Methods and reagents for reducing interference from drugs that bind CD47 in serological assays

Abstract

Provided are methods for reducing and / or preventing interference of a drug comprising (i) an antibody Fc region and (ii) a moiety that binds to human CD47 in a serological assay.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 858,871, filed on June 7, 2019, and U.S. Provisional Application No. 62 / 934,395, filed on November 12, 2019, the contents of each of which are incorporated herein by reference in their entirety.

[0003] Submission of sequence listings as ASCII text files

[0004] The contents of the following submission regarding an ASCII text file are incorporated herein by reference in their entirety: Computer Readable Form (CRF) of a Sequence Listing (File Name: 757972000940SEQL IST.TXT, Record Date: June 3, 2020, Size: 26KB). Technical Field

[0005] The present invention relates to methods and reagents for reducing interference of a drug comprising (i) an antibody Fc region and (ii) a moiety that binds to human CD47 in a serological assay. Background Art

[0006] An increasing number of antibody-based drugs are being developed as treatments for various diseases, including cancer. If the targets of therapeutic antibodies are also expressed on blood cells such as red blood cells (RBCs), white blood cells (WBCs), and / or platelets, such treatments may interfere with blood typing and serological assays.

[0007] For example, CD47 (a ubiquitously expressed cell surface protein that binds to signal regulatory protein-α (SIRPα) 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 variety of malignancies, including hematological tumors and solid tumors. Elevated CD47 expression is also associated with aggressive disease (Willingham et al., Proc Natl Acad Sci USA (2012) 109(17):6662-6667). Several cancer therapies targeting CD47 (e.g., antibodies and fusion proteins containing the Fc region of the antibody) have been developed to block the SIRPα-CD47 interaction, thereby allowing macrophages to perform their phagocytic function to eliminate tumor cells.

[0008] Importantly, since CD47 is also expressed on surface blood cells such as red blood cells (RBCs) and platelets (Oldenborg et al., Science (2000) 288(5473):2051-2054), drugs comprising an antibody Fc region targeting CD47 may interfere with blood typing and serological tests. In addition, since patients receiving CD47-targeted drugs (e.g., for the treatment of cancer) often require blood transfusions to treat concurrent anemia and / or thrombocytopenia, interference of anti-CD47 drugs with serological and blood typing assays is an important patient safety issue. Therefore, there is a need in the art to develop methods and reagents to reduce interference of CD47-targeted drugs comprising an antibody Fc region with serological assays. Summary of the invention

[0009] A method for reducing drug interference in a serological assay using reagent red blood cells (RBCs) or reagent platelets is provided, the method comprising: (a) adding a drug neutralizer to a plasma sample from a subject who has received drug treatment, the drug neutralizer binding to the drug and preventing the drug from binding to the reagent RBCs or the reagent platelets; and (b) performing a serological assay of the plasma sample using the reagent RBCs or the reagent platelets after step (a), wherein the drug comprises (i) a human antibody Fc region or a variant thereof and (ii) a portion that binds to human CD47.

[0010] In some embodiments, the portion of the drug that binds to human CD47 comprises wild-type SIRPα, a SIRPα variant, or a fragment of the wild-type SIRPα or the SIRPα variant. In some embodiments, the portion of the drug that binds to human CD47 comprises a SIRPα variant, and wherein the SIRPα variant comprises one or more amino acid substitutions, insertions, deletions, N-terminal extensions, and / or C-terminal extensions relative to wild-type SIRPα. In some embodiments, the portion of the drug that binds to human CD47 comprises a fragment of a SIRPα variant, and wherein the fragment comprises the extracellular domain of the SIRPα variant. In some embodiments, the drug neutralizer is an anti-SIRPα antibody capable of binding to wild-type SIRPα, a SIRPα variant, or a fragment of the wild-type SIRPα or the SIRPα variant.

[0011] In some embodiments, the portion of the drug that binds to human CD47 comprises wild-type SIRPγ, a SIRPγ variant, or a fragment of the wild-type SIRPγ or the SIRPγ variant. In some embodiments, the portion of the drug that binds to human CD47 comprises a SIRPγ variant, and wherein the SIRPγ variant comprises one or more amino acid substitutions, insertions, deletions, N-terminal extensions, C-terminal extensions, or any combination of the foregoing relative to wild-type SIRPγ. In some embodiments, the portion of the drug that binds to human CD47 comprises a fragment of a SIRPγ variant, and wherein the fragment comprises the extracellular domain of the SIRPγ variant. In some embodiments, the drug neutralizer is an anti-SIRPγ antibody capable of binding to wild-type SIRPγ, a SIRPγ variant, or a fragment of the wild-type SIRPγ or the SIRPγ variant.

[0012] In some embodiments, the portion of the drug that binds to human CD47 comprises a SIRPβ variant or a fragment of the SIRPβ variant. In some embodiments, the portion of the drug that binds to human CD47 comprises a SIRPβ variant, and wherein the SIRPβ variant comprises one or more amino acid substitutions, insertions, deletions, N-terminal extensions, C-terminal extensions, or any combination of the foregoing relative to wild-type SIRPγ. In some embodiments, the portion of the drug that binds to human CD47 comprises a fragment of a SIRPβ variant, and wherein the fragment comprises the extracellular domain of the SIRPβ variant. In some embodiments, the drug neutralizer is an anti-SIRPβ antibody capable of binding to a SIRPγ variant or a fragment of the SIRPβ variant.

[0013] In some embodiments, the drug is an anti-CD47 antibody. In some embodiments, the drug neutralizer is an anti-idiotypic antibody that binds to the antigen binding portion of the anti-CD47 antibody.

[0014] In some embodiments, the drug neutralizer is a CD47 polypeptide that is capable of binding to a portion of the drug that binds to human CD47. In some embodiments, the CD47 polypeptide is a monomer, dimer or oligomer. In some embodiments, the CD47 polypeptide is human CD47, mouse CD47, rat CD47, rhesus CD47 or cynomolgus CD47. In some embodiments, the CD47 polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the CD47 polypeptide is a CD47 variant, which comprises one or more amino acid substitutions, insertions, deletions, N-terminal extensions or C-terminal extensions relative to wild-type CD47. In some embodiments, the CD47 variant comprises an amino acid sequence listed in any one of SEQ ID NO: 2-5.

[0015] In some embodiments, the drug neutralizer has an affinity for the drug that is greater than the affinity of the drug for human CD47. In some embodiments, the drug neutralizer is added to the plasma sample in an amount in molar excess relative to the amount of drug in the plasma sample.

[0016] Also provided is a method for reducing drug interference in a serological assay of a blood sample containing red blood cells (RBCs) and / or platelets, the method comprising: (a) adding an anti-SIRPα antibody to a blood sample from a subject who has received drug treatment; and (b) performing a serological assay of the blood sample after step (a), wherein the drug comprises (i) an antibody Fc region and (ii) an extracellular domain of wild-type SIRPα or a variant thereof that binds to human CD47, and wherein the anti-SIRPα antibody fragment replaces the drug that binds to CD47 on the surface of RBCs in the blood sample. In some embodiments, the anti-SIRPα antibody comprises (a) a heavy chain variable domain (V H ) and a light chain variable domain (V L ); (b) a heavy chain variable domain (V H ) and a light chain variable domain (V L ); or (c) a heavy chain variable domain (V H ) and a light chain variable domain (V L ). In some embodiments, the drug comprises a variant of the extracellular domain of wild-type SIRPα. In some embodiments, the variant comprises one or more amino acid substitutions, insertions, deletions, N-terminal extensions and / or C-terminal extensions relative to the extracellular domain of wild-type SIRPα.

[0017] In some embodiments, the anti-SIRPα antibody is added to the blood sample in an amount in molar excess relative to the amount of drug in the blood sample.

[0018] Also provided is a method for reducing drug interference in a serological assay using reagent red blood cells (RBCs), reagent platelets, or a combination thereof, the method comprising: (a) adding a cell binding agent to the reagent red blood cells (RBCs), reagent platelets, or a combination thereof, wherein the cell binding agent binds to human CD47 and does not comprise an antibody Fc region; (b) performing a serological assay of a plasma sample using the reagent red blood cells (RBCs), reagent platelets, or a combination thereof of step (a), wherein the plasma sample is from a subject who has received drug treatment, and wherein the drug comprises (i) an antibody Fc region and (ii) a portion that binds to human CD47.

[0019] In addition, a method for reducing drug interference in a serological assay using reagent red blood cells (RBCs), reagent platelets, or a combination thereof is provided, the method comprising: (a) adding a cell binding agent to a plasma sample from a subject who has received drug treatment, wherein the cell binding agent binds to human CD47 and does not contain an antibody Fc region; and (b) performing a serological assay of the plasma sample using the reagent red blood cells (RBCs), reagent platelets, or a combination thereof after step (a), wherein the drug comprises (i) an antibody Fc region and (ii) a portion that binds to human CD47.

[0020] Also provided is a method for reducing drug interference in a serological assay of a blood sample containing reagent red blood cells (RBCs), reagent platelets, or a combination thereof, the method comprising: (a) adding a cell binding agent that binds to human CD47 and does not comprise an antibody Fc region to a blood sample from a subject who has received drug treatment; and (b) performing a serological assay of the blood sample after step (a), wherein the drug comprises (i) an antibody Fc region and (ii) a portion that binds to human CD47.

[0021] In some embodiments, the cell-binding agent comprises a wild-type SIRPα, wild-type SIRPγ, or a fragment of the wild-type SIRPα or the wild-type SIRPγ that can bind to human CD47. In some embodiments, the cell-binding agent comprises a SIRPα variant that can bind to human CD47 or its CD47 binding fragment. In some embodiments, the SIRPα variant comprises one or more amino acid substitutions, insertions, deletions, N-terminal extensions, C-terminal extensions, or any combination of the foregoing relative to wild-type SIRPα. In some embodiments, the cell-binding agent comprises a SIRPβ variant that can bind to human CD47 or its CD47 binding fragment. In some embodiments, the SIRPβ variant comprises one or more amino acid substitutions, insertions, deletions, N-terminal extensions, C-terminal extensions, or any combination of the foregoing relative to wild-type SIRPβ. In some embodiments, the cell-binding agent comprises a SIRPγ variant that can bind to human CD47 or its CD47 binding fragment. In some embodiments, the SIRPγ variant comprises one or more amino acid substitutions, insertions, deletions, N-terminal extensions, C-terminal extensions, or any combination of the foregoing relative to wild-type SIRPγ. In some embodiments, the variant comprises the amino acid sequence of any one of SEQ ID NOs: 8-16.

[0022] In some embodiments, the cell binding agent comprises an antigen binding fragment of an anti-CD47 antibody. In some embodiments, the antigen binding fragment is Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv or a bispecific antibody.

[0023] In some embodiments, the cell-binding agent has a higher affinity for human CD47 than the drug has for human CD47. In some embodiments, the cell-binding agent is added to the reagent RBCs and / or reagent platelets in an amount in molar excess relative to the amount of drug in the plasma sample. In some embodiments, the cell-binding agent is added to the plasma sample in an amount in molar excess relative to the amount of drug in the plasma sample. In some embodiments, the SIRPα agent is added to the blood sample in an amount in molar excess relative to the amount of drug in the blood sample.

[0024] In some embodiments, the antibody Fc region of the drug is a human IgG Fc region or a variant thereof. In some embodiments, the human IgG Fc region is an IgG1, IgG2 or IgG4 Fc region, or a variant of an IgG1, IgG2 or IgG4 Fc region.

[0025] In some embodiments, the serological assay is an ABO / Rh typing assay. In some embodiments, the serological assay is an immediate centrifugation (IS) assay. In some embodiments, the serological assay is a direct antiglobulin (DAT) assay using a multispecific reagent for detecting IgG and complement C3. In some embodiments, the serological assay is a direct antiglobulin (DAT) assay using a monospecific reagent for detecting complement C3. In some embodiments, the serological assay is a PEG enhanced serological assay. In some embodiments, the serological assay is an eluate test performed after the DAT assay.

[0026] Also provided is a polypeptide comprising any one of SEQ ID NOs: 11-20.

[0027] All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are hereby incorporated by reference in their entirety to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1A A serological assay is shown in which a plasma sample obtained from a subject is mixed with reagent red blood cells (i.e., red blood cells ("RBC") known to express a specific cell surface antigen or group of cell surface antigens) to detect the presence of antibodies in the plasma sample that bind to the RBC surface antigen. Alternatively, such a serological assay can be performed using reagent platelets (i.e., platelets known to express a specific cell surface antigen or group of cell surface antigens) instead of reagent RBCs.

