Multimers for reducing drug interference by CD47-binding agents in serological assays

Abstract

Methods are provided for reducing and / or preventing interference in serological assays by drugs that comprise (i) an antibody Fc region and (ii) a moiety that binds to human CD47.

Description

[Technical Field]

[0001] Cross-reference with related applications This application claims priority to U.S. Provisional Application No. 63 / 121,964, filed on December 6, 2020, the contents of which are incorporated in their entirety by reference.

[0002] Submission of sequence listings in ASCII text files. The following submission in ASCII text file is incorporated herein by reference in its entirety: Computer-Readable Format (CRF) of Sequence Listing (filename: 757972001440SEQLIST.TXT, date recorded: December 3, 2021, size: 96KB).

[0003] Field of Invention The present invention relates to a method and reagent used to reduce interference in serological assays by drugs comprising (i) an antibody Fc region and (ii) a portion that binds to human CD47. [Background technology]

[0004] The number of antibody-based drugs being developed to treat a wide variety of diseases, including cancer, is steadily increasing. When 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.

[0005] For example, CD47 (Jaiswal et al., Trends Immunol (2010) 31(6):212-219; Brown et al., Trends Cell Biol (2001) 11(3):130-135), a widely expressed cell surface protein that binds to signal regulatory protein α (SIRPα) and inhibits phagocytosis, is highly expressed on a wide variety of malignant tumors, including hematological and solid tumors. Elevated CD47 expression is also correlated with progressive disease (Willingham et al., Proc Natl Acad Sci USA (2012) 109(17):6662-6667). Several cancer therapies targeting CD47, such as antibodies and fusion proteins containing the antibody Fc region, have been developed to block the SIRPα-CD47 interaction, thereby enabling macrophages to perform phagocytic functions and eliminate tumor cells.

[0006] CD47 is also expressed on the surface of blood cells such as red blood cells (RBCs) and platelets (Oldenborg et al., Science (2000) 288(5473):2051-2054), and drugs containing the Fc region of CD47-targeting antibodies may interfere with blood typing and serological testing. Furthermore, since patients receiving CD47-targeted drugs (e.g., for cancer treatment) often require blood transfusions to treat concurrently occurring anemia and / or thrombocytopenia, interference with serological and blood typing assays by anti-CD47 drugs is a significant patient safety concern. Therefore, there is a need in the art to develop methods and reagents to reduce the interference of CD47-targeted drugs containing the Fc region of antibodies with serological assays. [Overview of the project]

[0007] In some embodiments, a method is provided for reducing drug interference in a serological assay using reagent red blood cells (RBCs) or reagent platelets, the method comprising: (a) adding a CD47 multimer that binds to a drug and blocks the drug from binding to reagent RBCs or reagent platelets to a plasma sample from a subject being treated with the drug; and (b) performing a serological assay of the plasma sample using reagent RBCs or 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, and the CD47 multimer comprises at least two CD47 polypeptide monomers.

[0008] In some embodiments, the CD47 multimer comprises 2 to 100 CD47 polypeptide monomers. In some embodiments, the CD47 multimer comprises a CD47 polypeptide monomer containing drug-binding wild-type CD47 or a fragment thereof. In some embodiments, the CD47 multimer comprises a CD47 polypeptide monomer containing drug-binding wild-type human CD47, wild-type mouse CD47, wild-type rat CD47, wild-type rhesus monkey CD47, wild-type cynomolgus monkey CD47, or a fragment of any one of the above. In some embodiments, the CD47 multimer comprises a CD47 polypeptide monomer containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the CD47 multimer comprises a CD47 polypeptide monomer containing a CD47 variant that includes one or more amino acid substitutions, insertions, deletions, N-terminal elongation, or C-terminal elongation compared to drug-binding wild-type CD47 or a fragment thereof. In some embodiments, the CD47 multimer comprises a CD47 polypeptide monomer containing the amino acid sequence described in any one of SEQ ID NOs: 2 to 6. In some embodiments, the CD47 multimer comprises a CD47 polypeptide monomer containing a fusion polypeptide. In some embodiments, the fusion polypeptide comprises a polymerizing domain. In some embodiments, the polymerizing domain comprises an Fc monomer, a c-Jun leucine zipper domain, or a c-Fos leucine zipper domain. In some embodiments, the Fc monomer is a mouse Fc monomer. In some embodiments, the mouse Fc monomer comprises the amino acid sequence described in any one of SEQ ID NOs. 81-83. In some embodiments, the fusion polypeptide comprises the amino acid sequence described in any one of SEQ ID NOs. 84-86. In some embodiments, the CD47 multimer comprises a CD47 polypeptide monomer containing an epitope tag or ligand (for example, further comprising). In some embodiments, the epitope tag comprises any one of SEQ ID NOs. 7-32 and 126, or the ligand comprises biotin. In some embodiments, the CD47 multimer comprises a soluble CD47 polypeptide monomer.

