sample is a sample derived from blood, such as plasma, serum, whole blood; preferably serum.
  • the subject has been exposed to (eg, has or has been administered) the cell surface receptor or a cell containing the cell surface receptor.
  • the ligand is coupled to a detectable label.
  • the detectable label is biotin or a fluorophore.
  • the present invention also provides a kit for detecting neutralizing antibodies against cell surface receptors in a sample, comprising: cells containing the cell surface receptors, and extracellular cells that specifically recognize the cell surface receptors. part of the ligand.
  • the cell surface receptor comprises an extracellular portion that is an extracellular ligand binding domain, such as an extracellular antigen binding domain, and a membrane anchoring region.
  • the extracellular antigen-binding region is an antibody or an antigen-binding fragment thereof, preferably a Nanobody.
  • the membrane anchoring region is a transmembrane region.
  • the cell surface receptor is a CAR.
  • the CAR comprises an extracellular antigen-binding region, a hinge region, a transmembrane region, and an intracellular region.
  • the cells are immune cells, preferably T cells, NK cells, MC cells.
  • the cell is a CAR-T cell expressing the CAR.
  • the ligand is an antigen specifically bound by the extracellular antigen-binding region of the CAR or a fragment of this antigen capable of binding to the CAR.
  • the antigen is a tumor associated antigen, such as a tumor surface antigen.
  • the sample is a sample derived from blood, such as plasma, serum, whole blood; preferably serum.
  • the sample is from a subject.
  • the subject has been exposed to (eg, has or has been administered) the cell surface receptor or a cell containing the cell surface receptor.
  • the ligand is coupled to a detectable label.
  • the detectable label is biotin or a fluorophore.
  • the kit further includes sample processing reagents, such as diluents.
  • the kit further includes reagents for detecting the binding of cell surface receptors to the ligand, such as buffers, flow antibodies, PBS, secondary antibodies, etc.
  • Fluorescently labeled tumor antigens or peptides and genetically modified immune cells are used as detection reagents, which are easy to prepare, have stable quality, and no obvious differences.
  • the preparation cycle is short, which ensures the efficient establishment of detection methods and improves the applicability of detection methods.
  • the flow cytometry method is used to detect the inhibitory effect of drug serum on the binding of chimeric antibodies expressed by genetically modified immune cells to tumor-related antigens or peptides, and the method is quantified by combining the positive rate and changes in fluorescence intensity.
  • the method is more direct and accurate.
  • the use of the invention can efficiently and conveniently detect the qualitative and quantitative neutralizing antibodies produced by organisms against genetically modified immune cells. It is especially suitable for the detection of neutralizing antibodies during the development of genetically modified immune cell therapy drugs. Improve the efficiency of cell therapy drug development and reduce the adverse risks of neutralizing antibodies caused by allogeneic components to the development of new drugs.
  • Figure 1 Schematic representation of fluorescently labeled tumor-associated antigens or peptides.
  • Figure 2 Principle of detection of neutralizing antibodies using fluorescently labeled tumor-associated antigens or peptides.
  • Figure 3 Flow cytometry results of MSLN_CAR-T cell preparation of patient S0105.
  • Figure 4 Flow cytometric detection results of serum neutralizing antibodies before the first reinfusion (D0) and after the first reinfusion (D28) of MSLN_CAR-T in patient S0105.
  • Figure 5 Flow cytometric detection results of serum neutralizing antibodies before (D35) and after (D63) secondary infusion of MSLN_CAR-T in patient S0105.
  • Figure 7 Flow cytometric detection results of serum neutralizing antibodies before the first reinfusion (D0) and after the first reinfusion (D21) of MSLN_CAR-T in patient S0106.
  • Figure 8 Flow cytometric detection results of serum neutralizing antibodies before the first reinfusion of MSLN_CAR-T in S0304 patient (D0) and after the reinfusion (D21).
  • neutralizing antibodies are specific antibodies produced when foreign substances enter the body. After a patient is infused with a cell therapy product (such as CAR-T), due to the immunogenicity of the therapeutic molecule (such as CAR), the body produces antibodies against the therapeutic molecule, especially neutralizing antibodies that directly block the recognition of the target protein by the therapeutic molecule. , reducing the tumor killing function of cell therapy products.
  • CAR-T a cell therapy product
  • the body produces antibodies against the therapeutic molecule, especially neutralizing antibodies that directly block the recognition of the target protein by the therapeutic molecule. , reducing the tumor killing function of cell therapy products.
  • the inventors discovered that there is a competitive relationship between the target protein of the therapeutic molecule and the neutralizing antibody in binding to the therapeutic molecule. Therefore, the target protein-binding sites on the therapeutic molecule are occupied by neutralizing antibodies, and the therapeutic molecule no longer binds to the target protein molecule.
