WO2022010806A1 - Procédés pour réduire l'interférence de médicaments qui se lient à des cibles thérapeutiques exprimées sur des cellules sanguines dans des dosages sérologiques - Google Patents

Procédés pour réduire l'interférence de médicaments qui se lient à des cibles thérapeutiques exprimées sur des cellules sanguines dans des dosages sérologiques Download PDF

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WO2022010806A1
WO2022010806A1 PCT/US2021/040374 US2021040374W WO2022010806A1 WO 2022010806 A1 WO2022010806 A1 WO 2022010806A1 US 2021040374 W US2021040374 W US 2021040374W WO 2022010806 A1 WO2022010806 A1 WO 2022010806A1
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variant
drug
antibody
human
binds
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PCT/US2021/040374
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Bang Janet Sim
Jaume Pons
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ALX Oncology Inc.
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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
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells

Definitions

  • This invention relates to methods of reducing (or eliminating) interference in serological assays by drugs that comprise (i) an antibody Fc region and (ii) a moiety that binds to a therapeutic target that is expressed on the surface of blood cells (e.g., CD47).
  • CD47 a widely-expressed cell surface protein that binds to signal regulatory protein- ⁇ (SIRP ⁇ ) and inhibits phagocytosis (Jaiswal et al., Trends Immunol (2010) 31(6):212-219; Brown et al., Trends Cell Biol (2001) 11(3):130-135), is expressed at high levels on a wide variety of malignant tumors, including hematological and solid tumors. Elevated CD47 expression also correlates with aggressive disease (Willingham et al., Proc Natl Acad Sci USA (2012) 109(17):6662-6667).
  • CD47 a cancer therapies targeting CD47, e.g., antibodies and fusion proteins comprising an antibody Fc region, have been developed to block the SIRP ⁇ -CD47 interaction, thereby permitting macrophages to carry out their phagocytic function to clear tumor cells.
  • CD47 is also expressed on the surface of blood cells, such as red blood cells (RBCs) and platelets (Oldenborg et al., Science (2000) 288(5473):2051-2054), drugs comprising antibody Fc regions that target CD47 could interfere with blood typing and serological tests.
  • RBCs red blood cells
  • platelets Oldenborg et al., Science (2000) 288(5473):2051-2054
  • a method of reducing drug interference in a serological assay using reagent red blood cells (RBCs) or reagent platelets comprising: combining the reagent RBCs or reagent platelets with a plasma sample from a subject who has received treatment with the drug; and adding an agglutination agent that does not bind an antibody Fc region to the combined plasma sample and reagent RBCs or reagent platelets, wherein the drug comprises (i) a human antibody Fc region or variant thereof and (ii) a moiety that binds to a therapeutic target that is expressed on the surface of RBCs and/or platelets.
  • a method of reducing drug interference in a serological assay using reagent plasma comprising: combining the reagent plasma with a sample of red blood cells (RBCs) from a subject who has received treatment with the drug; and adding an agglutination agent that does not bind an antibody Fc region to the combined reagent plasma and sample of RBCs, wherein the drug comprises (i) a human antibody Fc region or variant thereof and (ii) a moiety that binds to a therapeutic target that is expressed on the surface of RBCs and/or platelets.
  • RBCs red blood cells
  • the agglutination agent is an anti-light chain antibody, and anti-J chain antibody, Protein L, a peptide that binds an antibody Fab region, or a small molecule that binds an antibody Fab region.
  • the agglutination agent is an anti-kappa light chain antibody.
  • the agglutination agent is an anti-lambda light chain antibody.
  • the drug comprises (i) a human antibody Fc region or variant thereof and (ii) a moiety that binds to human CD47.
  • the moiety of the drug that binds to human CD47 comprises a wild type SIRP ⁇ , a SIRP ⁇ variant, or a fragment of the wild type SIRP ⁇ or the SIRP ⁇ variant.
  • the moiety of the drug that binds to human CD47 comprises the SIRP ⁇ variant, and wherein the SIRP ⁇ variant comprises one or more amino acid substitution(s), insertion(s), deletion(s), N-terminal extension(s), and/or C-terminal extension(s) relative to the wild type SIRP ⁇ .
  • the moiety of the drug that binds to human CD47 comprises the fragment of the SIRP ⁇ variant, and wherein the fragment comprises an extracellular domain of the SIRP ⁇ variant.