[0029] Figure 1B shows how the presence of a drug comprising (i) an antibody Fc region and (ii) a portion that binds human CD47 in a plasma sample interferes with Figure 1A Determination of.

[0030] Figure 1C A serological assay is shown in which a blood sample obtained from a subject is mixed with reagent plasma / antiserum (i.e., plasma or antiserum containing antibodies to a known RBC surface antigen or a known platelet surface antigen) in order to detect the presence of the antigen on the subject's RBCs and / or platelets.

[0031] Figure 1D shows how the presence of a drug comprising (i) an antibody Fc region and (ii) a portion that binds human CD47 in a blood sample interferes with Figure 1C Determination of.

[0032] Figure 2A method of reducing interference in a serological assay is shown, the method comprising adding a drug neutralizer to a plasma sample obtained from a subject who has been treated with a drug. Briefly, the drug neutralizer binds the drug in the plasma sample so that little or no free drug is available to bind to CD47 on the surface of a reagent RBC or a reagent platelet.

[0033] Figure 3A A method of reducing interference in a serological assay is shown, the method comprising adding an anti-SIRPα antibody to a plasma sample from a subject who has received a drug treatment. The anti-SIRPα antibody binds to the drug in the plasma sample so that little or no free drug is available to bind to CD47 on the surface of a reagent RBC or a reagent platelet.

[0034] Figure 3B A method for reducing interference in a serological assay is shown, the method comprising adding an anti-SIRPα antibody to a blood sample from a subject who has received drug treatment. The anti-SIRPα antibody displaces the drug bound to CD47 on the surface of the subject's RBCs and / or platelets, and minimizes the amount of drug-bound RBCs and / or drug-bound platelets in the patient's blood sample (or eliminates drug-bound RBCs and / or drug-bound platelets in the sample).

[0035] Figure 4A A method of reducing interference in a serological assay is shown, the method comprising adding a cell binding agent to a reagent RBC or a reagent platelet. Briefly, a cell binding agent that binds to human CD47 and does not contain an antibody Fc region binds to CD47 on the surface of the reagent RBC or the reagent platelet. Binding of the cell binding agent to the reagent RBC (or reagent platelet) prevents the drug from binding to the reagent RBC (or reagent platelet).

[0036] Figure 4B A method for reducing interference in a serological assay is shown, the method comprising adding a cell-binding agent to plasma from a subject who has received drug treatment. In brief, the cell-binding agent competes with the drug for binding to CD47 expressed on the surface of a reagent RBC or a reagent platelet, and minimizes the amount of drug-bound reagent RBC or drug-bound reagent platelets in the assay (or eliminates drug-bound reagent RBC and / or drug-bound reagent platelets in the assay).

[0037] Figure 4CA method of reducing interference in a serological assay is shown, the method comprising adding a cell-binding agent to a blood sample from a subject who has received drug treatment. In brief, the cell-binding agent competes with the drug for binding to CD47 expressed on the surface of the subject's RBCs and / or platelets, and minimizes (or eliminates) the amount of drug-bound RBCs and / or drug-bound platelets in the assay.

[0038] Figure 5A Shown are the results of a hemagglutination experiment conducted to determine whether Antibody A (an exemplary anti-SIRPα antibody that blocks the interaction between SIRPα and CD47) mitigates the interference of Drug A binding to CD47 on the surface of red blood cells.

[0039] Figure 5B Shown are the results of an experiment conducted to determine whether Antibody A displaces Drug A binding to CD47 on the surface of DLD-1 cells. DETAILED DESCRIPTION

[0040] Methods for mitigating interference in pre-transfusion serological assays

[0041] CD47 is a transmembrane protein that interacts with thrombospondin-1 (TSP-1) and several molecules on immune cells, including signal regulatory protein alpha (SIRPα). After binding to CD47, SIRPα initiates a signaling cascade that inhibits phagocytosis and prevents the phagocytic removal of healthy cells by the immune system. However, many cancers overexpress CD47 and escape phagocytic clearance. Therefore, drugs targeting CD47, such as anti-CD47 antibodies and fusion proteins comprising an antibody Fc region and a portion that binds to CD47, have important therapeutic implications. CD47 is also expressed on the surface of human red blood cells (RBCs) and platelets. Therefore, after administering to a subject a drug comprising (i) an antibody Fc region and (ii) a portion that binds to human CD47, the drug present in the subject's plasma or bound to the subject's RBCs and / or platelets may cause interference in conventional pre-transfusion serological assays.

[0042] For example, Figure 1AA serological assay is shown in which a plasma sample obtained from a subject is mixed with reagent RBCs or "reference RBCs" (i.e., RBCs known to express a particular cell surface antigen or group of cell surface antigens) or reagent platelets or "reference platelets" (i.e., platelets known to express a particular cell surface antigen or group of cell surface antigens) to detect the presence of antibodies in the plasma sample that bind to cell surface antigens known to be expressed on the reagent RBCs or reagent platelets. After the plasma sample is mixed with the reagent RBCs (or reagent platelets), anti-human globulin (AHG) is added, and if the plasma sample contains antibodies that bind to RBC surface antigens (or platelet surface antigens), agglutination (e.g., clotting) of the reagent RBCs (or reagent platelets) occurs. However, the presence of a drug that comprises (i) an antibody Fc region and (ii) a portion that binds to human CD47 in the subject's plasma may interfere with the assay and produce a false-positive result. As Figure 1B As shown, after the subject's plasma is mixed with the reagent RBC (or reagent platelets), the drug can bind to CD47 expressed on the surface of the reagent RBC (or reagent platelets). Adding AHG to the mixture results in agglutination of the reagent RBC (or reagent platelets).

[0043] Figure 1C A serological assay is shown in which a blood sample from a subject is mixed with reagent plasma / antiserum (i.e., plasma or antiserum containing antibodies to a known RBC surface antigen or a known platelet surface antigen) in order to detect the presence of the antigen on the subject's RBCs and / or platelets. After mixing the reagent plasma / antiserum and the sample from the subject, if the antigen recognized by the antibodies in the reagent plasma / antiserum is expressed on the subject's RBCs and / or platelets, the addition of AHG will result in agglutination. In a sample containing the subject's RBCs and / or platelets, the presence of a drug containing (i) an antibody Fc region and (ii) a portion that binds to human CD47 may interfere with the assay and produce a false positive result. As Figure 1D As shown, after AHG is added to a mixture comprising a subject's blood sample and reagent plasma / antiserum, the drug binding to CD47 on the subject's RBCs or platelets will cause agglutination.

[0044] The methods described below reduce (and, in some embodiments, eliminate) interference caused by drugs, i.e., Figure 1B and 1D shown.

[0045] A. Methods of using drug neutralizers to mitigate interference in pretransfusion serological assays

[0046] In some embodiments, the method comprises (a) adding a drug neutralizing agent that binds to the drug (i.e., binds to a portion of the drug that comprises a portion that binds to human CD47) to a plasma sample from a subject that has received drug treatment, and (b) performing a serological assay of the plasma sample after step (a) using reagent RBCs (i.e., RBCs known to express a particular cell surface antigen or group of cell surface antigens) and / or reagent platelets (i.e., platelets known to express a particular cell surface antigen or group of cell surface antigens), wherein the drug comprises (i) an antibody Fc region and (ii) a portion that binds to human CD47. Such embodiments are described herein. Figure 2 In general terms, Figure 2 As shown, the drug neutralizer binds to the drug in the subject's plasma sample (e.g., binds to the portion of the drug that binds to human CD47) and prevents the drug from binding to the reagent RBCs and / or reagent platelets. There is little or no free drug available to bind to CD47 on the surface of the reagent RBCs and / or reagent platelets. The interference (e.g., Figure 1B In some embodiments, the reagent RBC and / or the reagent platelet are also added to the reagent RBC and / or the reagent platelet before the serological determination is performed.

[0047] In some embodiments, the drug neutralizer is any agent that can bind to the drug and prevent the drug from binding to the reagent RBC and / or reagent platelets. In some embodiments, the drug neutralizer is added to the reagent RBC and / or reagent platelets (e.g., only to the reagent RBC and / or reagent platelets) before performing a serological assay.

[0048] In some embodiments, the method is performed in a solution, for example, where the drug neutralizer is soluble. In some embodiments, the drug neutralizer is fixed to a solid phase by adsorption to a matrix or surface, covalent coupling, or non-covalent coupling prior to performing the method. In some embodiments, the drug neutralizer is capable of binding to the drug after being fixed to the solid phase. The solid phase or surface used for fixation can be any inert support or carrier that is substantially insoluble in water and can be used in an immunoassay, including, for example, a surface, a particle, a porous matrix, a cellulose polymer sponge ( Phadia) and the like. Examples of commonly used supports include small pieces made of polyethylene, polypropylene, polystyrene, etc., Sephadex, polyvinyl chloride, plastic beads, microparticles, assay plates or test tubes. In some embodiments, the drug neutralizer is coated on a microtiter plate, such as a multi-well microtiter plate that can be used to analyze multiple samples simultaneously.

[0049] In some embodiments, the drug comprises (i) an antibody Fc region and (ii) a SIRPα variant, a SIRPβ variant, or a SIRPγ variant that binds to human CD47, and the drug neutralizer comprises a CD47 polypeptide that can bind to the SIRPα variant, SIRPβ variant, or SIRPγ variant. In some embodiments, the drug comprises an anti-CD47 antibody (e.g., an anti-human CD47 antibody), and the drug neutralizer comprises a CD47 polypeptide that can bind to an anti-human CD47 antibody. In some embodiments, the CD47 polypeptide comprises an extracellular domain of wild-type CD47 ("WT CD47-ECD"), or a portion of WT CD47-ECD that can bind to a drug and prevent the drug from binding to a reagent RBC and / or a reagent platelet. In some embodiments, the CD47 polypeptide comprises a CD47 monomer, a CD47 dimer, or a CD47 oligomer. In some embodiments, a CD47 oligomer is an oligomer that is spontaneously formed (e.g., in vitro). In some embodiments, the CD47 oligomer is an engineered oligomer, for example, a linking chain of CD47 polypeptides connected by peptide bonds or linkers, CD47 that has been engineered to contain a domain that promotes multimerization, or CD47 that has been engineered to contain a tag that promotes binding of CD47 to a solid support (e.g., beads, glass edges, etc.). In some embodiments, the CD47 polypeptide comprises a fusion polypeptide, for example, a fusion polypeptide comprising CD47 (or a fragment thereof). In some embodiments, the fusion polypeptide comprises CD47 (or a fragment thereof) and an antibody Fc region. In some embodiments, the CD47 polypeptide comprises human CD47, mouse CD47, rat CD47, rhesus CD47, cynomolgus CD47, or CD47 of any origin that is capable of binding to a drug and preventing the drug from binding to a reagent RBC and / or a reagent platelet. In some embodiments, the CD47 polypeptide comprises a fragment of human CD47, mouse CD47, rat CD47, rhesus CD47, cynomolgus CD47 or CD47 from any source, provided that the fragment is capable of binding to the drug and preventing the drug from binding to the reagent RBC and / or the reagent platelet. In some embodiments, the CD47 polypeptide is a variant of wild-type CD47 (or a fragment thereof, such as a variant of WT CD47-ECD), provided that the variant is capable of binding to the drug. In some embodiments, the variant (or its fragment) comprises one or more amino acid substitutions, deletions, insertions, N-terminal additions and / or C-terminal additions relative to wild-type CD47 (e.g., wild-type human, rat, mouse, rhesus or cynomolgus CD47). In some embodiments, one or more amino acid substitutions, deletions, insertions, N-terminal additions and / or C-terminal additions present in the variant (i.e., "CD47 variant") change the glycosylation pattern of the CD47 variant relative to wild-type CD47 (e.g., wild-type human, rat, mouse, rhesus or cynomolgus CD47).In some embodiments, one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions present in the CD47 variant increase the affinity of the CD47 variant for the drug relative to wild-type CD47 (e.g., wild-type human, rat, mouse, rhesus monkey, or cynomolgus monkey CD47).

[0050] In some embodiments, the CD47 variant comprises the amino acid sequence of any one of the following SEQ ID NOs: 1-5:

[0051]

[0052] Additional details regarding exemplary CD47 variants that can be used as drug neutralizers in the methods described herein are provided in Ho et al. (2015) "'Velcro' Engineering of High Affinity CD47 Ectodomain as SignalRegulatory Protein a (SIRPα) Antagonists That Enhance Antibody-Dependent Cellular Phagocytosis." J Biol Chem. 290: 12650-12663 and WO 2016 / 179399, the contents of which are incorporated herein by reference in their entirety.