  • the presence of neutralizing antibodies in serum samples can be rapidly detected by utilizing the competitive relationship between a detectable labeled target protein
  • a "therapeutic molecule” is a cell surface receptor with therapeutic efficacy.
  • the cell surface receptors herein are proteins that generally contain an extracellular ligand binding domain (extracellular part) and a transmembrane domain (membrane anchoring domain).
  • the extracellular ligand binding region is an anti-MSLN (Mesothelin, mesothelin) antibody, preferably an anti-MSLN Nanobody.
  • the cell surface receptor is a CAR whose extracellular antigen-binding region is the anti-MSLN Nanobody.
  • VHH or “Nanobody” refers to a single domain polypeptide or protein that specifically recognizes and binds to an antigen, which is the variable region of a heavy chain antibody.
  • Nanobodies contain three CDRs and four FRs and are the smallest functional antigen-binding fragments.
  • the "heavy chain antibody” described herein is a type of antibody derived from camelids or cartilaginous fishes. Compared with traditional 4-chain antibodies, heavy chain antibodies lack the light chain and heavy chain constant region 1 (CH1), and only contain 2 heavy chains composed of variable regions (VHH) and other constant regions. The variable region has a hinge-like structure. connected to the constant region.
  • Each heavy chain of camelid heavy chain antibodies contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of chondrichthyan heavy chain antibodies contains 1 variable region and 5 constant regions. Constant region (CH1-CH5).
  • Antigen-binding fragments of heavy chain antibodies include VHH and single-chain heavy chain antibodies. Heavy chain antibodies can have human IgG Fc by fusion to the constant region of human IgG Fc of CH2 and CH3.
  • the present invention provides a non-diagnostic or therapeutic method for detecting neutralizing antibodies against cell surface receptors in a sample, including the steps of: (1) combining the test sample, cells containing the cell surface receptors and specific Ligands that specifically recognize the extracellular part of the cell surface receptor are mixed and incubated, the neutralizing antibody competes with the ligand for binding to the cell surface receptor, (2) detecting the cell surface receptor and the ligand Binding, thereby detecting neutralizing antibodies against the cell surface receptor in the sample.
  • the non-diagnostic or therapeutic methods may also include sample pre-treatment steps such as centrifugation, dilution.
  • detecting the binding of the cell surface receptor to the ligand includes: detecting the binding complex between the ligand and the cell containing the cell surface receptor, and the total complex of the cell containing the cell surface receptor.
  • Level comparison allows for qualitative or quantitative detection of neutralizing antibodies against the cell surface receptor in a sample.
  • a sample cells containing the cell surface receptor (such as CAR-T cells) and a ligand (biotin or fluorescent label) that specifically recognizes the extracellular part of the cell surface receptor are After contact with the target protein), if the sample contains neutralizing antibodies, they will compete with the ligands for binding to cells.
  • ligand biological or fluorescent label
  • By detecting the binding complexes between cells and ligands it is possible to determine whether and how much neutralizing antibodies are contained in the sample by comparing the levels of the complexes with the levels of total cells.
  • total cellular level or “total cellular level” refers to the total level of cells containing cell surface receptors (eg, CAR-T).
  • level may be an absolute or relative value. Detection of the bound complex can be accomplished by detecting the absolute or relative amounts of ligand.
  • the density of the cells in the incubation system of step (1) is 1 to 5*10 7 cells/mL, preferably 1-2*10 7 cells/mL.
  • the volume ratio of the sample to be tested and the cell (for example, CAR-T cell) suspension can be 1:1.
  • the incubation in step (1) is at least 10 minutes, preferably at least 20 minutes.
  • the incubation in step (1) may also include the step of isolating the cells and incubating them with the ligand for at least 10 minutes (preferably 20 minutes).
  • the sample is from a subject.
  • the subject has been exposed to (eg, has or has been administered) the cell surface receptor or a cell containing the cell surface receptor. Therefore, what is obtained by detecting the binding complex between the ligand and the cell containing the cell surface receptor in step (2) is the level of labeled cells detected after the subject contacts the cell containing the cell surface receptor.
  • the "total level of cells" containing the cell surface receptor may be the level of labeled cells detected before the subject comes into contact with cells containing the cell surface receptor, that is, the subject's sample before contact is subjected to step (1) of the method. ) and then incubate the system with The cellular level of body binding.
  • the methods described herein may further comprise: prior to contacting the subject with the cell surface receptor or cells containing the cell surface receptor, subject a sample from the subject, cells containing the cell surface receptor and a specific Incubate a mixture of ligands that specifically recognize the extracellular portion of the cell surface receptor, and detect binding complexes of the ligand and cells containing the cell surface receptor (i.e., labeling of the subject sample after the incubation before exposure) cellular level).