  • the moiety of the drug that binds to human CD47 comprises a wild type SIRP ⁇ , a SIRP ⁇ variant, or a fragment of the wild type SIRP ⁇ or the SIRP ⁇ variant.
  • the moiety of the drug that binds to human CD47 comprises the SIRP ⁇ ⁇ variant, and wherein the SIRP ⁇ variant comprises one or more amino acid substitution(s), insertion(s), deletion(s), N-terminal extension(s), C-terminal extension(s), or any combination of the preceding, relative to the wild type SIRP ⁇ .
  • the moiety of the drug that binds to human CD47 comprises the fragment of the SIRP ⁇ variant, and wherein the fragment comprises an extracellular domain of the SIRP ⁇ variant. In some embodiments, the moiety of the drug that binds to human CD47 comprises a SIRPE variant or a fragment of the SIRPE variant. In some embodiments, the moiety of the drug that binds to human CD47 comprises the SIRPE ⁇ variant, and wherein the SIRPE ⁇ variant comprises one or more amino acid substitution(s), insertion(s), deletion(s), N- terminal extension(s), C-terminal extension(s), or any combination of the preceding, relative to the wild type SIRP ⁇ .
  • the moiety of the drug that binds to human CD47 comprises the fragment of the SIRPE variant, and wherein the fragment comprises an extracellular domain of the SIRPE variant.
  • the serological assay is an ABO/Rh typing assay.
  • the serological assay is an indirect antiglobulin test (IAT).
  • IAT indirect antiglobulin test
  • IS immediate spin
  • the serological assay is a direct antiglobulin (DAT) assay using a polyspecific reagent that detects IgG and complement C3.
  • the serological assay is a direct antiglobulin (DAT) assay using a monospecific reagent that detects complement C3.
  • the serological assay is a PEG-enhanced serological assay.
  • the serological assay is an eluate test that is performed following the DAT assay.
  • FIG.1A shows a serological assay in which a plasma sample obtained from a subject is mixed with reagent red blood cells (i.e., red blood cells (“RBCs”) that are known to express a particular cell surface blood group antigen, or group of cell surface antigens) to detect the presence of antibodies in the plasma sample that bind the blood group antigen expressed on the reagent RBCs.
  • RBCs red blood cells
  • such serological assay can be performed using reagent platelets (i.e., platelets that are known to express a particular cell surface blood group antigen, or group of cell surface blood group antigens) instead of reagent RBCs.
  • FIG 1B shows how the presence of a drug comprising (i) and antibody Fc region and (ii) a moiety that binds a therapeutic target that is also expressed on the surface of RBCs (and/or platelets) in a plasma sample interferes with the assay of FIG.1A.
  • FIG.1C shows a serological assay in which a blood sample obtained from a subject is mixed with reagent plasma/antisera (i.e., plasma or antisera known to contain antibodies against a specific RBC surface blood group antigen(s) or platelet surface antigen(s)) in order to detect the presence of the blood group antigen on the subject’s RBCs and/or platelets.
  • reagent plasma/antisera i.e., plasma or antisera known to contain antibodies against a specific RBC surface blood group antigen(s) or platelet surface antigen(s)
  • FIG 1D shows how the presence of a drug comprising (i) and antibody Fc region and (ii) a moiety that binds a therapeutic target that is also expressed on the surface of RBCs (and/or platelets) in a blood sample interferes with the assay of FIG.1C.
  • FIG.2A shows a method of reducing interference in a serological assay that comprises adding an agglutination agent that does not bind to an antibody Fc region to detect reagent RBCs (or reagent platelets) that have been bound by anti-RBC surface blood group antigen antibodies present in a plasma sample from a subject who has received treatment with a drug comprising (i) and antibody Fc region and (ii) a moiety that binds a therapeutic target that is also expressed on the surface of RBCs (and/or platelets). Agglutination occurs only in the presence of antibody-bound reagent RBCs (or antibody-bound reagent platelets).
  • FIG.2B shows a method of reducing interference in a serological assay that comprises adding an agglutination agent that does not bind to an antibody Fc region to detect RBCs in a sample from a subject (i.e., a subject who has received treatment with a drug comprising (i) and antibody Fc region and (ii) a moiety that binds a therapeutic target that is also expressed on the surface of RBCs and/or platelets) that have been bound by anti-RBC surface blood group antigen antibodies present in a reagent plasma.