[0053] In some embodiments, the drug comprises (i) an antibody Fc region and (ii) a SIRPα variant that binds to human CD47, and the drug neutralizer is an anti-SIRPα antibody (or an antigen-binding fragment thereof). In some embodiments, the method comprises (a) adding an anti-SIRPα antibody (or an antigen-binding fragment thereof) to a plasma sample from a subject receiving drug treatment, and (b) performing a serological assay of the plasma sample using reagent RBCs and / or reagent platelets after step (a). Figure 3A As shown, the anti-SIRPα antibody (or antigen-binding fragment thereof) binds to free drug present in the plasma of the subject and reduces (or in some embodiments, eliminates) the amount of free drug available for CD47 on the surface of the reagent RBC and / or reagent platelets. The binding of the drug to the reagent RBC and / or reagent platelets will cause interference (such as Figure 1BIn some embodiments, the presence of an antibody (or antigen binding fragment thereof) is minimized (or, in some embodiments, eliminated), thereby preventing false positive results in serological assays. In some embodiments, an anti-SIRPα antibody (or an antigen binding fragment thereof) is added to the reagent RBCs and / or reagent platelets and the subject's plasma before performing a serological assay. Since the extracellular domains of SIRPα, SIRPβ, and SIRPγ are highly homologous, anti-SIRPβ antibodies that cross-react with SIRPα and / or anti-SIRPγ antibodies may cross-react with SIRPα. Therefore, in some embodiments, anti-SIRPβ antibodies and / or anti-SIRPγ antibodies that cross-react with SIRPα are used in this method.

[0054] In some embodiments, the method comprises (a) adding an anti-SIRPα antibody (or an antigen-binding fragment thereof) to a blood sample from a subject receiving drug treatment, and (b) performing a serological assay of the blood sample using reagent plasma and / or antiserum after step (a). Figure 3B As generally depicted in , an anti-SIRPα antibody (or fragment thereof) displaces a drug that binds to CD47 on the surface of a subject's RBCs and / or platelets, thereby reducing (and in some embodiments eliminating) interference caused by the drug, e.g., Figure 1D As shown. In some embodiments, before performing a serological assay, an anti-SIRPα antibody (or a fragment thereof) is added to the reagent plasma / antiserum and the subject's RBC and / or platelets. Since the extracellular domains of SIRPα, SIRPβ, and SIRPγ are highly homologous, anti-SIRPβ antibodies and / or anti-SIRPγ antibodies may cross-react with SIRPα. Therefore, in some embodiments, an anti-SIRPβ antibody that cross-reacts with SIRPα and / or an anti-SIRPγ antibody that cross-reacts with SIRPα is used in the method. In some embodiments, an anti-SIRPα antibody (or an anti-SIRPβ antibody that cross-reacts with SIRPα or an anti-SIRPγ antibody that cross-reacts with SIRPα) comprises an anti-human globulin reagent (AHG) that does not bind to a serological assay. Further details on serological assays and reagents used in such assays are provided elsewhere herein.

[0055] In some embodiments, the anti-SIRPα antibody (e.g., an anti-SIRPβ antibody that cross-reacts with SIRPα and / or an anti-SIRPγ antibody that cross-reacts with SIRPα) is a full-length antibody. In some embodiments, the antigen-binding fragment of the anti-SIRPα antibody is, for example, but not limited to, Fab, Fab', F(ab')2, Fab'-SH, Fv, bispecific antibody, one-arm antibody, scFv, scFv-Fc, single domain antibody, single heavy chain antibody, etc. In some embodiments, the anti-SIRPα antibody (or its antigen-binding fragment) is an ADA (anti-drug antibody) or NAb (neutralizing antibody) that binds to a drug (i.e., a portion of the drug that contains a SIRPα variant). In some embodiments, the anti-SIRPα antibody (or its antigen-binding fragment) comprises a heavy chain variable domain (VH), the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 6; and a light chain variable domain (VL), the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-SIRPα antibody (or its antigen-binding fragment) comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 21; and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-SIRPα antibody (or its antigen-binding fragment) comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 23; and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 24.

[0056]

[0057] Other exemplary anti-SIRP antibodies (e.g., anti-SIRPα antibodies, anti-SIRPβ antibodies, and / or anti-SIRPγ antibodies that cross-react with SIRPα) that can be used as drug neutralizers in the methods described herein are known in the art. Further details about such antibodies are provided in, for example, WO 2018 / 057669; US-2018-0105600-A1; US20180312587; WO2018107058; WO2019023347; US20180037652; WO2018210795; WO2017178653; WO2018149938; WO2017068164; and WO2016063233, the contents of which are incorporated herein by reference in their entirety.

[0058] In some embodiments, the drug comprises (i) an antibody Fc region and (ii) a SIRPβ variant that binds to human CD47, and the drug neutralizer is an anti-SIRPβ antibody (or an antigen-binding fragment thereof). In some embodiments, the method comprises (a) adding an anti-SIRPβ antibody (or an antigen-binding fragment thereof) to a plasma sample from a subject receiving drug treatment, and (b) performing a serological assay of the plasma sample using a reagent RBC and / or a reagent platelet after step (a). In some embodiments, the anti-SIRPβ antibody (or an antigen-binding fragment thereof) is added to the reagent RBC and / or the reagent platelet and the subject's plasma before performing a serological assay. In some embodiments, the anti-SIRPβ antibody is a full-length antibody. In some embodiments, the antigen-binding fragment of the anti-SIRPβ antibody is, for example, but not limited to, Fab, Fab', F(ab')2, Fab'-SH, Fv, bispecific antibody, one-armed antibody, scFv, scFv-Fc, single domain antibody, single heavy chain antibody, etc. Since the extracellular domains of SIRPα, SIRPβ and SIRPγ are highly homologous, anti-SIRPα antibodies and / or anti-SIRPγ antibodies can cross-react with SIRPβ. Therefore, in some embodiments, anti-SIRPα antibodies and / or anti-SIRPγ antibodies that cross-react with SIRPβ are used in the method.

[0059] In some embodiments, the drug comprises (i) an antibody Fc region and (ii) a SIRPγ variant that binds to human CD47, and the drug neutralizer is an anti-SIRPγ antibody (or an antigen-binding fragment thereof). In some embodiments, the method comprises (a) adding an anti-SIRPγ antibody (or an antigen-binding fragment thereof) to a plasma sample from a subject receiving drug treatment, and (b) performing a serological assay of the plasma sample using a reagent RBC and / or a reagent platelet after step (a). In some embodiments, an anti-SIRPγ antibody (or an antigen-binding fragment thereof) is added to a reagent RBC and / or a reagent platelet and the subject's plasma before a serological assay is performed. In some embodiments, the anti-SIRPγ antibody is a full-length antibody. In some embodiments, the antigen-binding fragment of an anti-SIRPγ antibody is, for example, but not limited to, Fab, Fab', F(ab')2, Fab'-SH, Fv, bispecific antibody, one-armed antibody, scFv, scFv-Fc, single domain antibody, single heavy chain antibody, etc. Since the extracellular domains of SIRPα, SIRPβ and SIRPγ are highly homologous, anti-SIRPβ antibodies and / or anti-SIRPα antibodies can cross-react with SIRPγ. Therefore, in some embodiments, anti-SIRPα antibodies cross-reacting with SIRPγ and / or anti-SIRPβ antibodies cross-reacting with SIRPγ are used in the method.

[0060] In some embodiments, the drug comprises an anti-CD47 antibody, and the drug neutralizer is an anti-idiotypic antibody (or an antigen-binding fragment thereof) that binds to the antigen-binding portion of the anti-CD47 antibody. In some embodiments, the method comprises (a) adding the anti-idiotypic antibody (or an antigen-binding fragment thereof) to a plasma sample from a subject receiving anti-CD47 antibody treatment, and (b) performing a serological assay of the plasma sample using reagent RBCs and / or reagent platelets after step (a). In some embodiments, the anti-idiotypic antibody (or an antigen-binding fragment thereof) is added to the reagent RBCs and / or reagent platelets and the subject's plasma before performing a serological assay. In some embodiments, the anti-idiotypic antibody is a full-length antibody. In some embodiments, the antigen-binding fragment of the anti-idiotypic antibody is, for example, but not limited to, Fab, Fab', F(ab')2, Fab'-SH, Fv, bispecific antibody, one-armed antibody, scFv, scFv-Fc, single domain antibody, single heavy chain antibody, etc.

[0061] In some embodiments, the drug has an affinity for the drug neutralizer that is greater than the affinity of the drug for human CD47 (e.g., human CD47 expressed on surface reagent RBCs) for the drug. In some embodiments, the drug neutralizer has an affinity for the drug that is at least about 10 times, 25 times, 50 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, or 1000 times greater than the affinity of human CD47 for the drug.

[0062] In some embodiments, the amount of drug neutralizer added to the subject's plasma, a sample containing the subject's RBCs and / or platelets, reagent plasma / antiserum, reagent RBCs, and / or reagent platelets is an amount sufficient to achieve an excess of drug neutralizer relative to the amount of drug in the subject's plasma or in the sample containing the subject's RBCs and / or platelets. In some embodiments, the amount of drug neutralizer added to the subject's plasma, a sample containing the subject's RBCs and / or platelets, reagent plasma / antiserum, reagent RBCs, and / or reagent platelets is an amount sufficient to bind substantially all (e.g., all) of the drug in the subject's plasma or in the sample containing the subject's RBCs and / or platelets. In some embodiments, the amount of drug neutralizer added to the subject's plasma, a sample comprising the subject's RBCs and / or platelets, reagent plasma / antiserum, reagent RBCs and / or platelets is sufficient to achieve an increase in the amount of the drug relative to the amount of the drug in the subject's plasma or in the sample comprising the subject's RBCs and / or platelets, for example, 2-fold, 5-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 400-fold, 500-fold, 600-fold, 750-fold, 800-fold, 900-fold, 1000-fold, 1500-fold, 2000-fold, 2500-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7500-fold, 8000-fold, 9000-fold, 10000-fold, 15000-fold, 20000-fold, 25000-fold, 30000-fold, 40000-fold, 50000-fold, 60000-fold, 75000-fold, 80000-fold, 90000-fold, 50000-fold, Any one of 350-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold, 1000-fold, 1500-fold, 2000-fold, 2500-fold, 3000-fold, 3500-fold, 4000-fold, 4500-fold or 5000-fold (including any ranges between these values) molar excess (such as molar ratio or equivalent) of the drug neutralizer.In some embodiments, the amount of the drug neutralizer added to the subject's plasma, sample comprising the subject's RBCs and / or platelets, reagent plasma / antiserum, reagent RBCs and / or reagent platelets is sufficient to achieve about 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, 1 mg / ml, 1.25 mg / ml, 1.5 mg / ml, 1.75 mg / ml, 2 mg / ml, 2.25 mg / ml, 2.5 mg / ml, 2.75 mg / ml, 3 mg / ml, 3.25 mg / ml, 3.5 mg / ml, 3.75 mg / ml, 4 mg / ml 4.25mg / ml, 4.5mg / ml, 4.75mg / ml, 5mg / ml, 10mg / ml, 20mg / ml, 30mg / ml, 40mg / ml, 50mg / ml, 60mg / ml, 70mg / ml, 80mg / ml, 90mg / ml, 100mg / ml, 150 mg / ml, 200mg / ml, 250mg / ml, 300mg / ml, 350mg / ml, 400mg / ml, 450mg / ml, 500mg / ml, 550mg / ml, 600mg / ml, 650mg / ml, 700mg / ml, 750mg / ml, 800mg / The amount of the drug neutralizer at a concentration of any one of 150 mg / ml, 1600 mg / ml, 1650 mg / ml, 1700 mg / ml, 1750 mg / ml, 1800 mg / ml, 1850 mg / ml, 1900 mg / ml or 2000 mg / ml, including any ranges between these values.In some embodiments, at least about 5 μg, 10 μg, 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 1.25 mg, 1.5 mg, 1.75 mg, 2 mg, 2.25 mg, 2.5 mg, 2.75 mg, 3 mg, 3.25 mg, 3.5 mg, 3.75 mg, 4 mg, 4.25 mg, 4.5 mg, 4.75 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 1000mg, 1100mg, 1150mg, 1200mg, 1250mg, 1300mg, 1350mg, 1400mg, 1450mg, 1500mg, 1550mg, 1600mg, 1650mg, 1700mg, 1750mg, 1800mg, 1850mg, 1900mg or 2000mg of a drug neutralizer is added to the subject's plasma, a sample comprising the subject's RBCs and / or platelets, reagent plasma / antiserum, reagent RBCs and / or reagent platelets. In some embodiments, the drug neutralizer is added to achieve a K of the drug for human CD47. D In some embodiments, the drug neutralizing agent is added to the subject's plasma, a sample comprising the subject's RBCs and / or platelets, a reagent plasma / antiserum, and / or a reagent RBC in an amount that is at least about 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold excess of the drug. D at least about 500 times, 1000 times, 5000 times, 10 4 times, 10 5 times, 10 6 times, 10 7 times, 10 8 times, 10 9 times or 10 10In some embodiments, the drug neutralizer is added to the subject's plasma, a sample containing the subject's RBCs and / or platelets, a reagent plasma / antiserum, and / or a reagent RBC in an excess of any one of 5 to 10 times (including any range between these values). D Any of about 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold excess (including any ranges between these values) of the drug neutralizer is added to the subject's plasma, a sample comprising the subject's RBCs and / or platelets, a reagent plasma / antiserum, and / or a reagent RBC. In some embodiments, the drug neutralizer is added to achieve a K of the drug neutralizer to the drug. D About 500 times, 1000 times, 5000 times, 10 4 times, 10 5 times, 10 6 times, 10 7 times, 10 8 times, 10 9 times or 10 10 An amount any one of 5 to 10 times (including any range between these values) in excess is added to the subject's plasma, a sample containing the subject's RBCs and / or platelets, reagent plasma / antiserum and / or reagent RBCs.