  • the ligand is coupled to a detectable label.
  • Detectable labels include, but are not limited to, biotin, antibodies, fluorescent or luminescent labels, radioactive labels, MRI or CT contrast agents, or enzymes capable of producing detectable products.
  • the detectable label is biotin or a fluorophore (including flow fluorescein, such as PE).
  • the binding complex of a ligand and a cell described herein may be a labeled cell.
  • Label-conjugated ligands eg, PE-labeled mesothelin
  • the ligand and biotin can be coupled by direct binding; the ligand and the fluorophore can be coupled together by the binding of biotin and streptavidin.
  • Cells containing (including expressing) the cell surface receptors can be any cells used for cell therapy, such as immune cells, including but not limited to T cells, NK cells, and MC cells.
  • the cells are CAR-T cells expressing CAR as a cell surface receptor.
  • CAR expression vectors such as viral vectors, non-viral vectors
  • the ligand is an antigen (antigenic protein or polypeptide) specifically bound by the extracellular ligand binding region of a cell surface receptor or a fragment of the antigen capable of binding to the extracellular ligand binding region.
  • the antigen is a tumor associated antigen, such as a tumor surface antigen. Any tumor-associated antigen in the art is suitable for use in the present invention.
  • the antigen is MSLN (NCBI, Accession NO.: NP_001170826.1).
  • the sample is from a subject, which may be a healthy subject or a tumor patient.
  • the tumor patient's tumor may or may not have the above-mentioned tumor-associated antigens.
  • the sample may be a sample derived from blood, such as plasma, serum, or whole blood; serum is preferred.
  • Plasma, serum, and whole blood can be pretreated before performing the method of the present invention.
  • Those skilled in the art are aware of these pretreatment methods, such as blood
  • the slurry sample was centrifuged at 3000 g for 10 minutes at 4°C and the supernatant was taken.
  • any other sample that may contain neutralizing antibodies is also included within the scope of the invention.
  • CAR is a chimeric antigen receptor, and its structure includes: extracellular region, hinge region, transmembrane region and intracellular region.
  • the N segment of the CAR may also have a signal peptide.
  • the intracellular domain includes intracellular costimulatory domain and intracellular signaling domain.
  • the CAR can also have any structure known in the art that can be linked to a CAR sequence.
  • the extracellular region of the CAR is an MSLN antibody or antigen-binding fragment thereof, preferably an MSLN Nanobody.
  • Optional signal peptides on the CAR can be selected as desired.
  • CD8 signal peptide, CD28 signal peptide, CD4 signal peptide or light chain signal peptide the sequences of which are within the knowledge of those skilled in the art.
  • the CD8 signal peptide suitable for the present invention can be various human CD8 signal peptide sequences commonly used for CAR in the art.
  • the amino acid sequence of the human CD8 signal peptide can be as shown in amino acids 1-22 of SEQ ID NO: 1.
  • the hinge region of the CAR is selected from the group consisting of CD8 ⁇ hinge region, IgD hinge region, IgG1 Fc CH2CH3 hinge region or IgG4 Fc CH2CH3 hinge region, and its sequence is within the knowledge of those skilled in the art.
  • the CD8 hinge region suitable for the present invention can be various human CD8 hinge region sequences commonly used for CAR in the art.
  • the amino acid sequence of the human CD8 hinge region can be as shown in amino acids 142-196 of SEQ ID NO: 1.
  • the transmembrane region of the CAR is selected from one of the group consisting of CD28 transmembrane region, CD8 transmembrane region, CD3 ⁇ transmembrane region, CD134 transmembrane region, CD137 transmembrane region, ICOS transmembrane region and DAP10 transmembrane region; preferably it is CD28 transmembrane region.
  • membrane regions the sequences of which are within the knowledge of those skilled in the art.
  • the human CD28 transmembrane region suitable for the present invention can be various human CD28 transmembrane region sequences commonly used for CAR in the art.
  • the amino acid sequence of the human CD28 transmembrane region is as shown in SEQ ID NO: 1, amino acids 197-224.
  • intracellular costimulatory domains can be selected as needed, including intracellular domains with costimulatory signaling molecules, such as CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T Intracellular domains of cellular costimulator (ICOS) and DNAX-activating protein 10.
  • costimulatory signaling molecules such as CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T Intracellular domains of cellular costimulator (ICOS) and DNAX-activating protein 10.
  • CD28 costimulatory domains suitable for use in the present invention can be various human CD28 costimulatory domain sequences commonly used for CARs in the art.
  • the CD28 costimulatory domain has an amino acid sequence such as SEQ The amino acid sequence at positions 225-265 of ID NO:1 is shown.