  • a subject i.e., a subject who has received treatment with a drug comprising (i) and antibody Fc region and (ii) a moiety that binds a therapeutic target that is also expressed on the surface of RBCs and/or platelets
  • FIG.3 shows the results of experiments that were performed to assess the feasibility of using an anti-human kappa light chain antibody as an agglutination agent in serological assays, as depicted in e.g., FIGs 2A and 2B.
  • DETAILED DESCRIPTION OF THE INVENTION Methods of Mitigating Interference in Pre-Transfusion Serological Assays [0021] Many therapeutic targets are also expressed on red blood cells.
  • CD47 is a transmembrane protein that interacts with thrombospondin-1 (TSP-1) as well as several molecules on immune cells, including signal regulatory protein alpha (SIRP ⁇ ). Upon binding CD47, SIRP ⁇ initiates a signaling cascade that inhibits phagocytosis and prevents phagocytic removal of healthy cells by the immune system. However, many cancers overexpress CD47 and evade phagocytic clearance. Accordingly, drugs that target CD47 (such as anti-CD47 antibodies and fusion proteins comprising an antibody Fc region and a moiety that binds CD47) are of significant therapeutic interest. CD47 is also expressed on the surface of human red blood cells (RBCs) and platelets.
  • RBCs human red blood cells
  • the drug present in the subject’s plasma or bound to the subject’s RBCs and/or platelets may cause interference in routine pre- transfusion serological assays.
  • FIG 1A shows a serological assay in which a plasma sample obtained from a subject is mixed with reagent RBC or “reference RBC” (i.e., RBC that are known to express a particular cell surface blood group antigen, or group of cell surface blood group antigens) or with reagent platelets or “reference platelets” (i.e., platelets that are known to express a particular cell surface blood group antigen, or group of cell surface blood group antigens) to detect the presence of antibodies in the plasma sample that bind the known cell surface blood group antigen(s) expressed on the reagent RBCs or reagent platelets.
  • reference RBC i.e., RBC that are known to express a particular cell surface blood group antigen, or group of cell surface blood group antigens
  • AHG i.e., anti-human globulin
  • the presence of a drug comprising (i) an antibody Fc region and (ii) a moiety that binds to a therapeutic target that is also expressed on RBCs (e.g., on the surface of RBCs and/or platelets) in the subject’s plasma may interfere with the assay and produce a false positive result.
  • the drug may bind the therapeutic target that is expressed on the surface of reagent RBCs (or reagent platelets).
  • AHG i.e., anti-human globulin
  • FIG.1C depicts a serological assay in which a blood sample from a subject is mixed with reagent plasma/antisera (i.e., plasma or antisera containing antibodies against a known RBC surface blood antigen(s) or a known platelet surface blood group antigen(s)) in order to detect the presence of the blood group antigen(s) on the subject’s RBCs and/or platelets.
  • reagent plasma/antisera i.e., plasma or antisera containing antibodies against a known RBC surface blood antigen(s) or a known platelet surface blood group antigen(s)
  • the addition of AHG will lead to agglutination if the blood group antigen recognized by the antibodies in the reagent plasma/antisera is expressed on the subject’s RBCs and/or platelets.
  • a drug comprising (i) an antibody Fc region and (ii) a moiety that binds to a therapeutic target that is also expressed on the surface of RBCs (and/or platelets) in a sample comprising the subject’s RBCs and/or platelets may interfere with the assay and produce a false positive result.
  • the drug bound to the therapeutic target on the subject’s RBCs or platelets will cause agglutination after AHG is added to a mixture comprising the subject’s blood sample and reagent plasma/antisera.
  • the methods described below reduce (and, in some embodiments, eliminate) the interference caused by the drug, i.e., as illustrated in FIGs.1B and 1D.
  • reagent red blood cells i.e., RBCs that are known to express a particular cell surface blood group antigen, or group of cell surface blood group antigens
  • reagent platelets i.e., platelets that are known to express a particular cell surface blood group antigen, or group of cell surface blood group antigens
  • the method comprising: combining the reagent RBCs or reagent platelets with a plasma sample from a subject who has received treatment with the drug; and adding an agglutination agent that does not bind an antibody Fc region to the combined plasma sample and reagent RBCs or reagent platelets, wherein the drug comprises (i) a human antibody Fc region or variant thereof and (ii) a moiety that binds to a therapeutic target that is also expressed on RBCs and/or platelets (e.g., on the surface of RBCs and/
  • the drug comprises (i) an antibody Fc region and (ii) a moiety that binds to human CD47. In some embodiments, the drug comprises (i) an antibody Fc region and (ii) a SIRP ⁇ variant, a SIRPE variant, or a SIRP ⁇ variant that binds to human CD47. [0026] Such embodiments are generically depicted in FIG 2A.