[0009] In some embodiments, the CD47 polymer comprises at least two CD47 polypeptide monomers linked via peptide bonds. In some embodiments, the at least two CD47 polypeptide monomers are linked via a linker peptide. In some embodiments, the linker peptide comprises one of SEQ ID NOs: 85-109, 127-130, 140, and 141. In some embodiments, the linker peptide comprises one or more spacers. In some embodiments, the spacers comprise GS, GGS, or one of SEQ ID NOs: 52-70. In some embodiments, the CD47 polymer comprises at least two CD47 polypeptide monomers bound to a solid support. In some embodiments, the solid support is a gold nanosphere, gold nanoshell, magnetic beads, silica beads, dextran polymer, test tube, slide, gel column, or microtiter well. In some embodiments, each of at least two CD47 polypeptide monomers contains an epitope tag or ligand, and a scavenger that specifically binds to the epitope tag or ligand is immobilized on a solid support, and the CD47 polypeptide monomers are bound to the solid support by the specific binding of the epitope tag or ligand by the scavenger. In some embodiments, the ligand is biotin and the scavenger is streptavidin. In some embodiments, at least one of the at least two CD47 polypeptide monomers contains SEQ ID NO: 6. In some embodiments, the CD47 polymer contains streptavidin or avidin bound to two, three, or four biotinylated CD47 polypeptide monomers. In some embodiments, at least one of the two, three, or four biotinylated CD47 polypeptide monomers contains SEQ ID NO: 6. In some embodiments, the CD47 polymer is a homopolymer. In some embodiments, the CD47 polymer is a heteropolymer.

[0010] In some embodiments, methods are provided for reducing drug interference in serological assays using reagent red blood cells (RBCs), reagent platelets, or combinations thereof, the method comprising: (a) adding a SIRP multimer that specifically binds to human CD47 to reagent red blood cells (RBCs), reagent platelets, or combinations thereof; and (b) performing a serological assay of a plasma sample using the reagent red blood cells (RBCs), reagent platelets, or combinations thereof from step (a), wherein the plasma sample is derived from a subject that has been treated with a drug, the drug comprises (i) an antibody Fc region and (ii) a portion that binds to human CD47, and the SIRP multimer comprises at least two SIRP polypeptide monomers. In some embodiments, methods are provided for reducing drug interference in serological assays using reagent red blood cells (RBCs), reagent platelets, or a combination thereof, the method comprising (a) adding a SIRP multimer that specifically binds to human CD47 to a plasma sample derived from a subject being treated with a drug, and, after step (a), performing a serological assay of the plasma sample using reagent red blood cells (RBCs), reagent platelets, or a combination thereof, wherein the drug comprises (i) an antibody Fc region and (ii) a portion that binds to human CD47, and the SIRP multimer comprises at least two SIRP polypeptide monomers. In some embodiments, a method is provided 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 SIRP macromer that specifically binds to human CD47 to a blood sample derived from a subject treated with a drug, 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, and the SIRP macromer comprises at least two SIRP polypeptide monomers.