  • the intracellular signaling domain of the CAR can be selected as needed, including but not limited to the CD3 ⁇ intracellular signaling domain or the Fc ⁇ RI ⁇ intracellular signaling domain.
  • the CD3 ⁇ intracellular signaling domain suitable for the present invention can be various CD3 ⁇ intracellular signaling domains known in the art for CAR.
  • the amino acid sequence of the CD3 ⁇ intracellular signaling domain is as shown in SEQ ID NO: 1 amino acid sequence 266-377.
  • the above-mentioned parts that form the chimeric antigen receptor of the present invention can directly interact with each other.
  • ligated or can be ligated via an adapter sequence.
  • the linker sequence may be one well known in the art and suitable for use with antibodies, such as a G and S-containing linker sequence.
  • linkers typically contain one or more repeating motifs.
  • the motif may be GGGS, GGGGS, SSSSG, GSGSA, and GGSGG.
  • the motifs are contiguous in the linker sequence, with no intervening amino acid residues between repeats.
  • Linker sequences can contain 1, 2, 3, 4 or 5 repeating motifs.
  • the length of the linker can be 3 to 25 amino acid residues, such as 3 to 15, 5 to 15, or 10 to 20 amino acid residues.
  • the linker sequence is a polyglycine linker sequence.
  • the number of glycines in the linker sequence is not particularly limited, but is usually 2 to 20, such as 2 to 15, 2 to 10, or 2 to 8.
  • the linker can also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine Acid (F), arginine (R), glutamine (Q), etc.
  • the linker sequence is a (GGGGS)n linkage, where n is an integer from 1 to 5.
  • the CAR contains CD8 signal peptide, anti-MSLN nanobody, CD8 hinge region, CD28 transmembrane region, CD28 costimulatory domain, and CD3 ⁇ intracellular signaling domain in sequence from N-terminus to C-terminus.
  • an exemplary CAR having the above structure is shown in SEQ ID NO: 1.
  • the amino acid sequence of the CAR of the present invention is as shown in SEQ ID NO: 1 amino acids 23-384.
  • the method of the present invention includes: (1) mixing and incubating samples derived from plasma or serum, CAR-T cells, biotin or fluorescently labeled ligands, and the plasma or serum is contacted with The plasma or serum of a subject who has passed the CAR-T cells, the extracellular portion of the CAR has an antibody against the ligand, (2) identify the level of labeled CAR-T cells, and the level of total CAR-T cells The comparison can qualitatively or quantitatively detect neutralizing antibodies against the CAR in a sample.
  • (2) Comprising: identifying the level of labeled CAR-T cells and comparing it with the level of labeled cells after the incubation of a sample derived from plasma or serum of the subject before administration of the CAR-T cells, thereby detecting the level of the labeled CAR-T cells in the sample.
  • CAR neutralizing antibodies
  • the method of the present invention includes: mixing and incubating samples derived from plasma, CAR-T cells with anti-MSLN Nanobodies on the cell surface, biotin or fluorescent substance-coupled MSLN, and neutralizing antibodies in the plasma. Compete with the MSLN for binding to CAR on CAR-T cells, and detect the binding complex between MSLN and CAR-T cells by flow cytometry. Compared with the total CAR-T cell level, the target in the plasma can be qualitatively or quantitatively detected. CAR neutralizing antibodies.
  • the method includes: (1) resuspending CAR-T cells in a buffer (such as PBS) with a cell density of 2 to 3*10 7 /mL, (2) centrifuging the plasma sample at 3000g for 10 seconds at 4°C. Take the supernatant after 30 minutes, (3) mix the CAR-T cell suspension and plasma supernatant at a ratio of 1:1 and incubate for 30 minutes, (4) mix the mixture with PE fluorescently labeled mesothelin and incubate it for 20 minutes, (5) separate The cells were then mixed with PE fluorescently labeled mesothelin and incubated for 20 minutes. (6) The level of fluorescently labeled cells was identified by flow cytometry and compared with the level of fluorescently labeled cells in the serum of patients who were not administered CAR-T cells, thereby detecting Neutralizing antibodies against the CAR in plasma.
  • a buffer such as PBS
  • detecting neutralizing antibodies against the cell surface receptor in the sample includes: assessing the relative content of neutralizing antibodies, or assessing the concentration of the cell surface receptor or cells containing the cell surface receptor.
  • the assessment includes using the method of detecting neutralizing antibodies of the present invention to detect neutralizing antibodies against the cell surface receptor in a sample of a subject who has been treated (e.g., administered with a cell surface receptor or cells containing a cell surface receptor). and antibodies.
  • the presence and amount of neutralizing antibodies in the sample is determined by comparing the levels of binding complexes (labeled cells) after treatment with the levels of complexes before treatment.