[0063] In some embodiments, the drug neutralizing agent is any agent capable of binding to the Fc region of the drug. In some embodiments, the drug neutralizing agent is SP-A (surfactant protein A) or SP-D (surfactant protein D).

[0064] In some embodiments, the method includes the use of two or more drug neutralizing agents described herein. For example, in some embodiments, the methods provided herein include adding, for example, two or more different CD47 variants (or fragments thereof capable of binding to a drug), two or more different anti-SIRPα antibodies (or antigen-binding fragments thereof), two or more different anti-SIRPβ antibodies (or antigen-binding fragments thereof), two or more different anti-SIRPγ antibodies (or antigen-binding fragments thereof), two or more different anti-idiotypic anti-CD47 antibodies (or antigen-binding fragments thereof), or two or more agents that bind the Fc region of a drug to a plasma sample from a subject who has received drug treatment. In some embodiments, the methods provided herein include adding a combination of different drug neutralizing agents, such as a soluble CD47 agent and / or an anti-SIRPα antibody (or an antigen-binding fragment thereof), and / or an anti-SIRPβ antibody (or an antigen-binding fragment thereof), and / or an anti-SIRPβ2 antibody (or an antigen-binding fragment thereof), and / or an anti-SIRPγ antibody (or an antigen-binding fragment thereof), and / or an anti-idiotypic anti-CD47 antibody (or an antigen-binding fragment thereof), and / or a combination of agents that bind the Fc region of the drug to a plasma sample from a subject who has received drug treatment. In some embodiments, the method includes adding two or more different anti-SIRPγ antibodies (or antigen-binding fragments thereof) to a subject's plasma sample, a sample containing a subject's RBCs and / or platelets, reagent plasma / antiserum, reagent RBCs, and / or reagent platelets.

[0065] B. Methods for Mitigating Interference Using Cell-Binding Agents in Pretransfusion Serology Assays

[0066] In some embodiments, the method comprises (a) adding a cell-binding agent that binds human CD47 and does not comprise an antibody Fc region to reagent RBCs (i.e., RBCs known to express a particular cell surface antigen or group of cell surface antigens) and / or reagent platelets (i.e., platelets known to express a particular cell surface antigen or group of cell surface antigens), and (b) performing a serological assay of a plasma sample using the reagent RBCs and / or reagent platelets after step (a), wherein the plasma sample is from a subject who has received a drug treatment, and wherein the drug comprises (i) an antibody Fc region and (ii) a portion that binds to human CD47. Such embodiments are described in Figure 4A In general terms, Figure 4A As shown, the cell-binding agent binds to CD47 expressed on the surface of the reagent RBC (and / or reagent platelets) and prevents the drug from binding to the reagent RBC (and / or reagent platelets), thereby minimizing (or, in some embodiments, eliminating) interference (such as Figure 1BIn some embodiments, a cell-binding agent is added to the subject's plasma and reagent RBCs and / or reagent platelets prior to performing a serological assay.

[0067] In some embodiments, the method comprises (a) adding a cell-binding agent that binds to human CD47 and does not comprise an antibody Fc region to a plasma sample from a subject that has received a drug treatment, and (b) performing a serological assay of the plasma sample using reagent RBCs and / or reagent platelets after step (a), wherein the drug comprises (i) an antibody Fc region and (ii) a portion that binds to human CD47. Figure 4B As shown, the cell-binding agent competes with the drug for binding to CD47 expressed on the surface of the reagent RBCs (and / or reagent platelets), thereby minimizing (or, in some embodiments, eliminating) interference that would result from binding of the drug to the reagent RBCs and / or reagent platelets (e.g., Figure 1B shown).

[0068] In some embodiments, the method comprises (a) adding a cell-binding agent that binds to human CD47 and does not comprise an antibody Fc region to a blood sample from a subject who has received drug treatment, and (b) performing a serological assay of the blood sample using reagent plasma / antiserum after step (a), wherein the drug comprises (i) an antibody Fc region and (ii) a portion that binds to human CD47. Figure 4C As shown, the cell-binding agent competes with the drug for binding to CD47 expressed on the surface of the subject's RBCs and / or platelets, thereby minimizing (or, in some embodiments, eliminating) interference that would result from binding of the drug to the subject's RBCs and / or platelets (e.g., Figure 1D In some embodiments, the cell-binding agent is added to the reagent plasma / antiserum and blood sample from the subject prior to performing the serological assay.

[0069] In some embodiments, the cell-binding agent is any agent that binds to CD47 and prevents the drug from binding to CD47. As described above, the cell-binding agent does not comprise an antibody Fc region. In alternative embodiments, the cell-binding agent comprises an antibody Fc region variant that does not bind AHG.

[0070] In some embodiments, the method is performed in solution, for example, where the cell binding agent is soluble. In some embodiments, the cell binding agent is fixed to a solid phase by adsorption to a matrix or surface, covalent coupling, or non-covalent coupling prior to performing the method. In some embodiments, the cell binding agent is capable of binding CD47 after being fixed to the solid phase. The solid phase or surface used for fixation can be any inert support or carrier that is substantially insoluble in water and can be used in an immunoassay, including, for example, a surface, a particle, a porous matrix, a cellulose polymer sponge ( Phadia) or the like. Examples of commonly used supports include small pieces made of polyethylene, polypropylene, polystyrene, etc., Sephadex, polyvinyl chloride, plastic beads, microparticles, assay plates or test tubes. In some embodiments, the cell binding agent is coated on a microtiter plate, such as a multi-well microtiter plate that can be used to analyze multiple samples simultaneously.

[0071] In some embodiments, the cell-binding agent comprises SIRPα or SIRPγ. In some embodiments, the cell-binding agent comprises a fragment of SIRPα or SIRPγ that can bind to CD47 (e.g., the extracellular domain of wild-type SIRPα ("WT SIRPα-ECD") or its D1 domain, for example, the extracellular domain of wild-type SIRPγ ("WT SIRPγ-ECD") or its D1 domain). In some embodiments, the cell-binding agent comprises human SIRPα, human SIRPγ, mouse SIRPα, mouse SIRPγ, rat SIRPα, rat SIRPγ, rhesus SIRPα, rhesus SIRPγ, cynomolgus SIRPα, cynomolgus SIRPγ, SIRPα of any origin, or SIRPγ of any origin, provided that SIRPα or SIRPγ can bind to CD47 (e.g., human CD47 expressed on the surface of reagent RBCs and / or reagent platelets). In some embodiments, the cell-binding agent comprises human SIRPα, human SIRPγ, mouse SIRPα, mouse SIRPγ, rat SIRPα, rat SIRPγ, rhesus monkey SIRPα, rhesus monkey SIRPγ, cynomolgus monkey SIRPα, cynomolgus monkey SIRPγ, SIRPα of any origin, or a fragment of SIRPγ of any origin, provided that the fragment is capable of binding to CD47 (e.g., human CD47 expressed on the surface of reagent RBCs and / or reagent platelets). In some embodiments, the cell-binding agent is a SIRPα variant (or a fragment thereof, such as a variant of WT SIRPα-ECD or its D1 domain), a soluble SIRPβ variant (or a fragment thereof, such as a variant of WT SIRPβ-ECD or its D1 domain), or a soluble SIRPγ variant (or a fragment thereof, such as a variant of WT SIRPγ-ECD or its D1 domain) capable of binding to human CD47. In some embodiments, SIRPα variant, SIRPβ variant or SIRPγ variant (or a fragment of any of the foregoing capable of binding to CD47) comprises one or more amino acid substitutions, deletions, insertions, N-terminal additions and / or C-terminal additions relative to wild-type SIRPα, wild-type SIRPβ or wild-type SIRPγ, respectively. In some embodiments, one or more amino acid substitutions, deletions, insertions, N-terminal additions and / or C-terminal additions present in SIRPα variant, SIRPβ variant or SIRPγ variant (or a fragment of any of the foregoing capable of binding to CD47) change the glycosylation pattern of SIRPα variant, soluble SIRPβ variant or soluble SIRPγ variant relative to wild-type SIRPα, wild-type SIRPβ or wild-type SIRPγ, respectively.In some embodiments, one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions present in a SIRPα variant, a SIRPβ variant, or a SIRPγ variant (or a fragment of any of the foregoing capable of binding to CD47) increase the affinity of the SIRPα variant, SIRPβ variant, or SIRPγ variant for human CD47 relative to wild-type SIRPα, wild-type SIRPβ, or wild-type SIRPγ, respectively. In some embodiments, the cell-binding agent is a monomer (e.g., a wild-type SIRPα monomer, a wild-type SIRPγ monomer, a SIRPα variant monomer, a SIRPβ variant monomer, a SIRPγ monomer, or a fragment of any of the foregoing capable of binding to CD47). In some embodiments, the cell-binding agent is a dimer (e.g., a homodimer or heterodimer comprising any combination of, for example, wild-type SIRPα, wild-type SIRPγ, a SIRPα variant, a SIRPβ variant, a SIRPγ variant, or a fragment of any of the foregoing capable of binding to CD47). In some embodiments, the cell-binding agent is an oligomer (e.g., a homo-oligomer or hetero-oligomer comprising, for example, one or more wild-type SIRPα, wild-type SIRPγ, SIRPα variants, SIRPβ variants, SIRPγ variants, and / or any combination of fragments of any of the foregoing that are capable of binding to CD47.

[0072] In some embodiments, the cell-binding agent comprises a SIRPα variant, a SIRPβ variant, or a SIRPγ variant. In some embodiments, the SIRPα variant, the SIRPβ variant, or the SIRPγ variant comprises the amino acid sequence of any one of the following SEQ ID NOs: 8-16.

[0073]

[0074] Other exemplary soluble SIRPα variants, soluble SIRPβ variants, and SIRPγ 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 entirety.

[0075] In some embodiments, the cell binding agent is a fragment of an anti-CD47 antibody that does not include an Fc region. In some embodiments, such fragments include, but are not limited to, for example, Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, or bispecific antibodies. Exemplary anti-CD47 antibody fragments that can be used with the methods provided herein include, but are not limited to, for example, mouse 5F9 (see Liu et al. (2015) PLoS One.10(9):e0137345), Hu5F9-G4 (Forty Seven, Inc.), B6H12.2, CC2C6, 8H7, BRIC 126, and others. In some embodiments, the anti-CD47 antibody includes an Fc region that does not bind AHG.

[0076] In some embodiments, the affinity of the cell-binding agent for human CD47 is higher than the affinity of the drug for human CD47 (e.g., human CD47 expressed on surface reagent RBCs and / or platelets). In some embodiments, the affinity of the cell-binding agent for human CD47 is at least about 10 times, 25 times, 50 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, or 1000 times the affinity of the drug for CD47 (including any ranges between these values).