  • agglutination agent that does not bind an antibody Fc region is added, and agglutination (e.g., clumping) of the reagent RBC (or reagent platelets) occurs if the plasma sample from the subject who has received treatment with the drug contains an antibody that binds an RBC surface blood group antigen (or platelet surface blood group antigen).
  • the interference that would result from using AHG to detect antibody-bound reagent RBCs and/or reagent platelets is minimized (or, in some embodiments, eliminated), thus preventing a false positive result in the serological assay.
  • reagent plasma i.e., plasma or antisera containing antibodies against a known RBC surface blood group antigen(s) or a known platelet surface blood group antigen(s)
  • said method comprising: combining the reagent plasma with a sample of red blood cells (RBCs) from a subject who has received treatment with the drug; and adding an agglutination agent that does not bind an antibody Fc region to the combined reagent plasma and sample of RBCs, wherein the drug comprises (i) a human antibody Fc region or variant thereof and (ii) a moiety that binds to a therapeutic target that is also expressed on RBCs and/or platelets (e.g., on the surface of RBCs and/or platelets).
  • the drug comprises (i) an antibody Fc region and (ii) a moiety that binds to human CD47. In some embodiments, the drug comprises (i) an antibody Fc region and (ii) a SIRP ⁇ variant, a SIRPE variant, or a SIRP ⁇ variant that binds to human CD47. [0028] Such embodiments are generically depicted in FIG 2B.
  • an agglutination agent that does not bind an antibody Fc region is added to the mixture, and agglutination (e.g., clumping) of the subject’s RBC occurs if the reagent plasma sample contains an antibody that binds an antigen on the surface of the subject’s RBC.
  • the interference that would result from using AHG to detect RBCs bound with antibodies present in the reagent plasma is minimized (or, in some embodiments, eliminated), thus preventing a false positive result in the serological assay.
  • the method is performed in solution, e.g., wherein the agglutination agent that does not bind an antibody Fc region is soluble.
  • the agglutination agent that does not bind an antibody Fc region is immobilized to a solid phase before the method is performed via adsorption to a matrix or surface, covalent coupling, or non- covalent coupling.
  • agglutination agent that does not bind an antibody Fc region is capable of binding drug following immobilization to the solid phase.
  • the solid phase or surface used for immobilization can be any inert support or carrier that is essentially water insoluble and useful in immunoassays, including supports in the form of, for example, surfaces, particles, porous matrices, cellulose polymer sponge (ImmunoCAP®, Phadia), and the like.
  • supports in the form of, for example, surfaces, particles, porous matrices, cellulose polymer sponge (ImmunoCAP®, Phadia), and the like.
  • commonly used supports include small sheets, Sephadex, polyvinyl chloride, plastic beads, microparticles, assay plates, or test tubes manufactured from polyethylene, polypropylene, polystyrene, and the like.
  • the agglutination agent that does not bind an antibody Fc region is coated on a microtiter plate, such as a multi-well microtiter plate that can be used to analyze multiple samples simultaneously.
  • the agglutination agent that does not bind an antibody Fc region is an anti-light chain antibody.
  • anti-light chain antibodies that find use with the methods described herein include, but are not limited to, e.g., RM129 (i.e., a rabbit anti- human Ig light chain monoclonal antibody clone that recognizes both human kappa and lambda light chains, available from ThermoFisher, Catalog #SA5-10224, and from RevMAb Bioscience, Catalog # 32-1031-00); goat anti-human kappa light chain polyclonal antibody (available from BioRad, Catalog # STAR127, and from Novus Biologicals, Catalog # NBP1-75060); IS7-24C7 (i.e., a mouse anti-human lambda light chain monoclonal antibody, available from Miltenyi Biotec, Catalog #
  • the agglutination agent that does not bind an antibody Fc region is an anti-J chain antibody.
  • the J chain is a 137-amino acid protein component of IgM and IgA antibodies.
  • the J chain exhibits a standard immunoglobulin folding structure of two ⁇ -pleated sheets of four ribbons folded against one another.