  • the assessment may also include prior to treatment (e.g., administration of a cell surface receptor or A sample from the subject is tested for neutralizing antibodies directed against the cell surface receptor before the cells containing the cell surface receptor and compared with the results after treatment (eg, after said administration). Evaluated by comparing changes in neutralizing antibodies in subjects before and after treatment.
  • prior to treatment e.g., administration of a cell surface receptor or A sample from the subject is tested for neutralizing antibodies directed against the cell surface receptor before the cells containing the cell surface receptor and compared with the results after treatment (eg, after said administration). Evaluated by comparing changes in neutralizing antibodies in subjects before and after treatment.
  • Neutralizing antibody effects can be indicated by neutralization efficiency. Detect ligand and cell binding for different days The level of the complex (such as CAR positivity rate), and then the neutralizing antibody neutralizing efficiency is calculated by the following formula. The neutralizing antibody neutralizing efficiency represents the level of neutralizing antibody expression:
  • Neutralizing antibody neutralization efficiency (Day0 positive rate - DayN positive rate)/Day0 positive rate
  • the Day0 positivity rate represents the level of binding complexes detected before the subject comes into contact with cells containing cell surface receptors (for example, the CAR positivity rate under blocking serum from patients who have not infused CAR-T cells).
  • the DayN positivity rate represents the level of binding complexes detected before the subject comes into contact with cells containing cell surface receptors.
  • the level of binding complexes detected later in the recipient cells (for example, the CAR positivity rate under serum blocking using CAR-T infused back for N days), N can be any number of days.
  • the present invention also provides a kit for detecting neutralizing antibodies against cell surface receptors in a sample, including: (1) cell surface receptors (such as CAR) or coding sequences thereof, or cells containing the A cell surface receptor (eg, CAR-T), and (2) a ligand that specifically recognizes the extracellular portion of the cell surface receptor (eg, a tumor surface antigen) or its coding sequence.
  • cell surface receptors such as CAR
  • CAR-T cell surface receptor
  • a ligand that specifically recognizes the extracellular portion of the cell surface receptor eg, a tumor surface antigen
  • the samples, cell surface receptors, cells, ligands, etc. are characterized as described elsewhere herein.
  • the kit also contains sample processing reagents, such as diluents for diluting the plasma.
  • Fluorescently labeled MSLN can be obtained by: indirect labeling, such as biotin-:streptavidin (streptavidin) to achieve flow cytometry fluorescein and MSLN antigen binding; it can also be obtained by direct labeling, that is, flow cytometry fluorescence The protein label is directly coupled to the MSLN antigen.
  • indirect labeling such as biotin-:streptavidin (streptavidin) to achieve flow cytometry fluorescein and MSLN antigen binding
  • direct labeling that is, flow cytometry fluorescence
  • the protein label is directly coupled to the MSLN antigen.
  • the kit may further comprise one or more selected from the group consisting of reagents for introducing the coding sequence of a cell surface receptor (eg CAR) into cells (eg immune cells), reagents for expressing the ligand (eg tumor surface antigen) cells (e.g., mammalian cells), reagents for purifying the ligand, a detectable label (e.g., biotin or PE), and reagents for conjugating the detectable label to the ligand (e.g., coupling buffer liquid).
  • CAR cell surface receptor
  • a detectable label e.g., biotin or PE
  • reagents for conjugating the detectable label to the ligand e.g., coupling buffer liquid.
  • These agents can be selected based on the nature of the cell surface receptors and ligands, which is within the knowledge of those skilled in the art.
  • the kit may also contain reagents for detecting binding of cell surface receptors to the ligand.
  • reagents are, for example, reagents that detect detectable labels, including but not limited to, for example, streptavidin-conjugated fluorophores or antibodies, anti-biotin antibodies, and fluorescently labeled secondary antibodies.
  • Optional reagents depend on the selected detection method. Such methods include but are not limited to flow detection, and may also be ELISA and MSD. The required reagents are within the knowledge of those skilled in the art. For example, flow cytometry can be used to detect The above-mentioned binding complex requires reagents such as buffer, flow antibody, PBS, secondary antibody, etc.
  • the present invention also provides the use of cells containing cell surface receptors and ligands that specifically recognize the extracellular part of the cell surface receptors in the preparation of kits for evaluating cell surface receptors or cells containing them. In vivo effects of neutralizing antibodies on cells that compete with the ligand for binding to the cell surface receptor.
  • the present invention also provides the use of the methods, cells, ligands or kits described herein for the detection of neutralizing antibodies produced by the human body before and after reinfusion of genetically modified immune cells, early screening of cell therapy drugs, and drug safety. Evaluation, detection and pre-screening of other competing antibodies present in the human body for genetically modified antigen-targeted cell therapy drugs, and detection of the presence of immunogenicity.