[0077] In some embodiments, the amount of cell-binding agent added to the subject's plasma, sample containing the subject's RBC / platelets, reagent plasma / antiserum, reagent RBCs, and / or reagent platelets is an amount sufficient to achieve an excess amount of the cell-binding agent relative to the amount of CD47 expressed on the subject's RBC / platelets, reagent RBCs, and / or reagent platelets. In some embodiments, the amount of cell-binding agent added to the subject's plasma, sample containing the subject's RBC / platelets, reagent plasma / antiserum, reagent RBCs, and / or reagent platelets is an amount sufficient to bind substantially all (e.g., all) of the CD47 expressed on the subject's RBC / platelets, on the reagent RBCs, and / or reagent platelets. In some embodiments, the amount of cell-binding agent added to the subject's plasma, sample containing the subject's RBCs / platelets, reagent plasma / antiserum, reagent RBCs, and / or reagent platelets is sufficient to achieve an increase in the amount of CD47 expressed on the subject's RBCs / platelets, on reagent RBCs, and / or reagent platelets by about, for example, 2-fold, 5-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, or more relative to the amount of CD47 expressed on the subject's RBCs / platelets, on reagent RBCs, and / or reagent platelets. fold, 350-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold, 1000-fold, 1500-fold, 2000-fold, 2500-fold, 3000-fold, 3500-fold, 4000-fold, 4500-fold, or 5000-fold (including any ranges between these values) in molar excess (e.g., molar ratio or equivalent) of the cell-binding agent.In some embodiments, the amount of cell-binding agent added to the subject's plasma, sample containing the subject's RBCs / platelets, reagent plasma / antiserum, reagent RBCs, and / or reagent platelets is sufficient to achieve about 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, 1 mg / ml, 1.25 mg / ml, 1.5 mg / ml, 1.75 mg / ml. l, 2mg / ml, 2.25mg / ml, 2.5mg / ml, 2.75mg / ml, 3mg / ml, 3.25mg / ml, 3.5mg / ml, 3.75mg / ml, 4mg / ml, 4.25mg / ml, 4. 5mg / ml, 4.75mg / ml, 5mg / ml, 10mg / ml, 20mg / ml, 30mg / ml, 40mg / ml, 50mg / ml, 60mg / ml, 70mg / ml, 80mg / ml, 90mg / m l, 100mg / ml, 150mg / ml, 200mg / ml, 250mg / ml, 300mg / ml, 350mg / ml, 400mg / ml, 450mg / ml, 500mg / ml, 550mg / ml, 6 00mg / ml, 650mg / ml, 700mg / ml, 750mg / ml, 800mg / ml, 850mg / ml, 900mg / ml, 1000mg / ml, 1100mg / ml, 1150mg / ml, 12 In some embodiments, the present invention relates to an amount of the cell-binding agent to a concentration of at least 200 mg / ml, 1250 mg / ml, 1300 mg / ml, 1350 mg / ml, 1400 mg / ml, 1450 mg / ml, 1500 mg / ml, 1550 mg / ml, 1600 mg / ml, 1650 mg / ml, 1700 mg / ml, 1750 mg / ml, 1800 mg / ml, 1850 mg / ml, 1900 mg / ml or 2000 mg / ml, including any ranges between these values.In some embodiments, at least about 5 μg, 10 μg, 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 1.25 mg, 1.5 mg, 1.75 mg, 2 mg, 2.25 mg, 2.5 mg, 2.75 mg, 3 mg, 3.25 mg, 3.5 mg, 3.75 mg, 4 mg, 4.25 mg, 4.5 mg, 4.75 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg , 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 1000 mg, 1100 mg, 1150 mg, 1200 mg, 1250 mg, 1300 mg, 1350 mg, 1400 mg, 1450 mg, 1500 mg, 1550 mg, 1600 mg, 1650 mg, 1700 mg, 1750 mg, 1800 mg, 1850 mg, 1900 mg or 2000 mg of a cell binding agent is added to the subject's plasma, a sample containing the subject's RBCs, reagent plasma / antiserum and / or reagent RBCs. In some embodiments, the amount of cell-binding agent added to the subject's plasma, sample containing the subject's RBCs / platelets, reagent plasma / antiserum, reagent RBCs, and / or reagent platelets is the K of the drug for human CD47. D In some embodiments, the amount of cell-binding agent added to the subject's plasma, sample comprising the subject's RBCs / platelets, reagent plasma / antiserum, reagent RBCs, and / or reagent platelets is at least about 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold excess of the subject's plasma, sample comprising the subject's RBCs / platelets, reagent plasma / antiserum, reagent RBCs, and / or reagent platelets. D at least about 500 times, 1000 times, 5000 times, 10 4 times, 10 5 times, 10 6 times, 10 7 times, 10 8 times, 10 9 times or 10 10In some embodiments, the amount of cell-binding agent added to the subject's plasma, sample containing the subject's RBCs / platelets, reagent plasma / antiserum, reagent RBCs, and / or reagent platelets is a K of the cell-binding agent for human CD47. D In some embodiments, the amount of cell-binding agent added to the subject's plasma, a sample comprising the subject's RBCs / platelets, reagent plasma / antiserum, reagent RBCs, and / or reagent platelets is at least about 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold excess of the subject's plasma, a sample comprising the subject's RBCs / platelets, reagent plasma / antiserum, reagent RBCs, and / or reagent platelets. D at least about 500 times, 1000 times, 5000 times, 10 4 times, 10 5 times, 10 6 times, 10 7 times, 10 8 times, 10 9 times or 10 10 Any of the above values ​​(including any range between these values) is excessive.

[0078] C. Exemplary Drugs

[0079] The methods provided herein reduce (or, in some embodiments, eliminate) interference in serological assays caused by the presence of a drug that contains (i) an antibody Fc region and (ii) a portion that binds to human CD47 in a sample containing plasma or RBC / platelets obtained from a subject who has received drug treatment. In some embodiments, the drug contains an IgG Fc region, such as a human IgG Fc region, for example, an IgG1, IgG2, or IgG4 Fc region. In some embodiments, the drug contains a modified Fc region (such as a modified IgG Fc region) that contains one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions relative to a wild-type human Fc region (e.g., a wild-type human IgG Fc region). Exemplary 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.

[0080] In some embodiments, the portion that binds to human CD47 is a wild-type SIRPα lacking a transmembrane domain (e.g., an extracellular domain of any wild-type SIRPα that can bind to human CD47). In some embodiments, the portion that binds to human CD47 is a SIRPα variant that can bind to human CD47 and lacks a transmembrane domain. In some embodiments, the SIRPα variant comprises one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions relative to the extracellular domain of wild-type SIRPα. In some embodiments, the SIRPα variant is a SIRPα-d1 domain variant. In some embodiments, the SIRPα variant has a higher affinity for human CD47 than the affinity of wild-type SIRPα for human CD47.

[0081] In some embodiments, the portion that binds to human CD47 is a wild-type SIRPγ lacking a transmembrane domain (e.g., an extracellular domain of any wild-type SIRPγ that can bind to human CD47). In some embodiments, the portion that binds to human CD47 is a SIRPγ variant that can bind to human CD47 and lacks a transmembrane domain. In some embodiments, the SIRPγ variant comprises one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions relative to the extracellular domain of wild-type SIRPγ. In some embodiments, the SIRPγ variant is a SIRPγ-d1 domain variant. In some embodiments, the SIRPγ variant has a higher affinity for human CD47 than the affinity of wild-type SIRPγ for human CD47.

[0082] In some embodiments, the portion that binds to human CD47 is a SIRPβ variant that is capable of binding to human CD47 and lacks a transmembrane domain. In some embodiments, the SIRPγβ variant comprises one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions relative to the extracellular domain of wild-type SIRPβ. In some embodiments, the SIRPβ variant is a SIRPβ-d1 domain variant.

[0083] Exemplary SIRPα variants, SIRPβ variants, and SIRPγ variants are known in the art and 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 entirety.

[0084] In some embodiments of any of the methods described above, the drug is an anti-CD47 antibody. In some embodiments, the anti-CD47 antibody is AO-176, CC-90002, Hu5F9-G4 (also known as 5F9), SHR-1603, NI-1701, SRF231, TJC4, or IBI188. Detailed information about these and other therapeutic anti-CD47 antibodies is provided in WO2018175790A1; US20180142019; US20180171014; US20180057592; US20170283498; US9,518,116; US9,518,117; US20150274826; US20160137 733; US9,221,908; US20140161799; US20160137734; WO2015191861; WO2014093678; WO 2014123580; WO2013119714; US9,045,541; WO2016109415; WO2018183182; WO20180094 99; WO2017196793; US9663575; US20140140989; WO2018237168; US20180037652; US201 90023784; WO2018095428; EP3411071; WO2019042285; WO2016081423; WO2011076781; W O2012172521; WO2014087248; US20140303354; WO2016156537; US20160289727; US20190062428; US20180201677; US9352037; US20170044258; US9650441; and US20180105591.

[0085] Serological assays for pre-transfusion testing

[0086] Pre-transfusion testing is performed to ensure that the blood products intended for transfusion are compatible with the blood of the subject (i.e., transfusion recipient). Pre-transfusion testing covers serological determinations for confirming the ABO compatibility between donor blood and recipient blood, and serological determinations for detecting the most clinically significant RBC / platelet alloantibodies reacting with donor RBC and / or donor platelets (with reference to Technical Manual, 18th edition, AABB, Bethesda, MD, 2014). Other exemplary blood group antigens for serological determinations to determine donor / recipient transfusion compatibility include, but are not limited to, for example, 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 monkey blood group antigens, Dombrock blood group antigens, Landsteiner-Wiener blood group antigens, and VEL blood group antigens. The methods provided herein reduce or prevent drug interference in many serological assays known in the art (e.g., interference caused by a drug comprising (i) an antibody Fc region and (ii) a portion that binds to human CD47). Exemplary serological assays for which the methods may be used include, but are not limited to, those described in more detail below.

[0087] Typically, a sample containing, for example, non-hemolyzed blood, plasma (e.g., a plasma sample treated with an anticoagulant in EDTA), coagulated blood, or serum from a subject in need of a transfusion (e.g., a subject who has received a drug comprising (i) an antibody Fc region and (ii) a portion that binds to human CD47) is used. In general, the ABO blood type and Rh type of the subject are first determined. Next, an antibody screening method is used to detect any unexpected non-ABO blood type antibodies of clinical significance that may be present in the subject's plasma. If the screening test reveals the presence of such an antibody, an antibody identification panel is used to determine the specificity of the antibody. After determining the specificity of the antibody, a donor unit of the appropriate ABO blood type and Rh type is screened for the corresponding antigen. The antigen-negative unit is cross-matched with the subject in need of a transfusion to ensure compatibility.

[0088] Serological determination can be carried out in a test tube, on a glass slide, on a gel column or in a microtiter well plate, and hemolysis and agglutination are signals indicating a positive (incompatible) test result. Agglutination (a reaction reflecting the connection between adjacent RBCs coated with antibodies) can be scored macroscopically and / or microscopically and with a scale of 0-4+ in the most commonly used test tube method. Zero points indicate no reactivity, and are characterized by smooth and easily dispersed cells. 4+ scores indicate strong reactivity, and are characterized by a solid agglutinant that is not easily dispersed. 1+, 2+ or 3+ scores indicate a moderate level of reactivity, characterized by the gradual increase in the size of the agglutinant with a higher score. When using a gel column (gel card) containing anti-IgG antibodies in a column or a microtiter well plate (solid phase) containing a combined red blood cell antigen to carry out serological determination, similar agglutination scoring principles can be applied. Currently, there are multiple technologies that can be used to detect antibody-RBC antigen interactions with different sensitivities. In some embodiments, serological determination is performed manually. In some embodiments, serological assays are performed by automated machines.

[0089] For example, immediate centrifugation (IS) (also known as "immediate centrifugation crossmatch") is a direct agglutination assay that requires, for example, mixing reagent plasma / antiserum (i.e., plasma containing antibodies to known RBC and / or platelet surface antigens) and the subject's blood cells, immediately centrifuging the mixture at room temperature or 37°C for about 15-30 seconds, and visually inspecting the tube. Direct agglutination indicates that there is a strong interaction between the antibodies in the plasma and the RBC surface antigens. Alternatively, the subject's plasma and reagent RBCs (i.e., RBCs known to express a specific cell surface antigen or group of cell surface antigens) and / or reagent platelets (i.e., platelets known to express a specific cell surface antigen or group of cell surface antigens) can be mixed, centrifuged, and visually assessed for direct agglutination.