[0011] In some embodiments, the SIRP multimer comprises 2 to 100 SIRP polypeptide monomers. In some embodiments, the SIRP multimer comprises SIRP polypeptide monomers comprising wild-type SIRPα or a fragment thereof that can bind to human CD47. In some embodiments, the SIRP multimer comprises SIRP polypeptide monomers comprising wild-type human SIRPα, wild-type mouse SIRPα, wild-type rat SIRPα, wild-type rhesus monkey SIRPα, wild-type cynomolgus monkey SIRPα, or a fragment of any one of the above that can bind to human CD47. In some embodiments, the SIRP multimer comprises SIRP polypeptide monomers comprising SIRPα variants comprising one or more amino acid substitutions, insertions, deletions, N-terminal elongation, or C-terminal elongation compared to wild-type SIRPα or a fragment thereof that can bind to CD47. In some embodiments, the SIRP multimer comprises SIRP polypeptide monomers comprising wild-type SIRPγ or a fragment thereof that can bind to human CD47. In some embodiments, the SIRP multimer comprises a SIRP polypeptide monomer containing a fragment of wild-type human SIRPγ, wild-type mouse SIRPγ, wild-type rat SIRPγ, wild-type rhesus monkey SIRPγ, wild-type cynomolgus monkey SIRPγ, or any one of the above, which is bindable to human CD47. In some embodiments, the SIRP multimer comprises a SIRP polypeptide monomer containing a SIRPγ variant that includes one or more amino acid substitutions, insertions, deletions, N-terminal elongation, or C-terminal elongation compared to wild-type SIRPγ or a fragment thereof, which is bindable to CD47. In some embodiments, the SIRP multimer comprises a SIRP polypeptide monomer containing a SIRPβ variant that includes one or more amino acid substitutions, insertions, deletions, N-terminal elongation, or C-terminal elongation compared to wild-type SIRPβ or a fragment thereof, and the SIRPβ variant or a fragment thereof is bindable to CD47. In some embodiments, the SIRP multimer comprises a SIRP polypeptide monomer containing any one of the amino acid sequences of SEQ ID NOs. 33 to 45. In some embodiments, the SIRP polymer comprises a SIRP polypeptide monomer containing a fusion polypeptide.In some embodiments, the fusion polypeptide includes a polymerizing domain. In some embodiments, the polymerizing domain includes an Fc monomer, a c-Jun leucine zipper domain, or a c-Fos leucine zipper domain. In some embodiments, the Fc monomer is a mouse Fc monomer. In some embodiments, the mouse Fc monomer includes the amino acid sequence described in any one of SEQ ID NOs. 81-83. In some embodiments, the fusion polypeptide includes the amino acid sequence described in SEQ ID NO. 110. In some embodiments, the SIRP polymer includes a SIRP polypeptide monomer containing an epitope tag or ligand. In some embodiments, the epitope tag includes any one of SEQ ID NOs. 7-32 and 126, or the ligand includes biotin. In some embodiments, the SIRP polymer includes a soluble SIRP polypeptide monomer.

[0012] In some embodiments, the SIRP polymer comprises at least two SIRP polypeptide monomers linked via peptide bonds. In some embodiments, the SIRP polymer comprises at least two SIRP polypeptide monomers linked via a linker peptide. In some embodiments, the linker peptide comprises one of SEQ ID NOs: 85-109, 127-130, 140, and 141. In some embodiments, the linker peptide comprises one or more spacers. In some embodiments, the spacers comprise GS, GGS, or one of SEQ ID NOs: 52-70. In some embodiments, the SIRP polymer comprises at least two SIRP polypeptide monomers bound to a solid support. In some embodiments, the solid support is a gold nanosphere, gold nanoshell, magnetic beads, silica beads, dextran polymer, test tube, slide, gel column, or microtiter well. In some embodiments, each of at least two SIRP polypeptide monomers contains an epitope tag or ligand, and a scavenger that specifically binds to the epitope tag or ligand is immobilized on a solid support, and the SIRP polypeptide monomer is bound to the solid support by the specific binding of the epitope tag or ligand by the scavenger. In some embodiments, the ligand is biotin and the scavenger is streptavidin. In some embodiments, at least one of the at least two SIRP polypeptide monomers contains SEQ ID NO: 111. In some embodiments, the SIRP polymer contains streptavidin or avidin bound to two, three, or four biotinylated SIRP polypeptide monomers. In some embodiments, at least one of the two, three, or four biotinylated SIRP polypeptide monomers contains SEQ ID NO: 111. In some embodiments, the SIRP polymer is a homopolymer. In some embodiments, the SIRP polymer is a heteropolymer.

[0013] In some embodiments, a method for reducing drug interference in serological assays using reagent red blood cells (RBCs) or reagent platelets is provided, the method comprising: (a) adding an anti-SIRP multimer that binds to the drug and blocks the drug from binding to the reagent RBCs or reagent platelets to a plasma sample derived from a subject being treated with the drug; and (b) after step (a), performing a serological assay on the plasma sample using the reagent RBCs or reagent platelets, wherein the drug comprises (i) a human antibody Fc region or a variant thereof and (ii) a moiety that binds to human CD47, and the anti-SIRP multimer comprises one or more anti-SIRP antibodies or drug-binding fragments thereof.