  • various tumor-related antigens or polypeptides, special proteins, glycoproteins, etc. are labeled through fluorescent labeling technology as a key reagent for the detection of neutralizing antibodies, which is convenient to prepare and has stable quality. There is no need to prepare positive control antibodies for conventional ADA testing, which requires a long development cycle, large batch-to-batch differences, and unstable detection.
  • Cells that express specific chimeric antibodies and chimeric antigen receptors of various antigens, proteins, and polypeptides are edited through genetic modification technology (viral vectors, non-viral vectors and other genetic modification technologies). The preparation cycle is short, convenient and fast, and the detection results are more direct and have clear indicative significance.
  • Flow cytometry technology is used to detect the signal from the combination of cells and labeled key reagents, including the positive ratio and intensity value.
  • the method is direct, easy to operate, has large detection throughput, and can be accurately and directly quantified. Rapid detection of samples can be achieved.
  • a non-diagnostic or therapeutic method for detecting neutralizing antibodies against cell surface receptors in a sample including the steps:
  • the sample is from a subject who has been exposed to the cell surface receptor or cells containing the cell surface receptor, and/or
  • the cell surface receptor comprises an extracellular portion and a membrane anchoring region, the extracellular portion being an extracellular ligand binding region, and/or
  • the cells are somatic cells.
  • Step (2) includes: detecting the binding complex of the ligand and the cells containing the cell surface receptor, compared with the total level of cells containing the cell surface receptor, thereby detecting the target of the cell surface receptor in the sample. of neutralizing antibodies,
  • the method further comprises: before contacting the subject with the cell surface receptor or cells containing the cell surface receptor, combining a sample from the subject, the cell containing the cell surface receptor and the specific A mixture of ligands that recognize the extracellular portion of the cell surface receptor is incubated, and the binding complex of the ligand to the cell containing the cell surface receptor is detected.
  • Step (2) includes determining the neutralizing efficiency of the neutralizing antibody by the following formula:
  • Neutralizing antibody neutralization efficiency (Day0 positive rate - DayN positive rate)/Day0 positive rate
  • the Day0 positivity rate represents the level of binding complexes detected before the subject comes into contact with cells containing cell surface receptors
  • the DayN positivity rate represents the level of binding complexes detected after the subject comes into contact with cells containing cell surface receptors
  • N is the number of days.
  • the cell surface receptor is a CAR
  • the ligand is an antigen specifically bound by the extracellular antigen-binding region of the CAR or a fragment of the antigen capable of binding to the CAR;
  • the antigen is a tumor associated antigen, such as a tumor surface antigen, and/or
  • the sample is a sample derived from blood, such as plasma, serum, whole blood, and/or
  • the ligand is coupled to a detectable label.
  • the assessment includes detecting neutralizing antibodies directed against the cell surface receptor in a sample from a subject administered the cell surface receptor or cells containing the cell surface receptor; and/or
  • the assessment further includes: detecting neutralizing antibodies against the cell surface receptor in a sample from the subject prior to administering the cell surface receptor or cells containing the cell surface receptor to the subject and comparing the results after administration; and /or
  • the detection includes the steps of mixing the sample to be tested, the cells and the ligand, and detecting the binding of cell surface receptors to the ligand.
  • Neutralizing antibody neutralization efficiency (Day0 positive rate - DayN positive rate)/Day0 positive rate
  • the Day0 positivity rate represents the level of binding complexes detected before the subject comes into contact with cells containing cell surface receptors
  • the DayN positivity rate represents the level of binding complexes detected after the subject comes into contact with cells containing cell surface receptors
  • N is the number of days.
  • the cell surface receptor comprises an extracellular portion and a membrane anchoring region, the extracellular portion being an extracellular ligand binding region; and/or
  • the cells are somatic cells.
  • the cell surface receptor is a CAR
  • the ligand is an antigen specifically bound by the extracellular antigen-binding region of the CAR or a fragment of the antigen capable of binding to the CAR;
  • the antigen is a tumor associated antigen, such as a tumor surface antigen, and/or
  • the sample is a sample derived from blood, such as plasma, serum, whole blood, and/or
  • the ligand is coupled to a detectable label.
  • a kit for detecting neutralizing antibodies against cell surface receptors in a sample including: A cell containing the cell surface receptor, and a ligand that specifically recognizes the extracellular portion of the cell surface receptor;
  • the cell surface receptor comprises an extracellular portion and a membrane anchoring region, the extracellular portion being an extracellular ligand binding region, and/or
  • the cells are somatic cells.