[0090] Antihuman globulin (AHG) is used to detect RBCs that are bound by antibodies that do not produce direct agglutination. AHG is a secondary antihuman globulin antibody produced in another species. AHG reagents can be specific for a single class of human Ig (e.g., IgG), or can be multispecific, i.e., able to bind to multiple human Ig classes (e.g., IgG, IgM, IgA) and complement. AHG serum can be used for direct antiglobulin testing (DAT) and / or indirect antiglobulin testing (IAT). The DAT demonstrates sensitization of red blood cells in vivo and is performed by directly testing a washed patient red blood cell sample with AHG. The IAT demonstrates in vitro reactions between red blood cells and antibodies. In the IAT, serum (or plasma) is incubated with red blood cells and then washed to remove unbound globulins. The presence of agglutination in the presence of AHG indicates binding of antibodies to specific red blood cell antigens. Some methods involve adding potentiator agents (enhancement) such as saline, albumin, low ionic strength saline (LISS), or polyethylene glycol (PEG) and then incubating the sample at 37°C for 10-60 minutes prior to AHG testing.

[0091] 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 typing). Testing the recipient's plasma for the presence of anti-A and anti-B antibodies using red blood cells of known A and B types (reverse typing) is also part of routine ABO blood typing.

[0092] The Rh(D) type of a transfusion recipient is determined by testing the recipient's red blood cells with anti-D. ABO typing is usually performed using an immediate centrifugation (IS) test.

[0093] Alloantibodies to antigens not present on an individual's red blood cells may develop in any person exposed to foreign red blood cell antigens through pregnancy or blood transfusion. To detect antibodies to antigens other than group A or group B, a patient's plasma or serum sample is tested against selected commercial type O red blood cells that express most of the clinically significant antigens other than A and B.

[0094] In the case of a positive antibody screen, further serological testing using an extended panel of commercial O-type reagent RBCs is required to identify clinically significant antibodies. Then, once the specificity of the antibody is known, donor units must be screened against the corresponding antigen to select those units that lack the antigen.

[0095] Antigenic typing (phenotyping) of the recipient's red blood cells may also be performed to determine which red blood cell antibodies the individual may produce. Serological tests for RBC phenotyping involve mixing the recipient cells with a commercial reagent antiserum containing specific antibodies.

[0096] IAT with or without enhancement (eg, saline, LISS, PEG) is used for antibody detection and antibody characterization.

[0097] "Crossmatching" refers to the process of confirming the compatibility between the patient's blood (plasma) and the donor's red blood cells. Crossmatching is primarily intended to detect and prevent ABO incompatibilities. Serum crossmatching assays (IS crossmatch or AHG phase crossmatch) involve mixing donor red blood cells directly with recipient plasma and scoring for hemolysis and agglutination after immediate centrifugation or AHG testing.

[0098] This description is considered sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and are within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0099] Example

[0100] Example 1: Drugs comprising (i) an antibody Fc region and (ii) a portion that binds to human CD47 interfere with conventional serological assays

[0101] CD47 is a ubiquitously expressed cell surface protein that binds to signal regulatory protein-α (SIRPα) and inhibits phagocytosis (Jaiswal et al., Trends Immunol (2010) 31(6):212-219; Brown et al., Trends Cell Biol (2001) 11(3):130-135). A variety of malignancies, including hematological and solid tumors, show elevated CD47 expression, which is associated with aggressive disease (Willingham et al., Proc Natl Acad Sci USA (2012) 109(17):6662-6667). Several cancer therapies targeting CD47 have been developed to block SIRPα-CD47 interactions, thereby allowing macrophages to perform their phagocytic function to clear tumor cells. Importantly, since CD47 is also expressed on surface blood cells such as red blood cells (RBCs) and platelets (Oldenborg et al., Science (2000) 288(5473):2051-2054), CD47 binding drugs may interfere with blood typing and serological testing.

[0102] For example, CD47-targeted drugs can interfere with blood typing and serological tests by blocking and / or causing steric interference on the surface of blood cells, including RBCs and platelets. In addition, anti-CD47 drugs containing an Fc region (such as anti-CD47 antibodies) can interact with reagents used in serological tests, resulting in hemagglutination and erroneous reactivity readings.

[0103] Drug A is used to determine the extent to which antibody-based drugs targeting CD47 interfere with blood typing and serological assays. Drug A is an exemplary CD47 binding drug that comprises a SIRPα variant (i.e., a CD47 binding domain derived from human SIRPα) and an Fc variant derived from the Fc region of human immunoglobulin IgG1.

[0104] Materials and methods

[0105] Detection of drug A interference in blood typing and serological assays

[0106] Drug A was mixed with 5 mL of blood samples of type A, B, and O donors at concentrations of 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, or 10 μg / mL. The mixture was incubated overnight at 2-8°C. The interference of Drug A on the following blood typing and serological tests was then determined using the test tube method according to the Bonfils reference laboratory procedure:

[0107] Blood typing

[0108] Donor blood samples incubated with Drug A (as described above) were recovered and typed for ABO and RhD using a slide test with reagent RBCs known to express specific cell surface antigens and agglutination was scored.

[0109] Antiimmunoglobulin and complement assays

[0110] Recover plasma samples from each donor blood sample incubated with drug A. Then use plasma samples for serological tests together with reagent RBC (Immucor). Immediate centrifugal crossmatching (or immediate centrifugal stage) is performed by mixing plasma samples with reagent RBC, centrifuging the mixture immediately and checking the centrifuged mixture to detect agglutination. Antibody tests are also performed on plasma samples using PEG enhancement (11.5% PEG w / v) and then adding anti-human globulin (AHG) reagent. Another set of antibody tests are performed on plasma samples using PEG enhancement (11.5% PEG w / v) and then adding AHG reagent to determine the effect of dithiothreitol (DTT), ficin and / or alloabsorption treatment on readings. Standard protocols are used for each assay, and automatic controls (i.e., where serum from donor samples containing drug A is mixed with RBC from donor samples containing drug A) are performed in parallel.

[0111] In addition, RBC samples were recovered from each donor blood sample incubated with drug A. The RBC samples were then used for direct antiglobulin testing (DAT), which requires combining RBCs with a multispecific AHG reagent (which binds IgG and complement) or an AHG reagent (which binds only to complement). In addition, antibodies bound to RBCs in each sample were eluted and tested with reagent RBCs, i.e., detection / identification of autoantibodies and / or alloantibodies. Standard protocols were used.

[0112] result

[0113] As shown in Table 1, at any concentration of drug A tested (up to 10 μg / mL), drug A did not interfere with ABO / RhD typing or antibody screening using immediate centrifugal crossmatch (IS). However, at the highest dose of 10 μg / mL, drug A did interfere with antibody screening using AHG reagent and PEG enhancement, showing a reactivity level of 3+. Antibody screening assays using AHG reagent and PEG enhancement were also performed using an automated control antibody, revealing that drug A increased the reactivity level in a dose-dependent manner (from weak reactivity at 0.1 μg / mL drug A to 4+ reactivity at 10 μg / mL drug A).

[0114] In the antibody screening assay using the AHG reagent and PEG enhancement, neither DTT nor ficin could resolve the reactivity caused by 10 μg / mL drug A. In contrast, the allosorption test resolved the reactivity caused by drug A in the antibody screening assay using PEG enhancement, indicating that interference of drug A on serological assays can be reduced or resolved by saturation binding of the reagent on blood cells.

[0115] DAT testing for IgG showed that drug A caused reactivity in a dose-dependent manner (from weak reactivity at 0.1 μg / mL drug A to 3+ reactivity at 10 μg / mL drug A). In contrast, no reactivity was observed during DAT testing for complement at any concentration of drug A tested (up to 10 μg / mL), indicating that drug A interference was caused by IgG antibody-antigen interactions rather than complement interactions. Moderate reactivity (2+ to 3+ reactivity) was observed in the eluate testing of all tested drug A concentrations.

[0116] Table 1: Effect of the presence of drug A on the reactivity of serological tests

[0117]

[0118] The results discussed above and presented in Table 1 indicate that Drug A interferes with conventional serological assays. This interference is likely due to binding of plasma Drug A to CD47 on blood cells, which is then detected by IgG-specific reagents commonly used in serological tests, such as AHG.

[0119] Example 2: Mitigating interference in serological tests caused by Fc-containing proteins

[0120] The results presented in Example 1 indicate that drugs comprising (i) an antibody Fc region and (ii) a portion that binds to human CD47 can interfere with routine blood typing and serological assays. Such interference may compromise patient safety by causing delays in the release of donor blood products to patients in need of transfusions. The ability of Reagent B, an exemplary drug neutralizer comprising a CD47 monomer, to prevent or reduce the interference of Drug A in serological tests was evaluated as described below.

[0121] Materials and methods

[0122] Drug A is mixed with 5mL donor whole blood with a concentration of 10 μg / mL, and incubated overnight at about 2 ℃-8 ℃. Each plasma sample is obtained from the donor whole blood doped with drug A and aliquoted. Then reagent B is added to the plasma sample with a ratio of 1:9 (9 drops of blood plasma and 1 drop of reagent B) using a concentration of 2.75mg / mL or 0.275mg / mL. The final concentration of reagent B in each plasma sample is 17.5μM or 1.75μM respectively. Plasma sample / reagent B mixture is incubated at room temperature for about 30 minutes. Then the plasma sample treated with reagent B is carried out to the serological test described in Example 1.

[0123] result

[0124] As shown in Table 2, reagent B at a concentration of 17.5 μM mitigated the interference of drug A (10 μg / mL) in the serological assay described in Example 1 (see Table 1). However, at a lower concentration of 1.7 μM, reagent B did not eliminate the reactivity observed with drug A (see Table 2).

[0125] Table 2: Neutralization of Drug A with CD47 Agent B to Resolve Reactivity

[0126]

[0127]

[0128] Therefore, the results discussed above indicate that CD47 agent B can neutralize drug A and mitigate its interference with serological tests.

[0129] Example 3. Use of Agent B (an exemplary soluble CD47 monomer) or Agent C (an exemplary soluble SIRPα monomer) to mitigate interference of drugs containing (i) an antibody Fc region and (ii) a portion that binds to human CD4 in conventional serological tests.

[0130] introduction

[0131] CD47 is a ubiquitously expressed cell surface protein that acts as a self-marker and provides a "don't eat me" signal to inhibit phagocytosis by binding to signal regulatory protein-α (SIRPα), its natural receptor on macrophages (Jaiswal et al., Trends Immunol (2010) 31(6):212-219; Brown et al., Trends Cell Biol (2001) 11(3):130-135). Tumor cells overexpress CD47 to evade immune surveillance (Willingham et al., Proc Natl Acad Sci USA (2012) 109(17):6662-6667). Abundant CD47 expression has been observed in a variety of malignancies, including hematological and solid tumors, with elevated CD47 expression correlating with aggressive disease and decreased likelihood of survival (Willingham et al., Proc Natl Acad Sci USA (2012) 109(17):6662-6667; Chao et al. Cell. (2010) 142(4):699-713).

[0132] CD47 is widely expressed on the surface of human RBCs (Oldenborg et al., Science (2000) 288(5473): 2051-2054). In the presence of circulating drug A after administration in a patient, it is possible that drug A from the recipient patient can bind to CD47 on the reagent or donor RBC. As discussed in Examples 1 and 2, drug A is an exemplary CD47 binding drug that comprises a SIRPα variant (i.e., a CD47 binding domain derived from human SIRPα) and an Fc variant derived from the Fc region of human immunoglobulin IgG1. Since drug A contains the Fc region of IgG1, its binding to CD47 on the surface of RBCs can be similar to an antibody-antigen interaction, resulting in interference with the determination of conventional blood typing and screening serological tests for pre-transfusion blood bank testing. Previous in vitro studies have shown that the presence of drug A in plasma does not interfere with ABO / Rh typing and antibody screening using immediate centrifugation. However, at a concentration of 10 μg / mL, drug A in plasma interferes with antibody screening using PEG enhancement.

[0133] The following experiments were performed to assess whether the interference induced by drug A at higher concentrations could be neutralized by the addition of CD47 monomers (i.e., agent B described in Example 2 and below) or high affinity SIRPα monomers (i.e., agent C, which will be described in more detail below). Correlation flow cytometry analysis was performed to assess whether the binding of drug A to RBCs could be abolished by the addition of soluble CD47 monomers (agent B) or soluble high affinity SIRPα monomers (agent C).