  • the J chain comprises 8 cystine residues, two of which link the ⁇ chains of IgA or the ⁇ chains of IgM via disulfide bridges, effectively serving as the "glue” between two Fc regions of the IgA or IgM antibody (see, e.g., Kiyono, Hiroshi; Kunisawa, Jaw; McGhee, Jerry; Mestecky, Jiri (2008). "Chapter 31: The Mucosal Immune System". In Paul, William (ed.). Fundamental Immunology (Book) (6th ed.). Philadelphia, PA: Lippincott Williams & Wilkins. pp.983–1030). Anti-J chain antibodies are available from a variety of commercial sources.
  • anti-J chain antibodies that find use with the methods herein include, but are not limited to, e.g., SP105 (i.e., a monoclonal rabbit anti-human J chain antibody clone that is available from Millipore Sigma, Catalog # SAB5500121); OTI3E12 or 3E12 (i.e., a monoclonal mouse anti-human J chain antibody clone that is available from LS Bio, Catalog # LS-C786794-100); Mc19-9 (i.e., a monoclonal mouse anti-human J chain antibody clone available from Invitrogen, Catalog # MA1-80527, and from BioRad, Catalog # MCA693); OTI3B3 (i.e., a monoclonal mouse anti-human J chain antibody clone available from Invitrogen, Catalog # MA5-25840); OTI2B1 (i.e., a monoclonal mouse anti-human J chain antibody clone available from Invitrogen, Catalog
  • the agglutination agent that does not bind an antibody Fc region is Protein L.
  • Protein L is an immunoglobulin-binding protein that was originally derived from Peptostreptococcus magnus. Protein L binds kappa light chain without interfering with an antibody's antigen-binding site. Protein L is capable of binding IgG, IgM, IgA, IgE and IgD, as well as scFvs and Fabs (i.e., providing they comprise a kappa light chain). Protein L has been used to isolate antibody fragments that do not comprise an Fc domain (see, e.g., Rodrigo, et al.
  • Protein L is available from a variety of commercial sources, including, but not limited to, e.g., Prospec (Catalog # PRO-1790); Millipore Sigma (Catalog # P3101-); ThermoFisher (Catalog # 21189).
  • Other Agglutination Agents that Do Not Bind an Antibody Fc Region [0033]
  • the agglutination agent that does not bind an antibody Fc region is a small molecule that binds an antibody light chain.
  • the small molecule that binds an antibody light chain is a coumarin, an aryl cyanoacrylamide, a biaryl hydrazine, a hydantoin, or sulfone.
  • Exemplary small molecules that bind an antibody light chain include, but are not limited to, e.g., compounds 1-18 described in Morgan et al. (2019) PNAS U.S.A.116(17): 8360–8369, the contents of which are hereby incorporated by reference in their entirety.
  • the agglutination agent that does not bind an antibody Fc region is a Fab-binding peptide.
  • the Fab-binding peptide is a Protein G variant that has been engineered to bind an antibody Fab region with high affinity.
  • the Fab-binding peptide is a peptide disclosed in EP 3040344 A1, the contents of which are hereby incorporated by reference in their entirety.
  • the agglutination agent that does not bind an antibody Fc region is an anti-Fab fragment antibody.
  • Anti-Fab fragment antibodies are available from commercial sources. Exemplary anti-Fab fragment antibodies include, but are not limited to, e.g., 4A11 (i.e.
  • a monoclonal mouse anti-human IgG Fab fragment antibody clone available from Abcam, Catalog # ab771); polyclonal goat anti-human IgG Fab fragment antibody (available from Millipore Sigma, Catalog # 5260, and from Rockland Inc., Catalog # 809-1102).
  • Exemplary Drug Targets and Exemplary Drugs Exemplary Drug Targets reduce (or, in some embodiments, eliminate) interference in serological assays caused by the presence of a drug comprising (i) an antibody Fc region and (ii) a moiety that binds to a therapeutic target that is expressed (e.g., also expressed) on the surface of red blood cells (RBCs) and/or platelets.
  • RBCs red blood cells
  • the therapeutic target is, without limitation, Band 3 (also known as anion exchanger or AE1), ⁇ -spectrin, ⁇ - spectrin, ankyrin, ⁇ -adducin, ⁇ -adducin, protein 4.1, protein 4.2, p55, b-actin, tropomodulin, G3PD, stomatin, tropomyosin, glycophorin A, glycophorin B, glycophorin C, glycophorin D, glycophorin E, CD44, CD58, LW/ICAM-4, RAGE, B-CAM/LU (also known as CD239), LW (also known as intercellular adhesion molecule-4 (ICAM-4) or CD242), Indian (also known as In(Lu)-related p80 or CD44), lymphocyte-associated antigen-3 (also known as LFA-3 or CD58), CD99 (also known as MIC2), JMH (also known as CD10B or SEMA 7A), Ok (also known as neurothelin or CD
  • Band 3
  • the therapeutic target is PPBP (platelet basic protein precursor), RHOG (Ras homolog family member G), CLU (clusterin), ACTN1 (Actinin alpha 1 protein) or CALU (calumenin). Additional exemplary therapeutic targets that are expressed on platelets are described in Aloui et al. (2019) Journal of Proteomics.194: 25-36, the contents of which are incorporated herein by reference in their entirety.