  • kits according to item 9 characterized in that the extracellular ligand binding region is an antibody or an antigen-binding fragment thereof,
  • the cell surface receptor is a CAR
  • the ligand is an antigen specifically bound by the extracellular antigen-binding region of the CAR or a fragment of the antigen capable of binding to the CAR;
  • the antigen is a tumor associated antigen, such as a tumor surface antigen; and/or
  • the sample is a sample derived from blood, such as plasma, serum or whole blood; and/or
  • the ligand is coupled to a detectable label
  • the kit also contains sample processing reagents; and/or
  • the kit also contains reagents for detecting binding of cell surface receptors to the ligand.
  • the basic principle of the detection in this embodiment after the patient is infused back with the CAR-T cell therapy product, due to the immunogenicity of the CAR molecule, the human body produces antibodies against the CAR molecule, especially neutralizing antibodies that directly block the CAR molecule's ability to target the protein. Recognize and reduce the tumor killing function of CAR-T cells.
  • This experiment assumes that the antibodies produced are neutralizing antibodies.
  • the binding site between the CAR molecule and the target protein is occupied by the neutralizing antibody.
  • the CAR molecule no longer binds to the target protein molecule.
  • the competition between the labeled target protein and the neutralizing antibody for binding to the CAR molecule is used. relationship, detecting the presence of neutralizing antibodies in serum samples.
  • the cells After washing, resuspend the supernatant in complete medium and count. If the detection experiment is to be continued on the same day, the cells should be collected according to the amount of 2-3 million cells per flow cytometry test. After obtaining the total amount of cells according to 120% of the total required cells, centrifuge and discard the supernatant. Resuspend in PBS, let stand at room temperature for 30 minutes, centrifuge and discard the supernatant. Resuspend once in PBS, let stand at room temperature for 30 minutes, centrifuge and discard the supernatant. Finally, resuspend in PBS, and the cell density should be 20-30 million cells per ml.
  • the cells can be resuspended in complete culture medium at a concentration of 2 million per ml and placed in an incubator for culture. Collect the cells the next day, centrifuge and discard the supernatant, and resuspend the cells in PBS at a concentration of 20-30 million per ml for subsequent experiments.
  • the dosage of each plasma sample test is 200 microliters, and can be configured according to the dosage of no less than 250 microliters. It is recommended to use the gradient dilution method for series configuration, and to formulate the dilution strategy and operation steps in advance. Reduce the probability of errors while increasing the reliability of data.
  • the main contents of the experiment include various control and detection tubes.
  • Each control includes 3: blank or FMO control that only labels CD3, used to determine the negative value of CAR; detection of direct labeling of CD3 and CAR Control, compared with the cell data before recovery, the MSLN-CAR cells should be clearly divided into groups; the detection control of incubated PBS is used for experimental operations and system testing.
  • the detection tubes correspond to the design of plasma sample pretreatment, one-to-one correspondence.
  • PE fluorescently labeled mesothelin can be entrusted to Jinruix Company.
  • proteins to be labeled can also be processed through the following experimental operations:
  • Pretreatment of the protein to be labeled Use a 30KD ultrafiltration column to replace the MSLN protein buffer to be labeled to remove sodium azide, BSA, glycine, Tris or any other free amino acid additives contained in the protein sample;
  • Biotin labeling Dissolve Biotin: Weigh 1 portion (27.27 mg) of Biotin and dissolve it completely with 3 mL of fresh DMSO. Calculate the amount of biotin required for antibody labeling (calculate the required volume at a molar ratio of 20:1 with the antibody).
  • PE fluorescent labeling Streptavidin-labeled PE fluorescence is incubated with biotin-labeled MSLN protein to obtain PE fluorescent-labeled MSLN protein.
  • the CAR control detection tube is in good shape and can distinguish between negative and positive. Test each sample tube in turn, and calculate the number of CD3-positive cells that need to be collected according to the CAR positivity rate in the control tube while ensuring that the number of CAR-positive cells is no less than 1,000. Collect each test tube according to this standard.
  • flow cytometry software is used to analyze the relevant data.
  • the relative content of ADA or Nab in the plasma sample was evaluated based on the two dimensions of the change in the positive proportion of CAR-T cells and the signal intensity detected by the CAR molecules of the CAR-T cells, while taking into account the dilution factor of the plasma sample.
  • the relative content value can refer to the corresponding relationship between the ADA content of the existing sequence and the flow cytometry detection data.
  • CAR chimeric antibody CAR
  • the extracellular region is an alpaca-derived Nanobody that specifically binds to MSLN.
  • the sequence of the CAR is shown in SEQ ID NO: 1.
  • fluorescently labeled mesothelin is used to fully bind to the CAR, and the CAR is detected by flow cytometry.
  • Figure 3 32.7% of the T cells in the MSLN_CAR-T cell preparation (batch number: BZDS202012001) of patient S0105 expressed 1444-CAR on the surface.