[0134] Materials and methods

[0135] Agent B and agent C are expressed using Expi293 expression system (Thermofisher) based on the scheme of manufacturers. Both constructs contain C-terminal His6 tag, and are purified by immobilized metal affinity chromatography (IMAC). All purifications are carried out using GE AktaAvant25 or Avant150. The IMAC resin used is Ni Sepharose6FastFlow (GE catalog number 17-5318-01). First, balance buffer (20mM Tris pH7, 500mM NaCl, 5mM imidazoles) is used to balance resin. The thick supernatant of the protein containing his tag by resin load. The balance buffer of resin with about 20-30 column volumes is rebalanced, and then the washing buffer (20mM Tris pH7, 500mM NaCl, 40mM imidazoles) of 20-30 column volumes is rebalanced. The protein was eluted with about 10 column volumes of elution buffer (20 mM Tris pH 7, 500 mM NaCl, 250 mM imidazole). The eluted protein was immediately improved by gel filtration and resuspended in 1xPBS (137 mM sodium chloride, 2.7 mM potassium chloride, 4.3 mM sodium phosphate (dibasic, anhydrous), 1.4 mM potassium phosphate (monobasic, anhydrous)).

[0136] Patient serum samples used in this study were collected as part of routine care in a hospital setting.

[0137] Anti-E is a frequently identified alloantibody of clinical significance. Pooled patient sera with positive anti-E results were obtained from the hospital transfusion service for research purposes.

[0138] The RBC reagent cells used in this study were obtained from Biorad ID-DiaCell I-II-III.

[0139] Experimental protocol

[0140] Column agglutination test (CAT) in anti-human globulin (AHG) phase using BioRad Antibody Screening Cells I and II

[0141] The potential of drug A to interfere with serological blood bank tests at concentrations up to 500 μg / mL was evaluated. Antibody screening was performed on serum doped with drug A using RBC reagent cells ID-DiaCell I-II (Biorad) by gel IAT (ID-Card LISS / Coombs, BIO-RAD). Specifically, 25 μL of serum doped with drug A (0.1, 1.0, 10.0, 100.0 and 500.0 μg / mL) and 50 μL of 0.8% reagent RBC suspended in low ionic strength saline (LISS) were added to LISS / Coombs cards. After incubation at 37°C for 15 minutes, the card was centrifuged at 1,030 rpm for 10 minutes. The intensity of agglutination was graded as 0 (no agglutination), 0.5+ (very weak agglutination), 1+ (weak agglutination), 2+ (moderate agglutination), 3+ (strong agglutination) or 4+ (very strong agglutination).

[0142] Flow cytometric analysis of drug A binding to RBCs

[0143] Flow cytometry was performed to measure the binding of drug A to the reagent RBCs and the reduction of binding by using agent B or agent C. 25 μL of serum doped with drug A (0.1, 1.0, 10.0, 100.0 and 500.0 μg / mL) or normal serum that was negative for unexpected antibody tests was added to 50 μL of reagent RBCs (ID-DiaCell I). 25 μL of serum doped with drug A (500.0 μg / mL) pre-incubated with 10, 30 and 50 times molar excess of agent B was added to 50 μL of test RBCs (ID-DiaCell I). 25 μL of serum doped with drug A (500.0 μg / mL) was added to 50 μL of reagent RBCs (ID-DiaCell I) pre-incubated with 10, 50, 100, 300 and 500 times molar excess of agent C. The mixture was incubated at 37°C for 15 minutes and then washed four times with PBS. The washed mixture was incubated with 0.5 μL of FITC-conjugated F(ab')2 fragment goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA) at 4°C for 30 minutes and then washed twice with PBS. At least 20,000 events per sample were acquired on a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA), and RBCs were gated by forward and side scatter parameters.

[0144] result

[0145] Confirmation of interference due to drug A

[0146] Based on previous results on the potential of drug A to interfere with RBC antibody screening (see, e.g., Example 1), drug A was doped into normal pooled serum that had been confirmed to be free of red blood cell antibodies. Patient serum samples used in this study were collected as part of routine care in a hospital setting. The final concentrations of drug A were 0.1, 1, 10, 100, and 500 μg / mL. The highest concentration observed at the 10 mg / kg QW dose level observed in human patients exceeded the average C of 247 ± 32.5 μg / mL. max (See Jin et al., "Pharmacokinetic and Pharmacodynamic Characterization of DRUG A, a CD47 Blocker, in Patients with Advanced Malignancy and Non-Hodgkin Lymphoma." (Poster) Society for Immunotherapy of Cancer Annual Meeting, Washington, DC, November 7-11, 2018).

[0147] Column agglutination test (CAT) was performed under anti-human globulin (AHG) phase using BioRad Antibody Screening Cells I and II. Reaction intensities ranging from 2+ to 3+ on a scale of 0 to 4+ were observed in the concentrations of Drug A tested, indicating binding of Drug A to RBCs and interference due to the interaction of the Fc portion of Drug A with the AHG reagent (Table 3).

[0148] Table 3. Serum adulterated with Drug A tested positive in the column agglutination test under the anti-human globulin (AHG) phase

[0149]

[0150] Use Agent B to mitigate interference

[0151] Agent B is a CD47 monomer comprising an IgSF domain. It is assumed that the combination of agent B and drug A in solution will form an agent B-drug A complex. It is also assumed that agent B-drug A complex will not be able to bind to the endogenous CD47 expressed on RBC, thereby preventing drug A from interfering with RBC antibody screening test. In order to test this possibility, an agent B with a concentration corresponding to a 10X to 500X molar ratio of drug A is added to the serum doped with drug A. After incubation for 30 minutes at room temperature (RT), BioRad antibody screening cells I and II are used again to perform CAT under the AHG phase. The disappearance of drug A interference is achieved at a 40X to 100X molar ratio of agent B (Table 4).

[0152] Table 4. Agent B mitigates interference due to drug A in column agglutination test under anti-human globulin (AHG) phase

[0153]

[0154] *NT: Not tested 209.82 μM Agent B (3.32 mg / mL) was used for neutralization. Specifically, 25 μL of plasma doped with Drug A (500 μg / mL, 160 pmol) was incubated with 8, 16, 24, 32, and 40 μL of Agent B (10X, 1.6 nmol; 20X, 3.2nmol; 30X, 4.8nmol; 40X, 6.4nmol; 50X, 8.0nmol). 25 μL of plasma doped with Drug A (100 μg / mL, 32pmol) was incubated with 1.6, 3.2, 4.8, 6.4, and 8.0 μL of Agent B (10X, 320pmol; 20X, 640pmol; 30X, 960pmol; 40X, 1.28nmol; 50X, 1.6nmol). 25 μL of plasma spiked with drug A (10 μg / mL, 3.2 pmol) was incubated with 160, 320, 480, 640 and 800 nL of agent B (10X, 32 pmol; 20X, 64 pmol; 30X, 96 pmol; 40X, 128 pmol; 50X, 160 pmol). 25 μL of plasma spiked with drug A (1 μg / mL, 320 fmol) was incubated with 16, 32, 48, 64, 80, 160, 320 and 480 nL of agent B (10X, 3.2 pmol; 20X, 6.4 pmol; 30X, 9.6 pmol; 40X, 12.8 pmol; 50X, 16.0 pmol; 100X, 32.0 pmol; 200X, 64.0 pmol; 300X, 96.0 pmol). 25 μL of plasma spiked with drug A (0.1 μg / mL, 32 fmol) was incubated with 1.6, 3.2, 4.8, 6.4, 8.0, 16.0, 32.0 and 48.0 nL of agent B (10X, 320 fmol; 20X, 640 pmol; 30X, 960 fmol; 40X, 1.28 pmol; 50X, 1.60 pmol; 100X, 3.2 pmol; 200X, 6.4 pmol; 300X, 9.6 pmol).

[0155] Use Agent C to mitigate interference

[0156] Agent C is a recombinant high affinity SIRPα monomer (Kd = 14.4 pM for human CD47). It is hypothesized that the binding of the agent to CD47 expressed on RBCs will prevent drug A from binding to CD47 expressed on RBCs, thereby preventing drug A from interfering with the RBC antibody screening test. To test this possibility, BioRad antibody screening cells I and II were incubated with agent C at concentrations corresponding to 10x to 500x molar ratios with drug A. After incubation for 30 minutes at room temperature (RT), agent RBCs incubated with agent C were tested in the AHG phase of CAT using serum doped with drug A. Reduction of drug A interference was achieved at a 300X molar ratio of agent C (Table 5).

[0157] Table 5: Agent C mitigates interference due to drug A in column agglutination test under anti-human globulin (AHG) phase

[0158]

[0159] NT: Not tested

[0160] 345.66 μM Agent C (4.77 mg / mL) was used to mask the Drug A binding site on CD47 of the reagent RBCs. Specifically, 25 μL of plasma doped with Drug A (500 μg / mL, 160 pmol) was incubated with 5, 10, 15, 20, 25, 50 and 150 μL of Agent C (10X, 1.6 nmol; 20X, 3.2 nmol; 30X, 4.8 nmol; 40X, 6.4 nmol; 50X, 8.0 nmol; 100X, 16.0 nmol; 300X, 48.0 nmol). 25 μL of plasma spiked with drug A (100 μg / mL, 32 pmol) was incubated with 1, 2, 3, 4, 5, 10, 30, and 50 μL of agent C (10X, 320 pmol; 20X, 640 pmol; 30X, 960 pmol; 40X, 1.28 nmol; 50X, 1.6 nmol; 100X.3.2 nmol; 300X, 9.6 nmol; 500X, 16 nmol). 25 μL of plasma spiked with drug A (10 μg / mL, 3.2 pmol) was incubated with 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 3.0 and 5.0 μL of agent C (10X, 32 pmol; 20X, 64 pmol; 30X, 96 pmol; 40X, 128 pmol; 50X, 160 pmol; 100X, 320 pmol; 300X, 960 pmol; 500X, 1.6 nmol). 25 μL of plasma spiked with drug A (1 μg / mL, 320 fmol) was incubated with 10, 20, 30, 40, 50, 100, 300 and 500 nL of agent C (10X, 3.2 pmol; 20X, 6.4 pmol; 30X, 9.6 pmol; 40X, 12.8 pmol; 50X, 16.0 pmol; 100X, 32.0 pmol; 300X, 96.0 pmol; 500X, 160.0 pmol). 25 μL of plasma spiked with drug A (0.1 μg / mL, 32 fmol) was incubated with 1, 2, 3, 4, 5, 10, 30, and 50 nL of agent C (10X, 320 fmol; 20X, 640 pmol; 30X, 960 fmol; 40X, 1.28 pmol; 50X, 1.60 pmol; 100X, 3.2 pmol; 300X, 9.6 pmol; 500X, 16.0 pmol).

[0161] Mitigation of interference using Agent B or Agent C allows detection of RBC alloantibodies in patient sera

[0162] In order to study whether the mitigation of the interference of agent B or agent C to drug A will allow the detection of true RBC alloantibodies, the pooled human serum containing anti-E (an exemplary RBC alloantibody) is used in an experiment similar to the above-mentioned experiment. Anti-E is a commonly identified alloantibody with clinical significance, and the pooled patient serum with anti-E positive results is obtained from the hospital transfusion service for research. The patient serum samples used in this study are collected as a part of routine care. The patient serum containing anti-E is doped with 0.1 to 500 μg / mL of drug A. CAT under AHG phase is performed using Biorad I and II cells. Although Biorad II cells are E positive, at all drug A concentrations, pan-positive (Table 6A) is observed in the case of two reagent cells. However, after incubation with 32 μL of Agent B (3.32 mg / mL), which is 40x the molar ratio of 25 μL of Drug A (500 μg / ml), at room temperature for 30 minutes, CAT down AHG revealed only BioradII cells to be positive, indicating that only anti-E activity was detected and that Drug A interference was resolved at all Drug A concentrations tested (Table 6B).

[0163] Similar results were observed with Agent C. BioRad antibody screening cells I and II were incubated with 150 μL Agent C (4.77 mg / mL) (300x the molar ratio of 25 μL Drug A (500 μg / ml)) at room temperature for 30 minutes. CAT under the AHG phase was performed using patient serum doped with Drug A containing anti-E, and only Biorad II cells were revealed to be positive, indicating that only anti-E activity was detected, and Drug A interference was resolved at all tested Drug A concentrations (Table 6C).

[0164] Table 6A-6C. Mitigation of interference using Agent B or Agent C allows detection of RBC alloantibodies in patient serum

[0165] Table 6A.