  • the methods provided by the present application are used to reduce (or eliminate) interference in serological assays by drugs comprising (i) an antibody Fc domain and (ii) a moiety that binds to a therapeutic target that is expressed (e.g., also expressed) on the surface of blood cells (e.g., RBCs and/or platelets).
  • the drug comprises an IgG Fc region, such as a human IgG Fc region, e.g., an IgG1, IgG2, or an IgG4 Fc region.
  • the drug comprises a modified Fc region (such as a modified IgG Fc region) that comprises one or more amino acid substitution(s), deletion(s), insertion(s), N- terminal addition(s) and/or C-terminal addition(s) relative to a wild type human Fc region (e.g., a wild type human IgG Fc region).
  • modified Fc regions are described in WO2017177333; WO2014094122; US2015329616, WO 2017/027422; US 2017/0107270; and USP 10,259,859, the contents of which are incorporated herein by reference in their entirety.
  • any drug that binds to a therapeutic target expressed on RBCs and/or platelets and does not comprise a light chain finds use with the methods described herein.
  • the drug is a single-domain heavy chain antibody (also known as a nanobody).
  • Drugs that Target CD47 [0039] The methods provided herein reduce (or, in some embodiments, eliminate) interference in serological assays caused by the presence of a drug comprising (i) an antibody Fc region and (ii) a moiety that binds to human CD47 in a sample comprising plasma or RBCs/platelets obtained from a subject who has received treatment with the drug.
  • the drug comprises an IgG Fc region, such as a human IgG Fc region, e.g., an IgG1, IgG2, or an IgG4 Fc region.
  • the drug comprises a modified Fc region (such as a modified IgG Fc region) that comprises one or more amino acid substitution(s), deletion(s), insertion(s), N-terminal addition(s) and/or C-terminal addition(s) relative to a wild type human Fc region (e.g., a wild type human IgG Fc region).
  • the moiety that binds to human CD47 is a wild type SIRP ⁇ that lacks a transmembrane domain (e.g., the extracellular domain of any wild type SIRP ⁇ that is capable of binding human CD47).
  • the moiety that binds to human CD47 is a SIRP ⁇ variant that is capable of binding human CD47 and lacks a transmembrane domain.
  • the SIRP ⁇ variant comprises one or more amino acid substitution(s), deletion(s), insertion(s), N-terminal addition(s) and/or C-terminal addition(s) relative to extracellular domain of a wild-type SIRP ⁇
  • the SIRP ⁇ variant is a SIRP ⁇ -d1 domain variant.
  • the affinity of the SIRP ⁇ variant for human CD47 is higher than the affinity of a wild type SIRP ⁇ for human CD47.
  • the moiety that binds to human CD47 is a wild type SIRP ⁇ ⁇ that lacks a transmembrane domain (e.g., the extracellular domain of any wild type SIRP ⁇ that is capable of binding human CD47).
  • the moiety that binds to human CD47 is a SIRP ⁇ variant that is capable of binding human CD47 and lacks a transmembrane domain.
  • the SIRP ⁇ variant comprises one or more amino acid substitution(s), deletion(s), insertion(s), N-terminal addition(s) and/or C-terminal addition(s) relative to extracellular domain of a wild-type SIRP ⁇ ⁇
  • the SIRP ⁇ variant is a SIRP ⁇ - d1 domain variant.
  • the affinity of the SIRP ⁇ variant for human CD47 is higher than the affinity of a wild type SIRP ⁇ for human CD47.
  • the moiety that binds to human CD47 is a SIRPE variant that is capable of binding human CD47 and lacks a transmembrane domain.
  • the SIRP ⁇ ⁇ variant comprises one or more amino acid substitution(s), deletion(s), insertion(s), N- terminal addition(s) and/or C-terminal addition(s) relative to extracellular domain of a wild-type SIRPE ⁇
  • the SIRPE variant is a SIRPE-d1 domain variant.