  • CD8 signal peptide-(anti-MSLN)-CD8 hinge-CD28 TM-CD28IC-CD3z sequence is as follows:
  • Enrolled patient S0105 was reinfused with BZDS202012001 batch of MSLN_CAR-T cell products.
  • Peripheral blood samples were collected on D0 before reinfusion and serum was separated.
  • D28 days after reinfusion peripheral blood samples were collected and serum was separated.
  • the aforementioned method was used to detect the MSLN_CAR- Neutralizing antibodies against MSLN-CAR were produced in peripheral blood before T and on D28 after reinfusion. Confirmation by testing at different dilutions.
  • Table 1 The production and neutralization efficiency of neutralizing antibodies in the peripheral blood of S0105 patients after infusion of MSLN_CAR-T
  • Patient S0105 underwent a second infusion of BZDS202012001 batch of MSLN_CAR-T cell product on D35 after completing the first infusion.
  • Peripheral blood samples were collected on D35 before the infusion, and the serum was separated.
  • D63 peripheral blood samples were collected. Blood samples were collected, serum was separated, and neutralizing antibodies against MSLN-CAR produced in the peripheral blood of the patient before the second reinfusion of MSLN_CAR-T and on the 28th day after the reinfusion were detected using the aforementioned method. Confirmation by testing at different dilutions.
  • Table 2 The production and neutralization efficiency of neutralizing antibodies in the peripheral blood of patients with S0105 after the second infusion of MSLN_CAR-T
  • Collect peripheral blood mononuclear cells from patients with advanced solid tumors (patient numbers S0106 and S0304 respectively) who meet the clinical inclusion criteria, sort and purify T lymphocytes, and express chimeric cells on the surface of T cells through gene transduction and integration.
  • Antibody 2339-CAR (the antigen-binding domain of the CAR used is numbered 2339, a new antibody derived from alpaca that specifically binds to MSLN). Fluorescently labeled mesothelin was used for flow cytometric detection. The results are shown in Figure 6.
  • the MSLN_CAR-T cell preparation (batch number: BZDS202101001) of patient S0106 has 96.8% of T cells expressing 2339-CAR on the surface.
  • Patient S0106 was reinfused with BZDS202101001 batch of MSLN_CAR-T cell products.
  • Peripheral blood samples were collected on D0 before reinfusion and serum was separated.
  • D21 after reinfusion peripheral blood samples were collected and serum was separated.
  • the aforementioned method was used to detect the patient before MSLN_CAR-T was reinfused.
  • neutralizing antibodies against MSLN-CAR were produced in peripheral blood on D21 after reinfusion. Confirmation by testing at different dilutions.
  • Table 3 The production and neutralization efficiency of neutralizing antibodies in the peripheral blood of patients with S0106 who were reinfused with MSLN_CAR-T
  • Patient S0304 was reinfused with the same sequence (2339) MSLN_CAR-T cell product.
  • Peripheral blood samples were collected on D0 before reinfusion and serum was separated.
  • D21 after reinfusion peripheral blood samples were collected and serum was separated.
  • the method established by the present invention detects the production of neutralizing antibodies against MSLN-CAR in the peripheral blood of patients before reinfusion of MSLN_CAR-T and on D21 after reinfusion. Confirmation by testing at different dilutions.
  • Table 4 The production and neutralization efficiency of neutralizing antibodies in the peripheral blood of patients with S0304 who were reinfused with MSLN_CAR-T

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Abstract

La présente invention concerne un procédé de détection d'un anticorps neutralisant. La présente invention concerne un procédé non diagnostique ou non thérapeutique de détection d'un anticorps neutralisant contre un récepteur de surface cellulaire dans un échantillon. Le procédé comprend les étapes consistant à : (1) mélanger un échantillon à tester, une cellule contenant le récepteur de surface cellulaire et un ligand reconnaissant spécifiquement la partie extracellulaire du récepteur de surface cellulaire, puis incuber le mélange, l'anticorps neutralisant étant en concurrence avec le ligand pour se lier au récepteur de surface cellulaire; et (2) détecter la liaison du récepteur de surface cellulaire au ligand. Selon la présente invention, l'anticorps neutralisant produit par l'organisme contre le produit d'expression d'un gène cible sur la surface d'une cellule somatique génétiquement modifiée peut être détecté de manière efficace et pratique.
PCT/CN2023/085530 2022-03-31 2023-03-31 Procédé de détection d'anticorps neutralisant WO2023186106A1 (fr)

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CN112394179A (zh) * 2021-01-18 2021-02-23 北京百普赛斯生物科技股份有限公司 Fmc63 car-t细胞免疫原性elisa检测试剂盒

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