[0166]

[0167] *Biorad II cells are E positive.

[0168] Table 6B

[0169]

[0170] *Biorad II cells are E positive.

[0171] 6.4 nmol Agent B (40x molar ratio of 500 μg / mL Drug A) was used for all Drug A concentrations tested.

[0172] Table 6C

[0173]

[0174] *Biorad II cells are E positive.

[0175] 48 nmol Agent C (300x molar ratio of 500 μg / mL Drug A) was used for all Drug A concentrations tested.

[0176] Correlation analysis of drug A binding to RBC by flow cytometry

[0177] To determine whether the binding of drug A to CD47 expressed on the reagent RBCs was actually reduced by the presence of soluble high affinity SIRPα (agent C) or soluble CD47 (agent B), flow cytometry analysis was performed as follows: Drug A was spiked into normal serum at 0 (negative control), 0.1, 1.0, 10, 100, and 500 μg / mL. BioRAD I cells were incubated with serum samples spiked with drug A at 37°C for 15 minutes. FITC-conjugated anti-human IgG was used to stain drug A-bound RBCs. Cells were analyzed by flow cytometry and MFI (mean fluorescence intensity) was determined.

[0178] To evaluate the effect of Agent B on the binding of Drug A to RBCs, serum samples spiked with Drug A and unspiked negative control samples were incubated with Agent B (at concentrations ranging from 10x to 50x molar ratio of Drug A) for 15 min at room temperature. BioRAD I cells were incubated with serum samples at 37°C for 15 min prior to flow cytometric analysis.

[0179] To evaluate the effect of Agent C on the binding of Drug A to RBCs, Agent C (at concentrations ranging from 10x to 500x molar ratio of Drug A) was added to BioRAD I cells for 15 minutes at 37° C. Cells were further incubated with serum samples spiked with Drug A and unspiked negative control samples at 37° C. for 15 minutes before flow cytometric analysis.

[0180] RBC binds to drug A contained in serum, as indicated by increased FITC signal. As the concentration of drug A doped into serum increases, a concentration-dependent increase in MFI is observed (Table 7A). Both agent B and agent C = effective and reduce the binding of drug A to RBC in a concentration-dependent manner (Tables 7B and 7C).

[0181] Table 7A-7C. Agent B and Agent C Reduce Binding of Drug A to RBCs

[0182] Table 7A.

[0183]

[0184] Table 7B.

[0185]

[0186] Table 7C.

[0187]

[0188]

[0189] in conclusion

[0190] In the column agglutination test (CAT) under the anti-human globulin (AHG) phase using BioRad antibody screening cells I and II, drug A at concentrations up to 500 μg / mL in serum interfered with the test and produced false positive results. This interference was eliminated using recombinant CD47 monomer (agent B) or high-affinity SIRPα monomer (agent C). In addition, anti-E, a clinically relevant alloantibody, could be detected in serum after the interference of drug A was eliminated with agent B or agent C. Correlated flow cytometric analysis showed that drug A bound to RBCs in a concentration-dependent manner. Both agents B and C reduced the binding of drug A to RBCs, which is consistent with the neutralization of drug A interference binding to CD47 expressed on RBCs.

[0191] References

[0192] Brown EJ, Frazier WA. Integrin-associated protein (CD47) and its ligands. Trends Cell Biol. 2001 Mar; 11(3): 130-5.

[0193] Chao MP, Alizadeh AA, Tang C, Myklebust JH, Varghese B, Gill S, et al. Anti-CD47 antibody synergizes with rituximab to promote phagocytosis ad eradicatenon-Hodgkin lymphoma. Cell. 2010 Sep 3;142(4):699-713.

[0194] Jaiswal S,Chao MP,Mejeti R and Weissman IL.Macrophagesas mediatorsoftumor immunosurveillance.Trends Immmunol.2010June;31(6):212-219.

[0195] Oldenborg PA.Zheleznyak A,Fang YF,Lagenaur CF,Greshan HD et al.RoleofCD47 as a marker of self on red blood cells.Science 2000 Jun 16;288(5473):2051-4.

[0196] Weiskopf K,Ring AM,Ho CC,Volkmer J-P,Levin AM,Volkmer AK,etal.Engineered SIRPαvariants as immunotherapeutic adjuvants to anticancerantibodies.Science.2013 Jul 5;341(6141):88-91.

[0197] Willingham SB,Volkmer JP,Gentles AJ,Sahoo D,Dalerba P,Mitra SS,etal.The CD47-signal regulatory protein alpha(SIRPα)interaction is atherapeutic target for human solid tumors.Proc Natl Acad Sci USA.2012 Apr 24;109(17):6662-7.

[0198] Zhao XW, Van Beek EM, Schornagela K, Van der Maadenb H, Houdta MV; CD47-signal regulatory protein-α (SIRPα) interactions form a barrier for antioody-mediated tumor cell destruction. Proc Natl Acad Sci USA. 2011Nov 8; 108(45): 18342-7.

[0199] Technical Manual.Current Edition.Bethesda:AABB.

[0200] Summarize

[0201] In the column agglutination test (CAT) under the anti-human globulin (AHG) phase using BioRad antibody screening cells I and II, the presence of drug A in serum showed interference with the test and resulted in false positive results. This interference was resolved using recombinant CD47 monomer (agent B) or high affinity SIRPα monomer (agent C). In addition, anti-E, a clinically relevant alloantibody, was still detected after the interference was resolved by agent B or agent C. Correlated flow cytometric analysis showed that drug A bound to RBCs in a concentration-dependent manner. Both agents B and C reduced the binding of drug A to RBCs, consistent with neutralization of ALX interference.

[0202] Example 4. Use of anti-SIRPα antibodies (or antigen-binding fragments thereof) to mitigate interference of drugs containing (i) an antibody Fc region and (ii) a portion that binds to human CD4 in conventional serological tests The following experiment was performed to evaluate whether the interference induced by higher concentrations of drug A can be neutralized by adding anti-SIRPα antibodies (or Fab fragments thereof). Related flow cytometric analysis was performed to evaluate whether drug A binding to CD47 on the surface of RBCs can be displaced by adding anti-SIRPα antibodies (or Fab fragments thereof).

[0203] The hemagglutination assay was performed as follows: human erythrocytes pooled in 3%-3.4% modified Alsever's solution (Bio-Rad) were added to 96-well plates (Falcon) at 40 μl per well. The wells were incubated with 250 ng / mL of PBS (Gibco) or drug A at 37°C for 1 hour, and then washed 3 times in PBS, with sufficient decantation after each wash. The Fab fragments of antibodies A and C (i.e., exemplary anti-SIRPα antibodies that block the interaction between SIRPα and CD47) were titrated from 100 μg / mL or 625 μg / mL at a ratio of 1:2 and incubated with drug A-coated erythrocytes at 37°C for 1 hour, then washed 3 times with PBS, and the remaining liquid was fully decanted. Two drops of anti-human globulin anti-IgG (Bio-Rad) were then added to each well and centrifuged at 800G for 30 seconds, then the precipitate was gently removed and the jpeg image was captured. Antibody A comprises a heavy chain variable domain (V H ) and a light chain variable domain (V L Antibody C comprises a heavy chain variable domain (V H ) and a light chain variable domain (V L A parallel experiment was performed with antibody B (i.e., an exemplary anti-SIRPα antibody that does not block the interaction between SIRPα and CD47). Antibody B comprises a heavy chain variable domain (V H ) and a light chain variable domain (V L ). Each of antibodies A, B and C cross-reacts with SIRPβ and SIRPγ. Red blood cells incubated with PBS or drug A alone served as negative and positive controls for the assay, respectively.

[0204] like Figure 5A As shown, antibody C (which blocks the interaction between SIRPα and CD47) prevented hemagglutination when added to drug A-coated erythrocytes at 200-fold and 400-fold molar excesses relative to the amount of drug A. Antibody A (which also blocks the interaction between SIRPα and CD47) prevented hemagglutination when added to drug A-coated erythrocytes at 2500-fold molar excess relative to the amount of drug A. Addition of antibody B (an anti-SIRPα antibody that does not block the interaction between both SIRPα and CD47) prevented hemagglutination when added to drug A-coated erythrocytes at 400-fold molar excess relative to the amount of drug A. Figure 5A The results shown in indicate that the Fabs of Antibody A, Antibody B, and Antibody C each prevented hemagglutination by displacing Drug A from CD47 erythrocytes.CD47 on the surface of erythrocytes.

[0205] In order to confirm that the Fab of antibody A and antibody C can replace the drug A bound to CD47 on the surface of erythrocytes, flow cytometry experiments were performed as follows: according to the manufacturer's instructions, drug A was fluorescently labeled using Alexa Fluor 647 protein labeling kit (Thermo Fisher Scientific). DLD-1 human colon epithelial cells were washed once in staining buffer (PBS, 2% FBS) and stained with a fixable live / dead stain (Invitrogen) at 4°C for 1 hour in PBS. The cells were then washed in staining buffer and co-incubated in 96-well plates (Falcon) with 100ng / mL of labeled drug A and unlabeled antibody A Fab or unlabeled antibody C Fab starting with 50μg / mL at 1:2 titration. After incubation at 4°C for 1 hour, cells were washed twice in staining buffer and fixed in 0.5% paraformaldehyde. Cells were analyzed on Attune (ThermoScientific), and data analysis was subsequently performed using Flowjo10.6. The binding of drug A to CD47 on the surface of DLD-1 cells is indicated by the median fluorescence intensity of Alexa Fluor 647. Figure 5B As shown, adding antibody A and antibody C to cells that bind drug A reduces the binding of drug A to CD47 on the surface of DLD-1 cells. These results indicate that antibody A and antibody C are able to displace drug A bound to CD47 on the surface of DLD-1 cells.

[0206] In summary, the results in this example indicate that antibodies A, B, and C can prevent interference in conventional serological assays by replacing drug A that binds to CD47 on the surface of blood cells. These results also indicate that antibodies that cross-react with SIRPα, SIRPβ, and SIRPγ can prevent interference in conventional serological assays by replacing drug A that binds to CD47 on the surface of blood cells.

[0207] Unless clearly indicated to the contrary, each embodiment described herein can be combined with any other one or more embodiments. Specifically, unless clearly indicated to the contrary, any feature or embodiment indicated as preferred or advantageous can be combined with any one or more other features or embodiments indicated as preferred or advantageous.

[0208] All references cited in this application are expressly incorporated herein by reference.

Claims

1. A method for reducing drug interference in a pre-transfusion serological assay using reagent red blood cells (RBC) or reagent platelets for non-diagnostic purposes, the method comprising: (a) adding an anti-SIRPα antibody or an antigen-binding fragment thereof to a plasma sample from a subject who has been treated with a drug that interferes with the serological assay; as well as (b) performing said serological assay of said plasma sample using said reagent RBCs or said reagent platelets after step (a), The drug comprises (i) a human antibody Fc region and (ii) a SIRPα extracellular domain consisting of the amino acid sequence of SEQ ID NO: 9, and The anti-SIRPα antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable domain consisting of SEQ ID NO: 6 (V H ) and a light chain variable domain consisting of SEQ ID NO: 7 (V L ); (b) a heavy chain variable domain consisting of SEQ ID NO: 21 (V H ) and a light chain variable domain consisting of SEQ ID NO: 22 (V L );or (c) a heavy chain variable domain consisting of SEQ ID NO 23 (V H ) and a light chain variable domain consisting of SEQ ID NO: 24 (V L ). 2 . The method of claim 1 , wherein the anti-SIRPα antibody or antigen-binding fragment thereof is added to the plasma sample in an amount in molar excess relative to the amount of the drug in the plasma sample.

3. The method of any one of claims 1-2, wherein the human antibody Fc region of the drug is a human IgG Fc region.

4. The method of claim 3, wherein the human IgG Fc region is an IgG1, IgG2 or IgG4 Fc region.

5. The method of any one of claims 1-2, wherein the serological assay is an ABO / Rh typing assay.

6. The method of any one of claims 1-2, wherein the serological assay is an immediate centrifugation (IS) assay.

7. The method of claim 1, wherein the serological assay is a direct antiglobulin (DAT) assay using a multispecific reagent that detects IgG and complement C3.

8. The method of claim 1, wherein the serological assay is a direct antiglobulin (DAT) assay using a monospecific reagent that detects complement C3.

9. The method of any one of claims 1-2, wherein the serological assay is a PEG-enhanced serological assay.

10. The method of any one of claims 7-8, wherein the serological assay is an eluate test performed after the DAT assay.

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