  • SIRP ⁇ variants SIRPE ⁇ variants, and SIRP ⁇ variants are known in the art and are described in WO 2013/109752; US 2015/0071905; USP 9,944,911; WO 2016/023040; WO 2017/027422; US 2017/0107270; USP 10,259,859; US9845345; WO2016187226; US20180155405; WO2017177333; WO2014094122; US2015329616; US20180312563; WO2018176132; WO2018081898; WO2018081897; US20180141986A1; and EP3287470A1, the contents of which are incorporated herein by reference in their entireties.
  • Pre-transfusion testing is performed to ensure that the blood product intended for transfusion is compatible with the blood of the subject (i.e., the recipient of the transfusion).
  • Pre- transfusion testing encompasses the serological assays that are used to confirm ABO compatibility between donor blood and recipient blood, as well as those that are used to detect most clinically significant RBC / platelet alloantibodies that react with antigens on donor RBCs and/or donor platelets (ref. Technical Manual, 18th ed, AABB, Bethesda, MD, 2014).
  • exemplary blood group antigens for which serological assays are performed to determine donor / recipient transfusion compatibility include, without limitation, e.g., Kell blood group antigens, Duffy blood group antigens, Knops blood group antigens, Cartwright blood group antigens, Scianna blood group antigens, Indian blood group antigens, Rhesus blood group antigens, Dombrock blood group antigens, Landsteiner-Wiener blood group antigens, VEL blood group antigens, Rh blood group antigens, P blood group antigens, Lewis blood group antigens, Puffy blood group antigens, Kiddy blood group antigens, LW blood group antigens, li blood group antigens, Diego and Wright blood group antigens, Chido blood group antigens, and Rodgers blood group antigens.
  • Kell blood group antigens Duffy blood group antigens
  • Knops blood group antigens Cartwright blood group antigens
  • Scianna blood group antigens Indian blood
  • the methods provided herein reduce or prevent drug interference (e.g., interference by a drug comprising (i) an antibody Fc region and (ii) a moiety that binds to human CD47) in a number of serological assays known in the art.
  • drug interference e.g., interference by a drug comprising (i) an antibody Fc region and (ii) a moiety that binds to human CD47
  • Exemplary serological assays in which the methods can be used include (but are not limited to) those described in further detail below.
  • serological assays are performed using samples comprising, e.g., non- hemolyzed blood, plasma (e.g., a plasma sample that has been anticoagulated in EDTA), clotted blood, or serum from a subject who is in need of the transfusion (e.g., a subject who has received treatment with a drug comprising (i) an antibody Fc region and (ii) a moiety that binds to human CD47.
  • the subject s ABO group and Rh type are determined first.
  • an antibody screening method is used to detect any clinically significant unexpected non-ABO blood group antibodies that may be present in the subject’s plasma.
  • the specificity of that antibody is determined using an antibody identification panel. After the specificity of the antibody is identified, donor units of the appropriate ABO group and Rh type are screened for the corresponding antigen. Units that are negative for that antigen are crossmatched with the subject who is in need of the transfusion to ensure compatibility.
  • Serological assays can be performed in a tube, on a slide, on a gel column or in microtiter well plates, and hemolysis and agglutination are signals that indicate a positive (incompatible) test result.
  • Agglutination a reaction reflecting linkage of adjacent RBCs that are coated with antibody, can be scored macroscopically and/or microscopically and on scale from 0- 4+ in the most commonly used tube methods.
  • a score of zero indicates no reactivity and is characterized by smooth and easily dispersed cells.
  • a score of 4+ indicates strong reactivity and is characterized by one solid agglutinate that is not easily dispersed.
  • Scores of 1+, 2+, or 3+ indicate intermediate levels of reactivity, characterized by gradually increasing size of agglutinates with higher scores.
  • serological assays are performed manually. In some embodiments, serological assays are performed via automated machine.
  • immediate-spin is an assay that entails mixing, e.g., reagent plasma/antisera (i.e., plasma containing antibodies against a known RBC and/or platelet surface antigen) and the subject’s blood cells, immediately centrifuging the mixture for about 15-30 seconds at room temperature or at 37°C, and visually examining the tube for direct agglutination. Direct agglutination indicates that there is a strong interaction between an antibody in the plasma and an RBC surface antigen.
  • reagent plasma/antisera i.e., plasma containing antibodies against a known RBC and/or platelet surface antigen
  • plasma and reagent RBC i.e., RBC that are known to express a particular cell surface antigen, or group of cell surface antigens
  • regent platelets i.e., platelets that are known to express a particular cell surface antigen, or group of cell surface antigens
  • AHGs Anti-human globulins are typically used to detect antibody-bound RBC that do not produce direct agglutination.
  • AHG are secondary anti-human globulin antibodies that have been produced in another species.
  • AHG reagents can be specific for a single class of human Ig (such as IgG), or polyspecific, i.e., capable of binding to multiple human Ig classes (e.g., IgG, IgM, IgA) and to complement.
  • AHG sera may be used in a direct antiglobulin test (DAT) and/or in an indirect antiglobulin test (IAT).
  • DAT demonstrates in vivo sensitization of red cells and is performed by directly testing a sample of washed patient red cells with AHG.
  • An IAT demonstrates in vitro reactions between red cells and antibodies. In an IAT, serum (or plasma) is incubated with red cells, which are then washed to remove unbound globulins.
  • agglutination with the addition of AHG indicates antibody binding to a specific red cell antigen.
  • Some methods involve addition of potentiator reagents (enhancement) such as saline, albumin, low ionic strength saline (LISS), or polyethylene glycol (PEG), and the samples are then incubated at 37°C for 10-60 minutes prior to the AHG test.
  • potentiator reagents such as saline, albumin, low ionic strength saline (LISS), or polyethylene glycol (PEG)
  • an anti-light chain antibody is used in place of AHG to agglutinate antibody-bound RBC (i.e., “sensitized RBC”).
  • ABO typing involves testing the recipient’s red blood cells for the presence of A and B antigens using anti-A and anti-B antisera (forward grouping). Testing of the recipient plasma for the presence of anti-A and anti-B using known Type A and Type B red blood cells (reverse grouping) is also part of routine ABO blood group testing.
  • the Rh (D) type of the transfusion recipient is determined by testing recipient red blood cells with anti-D. ABO grouping is typically tested using immediate spin (IS).
  • Alloantibodies to antigens that are not present on an individual's red blood cells may develop in anyone who has been exposed to foreign red blood cell antigens through pregnancy or transfusion.
  • To detect antibodies to non- group A or B antigens a sample of the patient's plasma or serum is tested against selected commercial Type O red blood cells that express the majority of clinically significant antigens, other than A and B.
  • further serological testing is conducted with an expanded panel of commercial Type O reagent RBCs for the identification of clinically significant antibodies is required. Then, once the specificity of the antibody is known, donor units must be screened for the corresponding antigen to select those units that lack the antigen.
  • Antigen typing (phenotyping) of the recipient red blood cells may also be performed to determination of which red blood cell antibodies an individual is likely to develop.
  • Serological assay for RBC phenotyping involves mixing recipient cells with commercial reagent anti-sera containing specific antibodies.
  • An IAT without and with enhancement e.g. saline, LISS, PEG
  • Crossmatch refers to a method of confirming compatibility between the patient's blood (plasma) and the donor red blood cells. The crossmatch is meant primarily to detect and prevent ABO incompatibility.
  • a serological crossmatch assay involves the direct mixing of donor red blood cells with recipient plasma and scores for hemolysis and agglutination following immediate-spin method or AHG test.
  • Drug A is an exemplary drug that comprises a human antibody Fc region variant and a moiety that binds to CD47 (i.e., a therapeutic target that is expressed on the surface of red blood cells). Drug A does not contain an antibody light chain.
  • 5F9 also known as magrolimab, is a therapeutic monoclonal anti-human CD47 antibody.
  • interference in a serological assay using red blood cells or reagent platelets and a plasma sample from a subject who has received treatment with a drug comprising (i) a human antibody Fc region or variant thereof and (ii) a moiety that binds to a therapeutic target that is expressed on the surface of RBCs and/or platelets (e.g., CD47) could be reduced (or prevented) interference by using anti-kappa light chain antibody, rather than AHG, as an agglutination agent.

Abstract

L'invention concerne des procédés de réduction et/ou de prévention de l'interférence par un médicament comprenant (i) une région Fc d'anticorps et (ii) une fraction qui se lie à une cible thérapeutique qui est exprimée sur la surface de cellules sanguines (par exemple, des globules rouges et/ou des plaquettes).
PCT/US2021/040374 2020-07-06 2021-07-02 Procédés pour réduire l'interférence de médicaments qui se lient à des cibles thérapeutiques exprimées sur des cellules sanguines dans des dosages sérologiques WO2022010806A1 (fr)

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