[0014] In some embodiments, the anti-SIRP multimer comprises an anti-SIRP antibody or a drug-binding fragment thereof. In some embodiments, the anti-SIRP multimer comprises 1 to 100 anti-SIRP antibodies or drug-binding fragments thereof. In some embodiments, the anti-SIRP multimer comprises an anti-SIRP antibody or a drug-binding fragment thereof that binds to wild-type SIRPα, an SIRPα variant, an SIRPβ variant, wild-type SIRPγ, an SIRPγ variant, or any two or more of the foregoing. In some embodiments, the anti-SIRP multimer comprises an anti-SIRP antibody or a drug-binding fragment thereof comprising: (a) a heavy-chain variable domain (V H ) comprising SEQ ID NO: 46 and a light-chain variable domain (V L ) comprising SEQ ID NO: 47; (b) a heavy-chain variable domain (V H ) comprising SEQ ID NO: 48 and a light-chain variable domain (V L ) comprising SEQ ID NO: 49; (c) a heavy-chain variable domain (V H ) comprising SEQ ID NO: 50 and a light-chain variable domain (V L ) comprising SEQ ID NO: 51; (d) a heavy-chain variable domain (V H ) comprising SEQ ID NO: 113 and a light-chain variable domain (V L ) comprising SEQ ID NO: 114; (e) a heavy-chain variable domain (V H ) comprising SEQ ID NO: 115 and a light-chain variable domain (V L ) comprising SEQ ID NO: 116; H ) and a light-chain variable domain (V L ) comprising SEQ ID NO: 114; (e) a heavy-chain variable domain (V H ) comprising SEQ ID NO: 115 and a light-chain variable domain (V L ) comprising SEQ ID NO: 116; L); and / or (f) heavy chain variable domain (V) containing sequence number 133 H ) and light chain variable domain (V) containing sequence number 134 L ). In some embodiments, the anti-SIRP multimer comprises a full-length anti-SIRP antibody. In some embodiments, the anti-SIRP antibody comprises a mouse Fc domain. In some embodiments, the mouse Fc domain comprises the amino acid sequence described in any one of SEQ ID NOs: 81-83. In some embodiments, the anti-SIRP antibody comprises: (a) a heavy chain containing SEQ ID NO: 117 and a light chain containing SEQ ID NO: 118; (b) a heavy chain containing SEQ ID NO: 119 and a light chain containing SEQ ID NO: 118; (c) a heavy chain containing SEQ ID NO: 120 and a light chain containing SEQ ID NO: 121; or (d) a heavy chain containing SEQ ID NO: 122 and a light chain containing SEQ ID NO: 121. In some embodiments, the drug-binding fragment of the anti-SIRP antibody is Fab, Fab', F(ab')2, Fab'-SH, Fv, diabody, one-arm antibody, scFv, scFv-Fc, single-domain antibody, or single-heavy-chain antibody. In some embodiments, the drug-binding fragment comprises F(ab')2, where F(ab')2 comprises SEQ ID NOs: 131 and 132. In some embodiments, the anti-SIRP antibody or its drug-binding fragment includes an epitope tag or ligand. In some embodiments, the epitope tag includes one of SEQ ID NOs. 7-32 and 126, or the ligand includes biotin. In some embodiments, the epitope tag includes HHHHHHGLNDIFEAQKIEWHE (SEQ ID NO: 135) or GSGSHHHHHGLNDIFEAQKIEWHE (SEQ ID NO: 126).

[0015] In some embodiments, the anti-SIRP multimer comprises one or more anti-SIRP antibodies or drug-binding fragments thereof conjugated to a solid support. In some embodiments, the solid support is a gold nanosphere, gold nanoshell, magnetic beads, silica beads, dextran polymer, test tube, slide, gel column, or microtiter well. In some embodiments, one or more anti-SIRP antibodies or drug-binding fragments comprises an epitope tag or ligand, and a capture agent that specifically binds to the epitope tag or ligand is immobilized on the solid support, and the anti-SIRP antibody or drug-binding fragment is conjugated to the solid support by the specific binding of the epitope tag or ligand by the capture agent. In some embodiments, the ligand is biotin, and the capture agent is streptavidin. In some embodiments, the anti-SIRP multimer comprises two, three, or four biotinylated anti-SIRP antibodies or fragments thereof conjugated to streptavidin or avidin. In some embodiments, the anti-SIRP multimer comprises streptavidin or avidin bound to two, three, or four biotinylated F(ab')2 fragments, where two or more biotinylated F(ab')2 fragments include SEQ ID NOs. 131 and 132. In some embodiments, the anti-SIRP multimer is a homomultimer. In some embodiments, the anti-SIRP multimer is a heteromultimer.

[0016] In some embodiments of any of the methods described herein, the drug comprises an anti-CD47 antibody. In some embodiments, the drug portion that binds to human CD47 comprises wild-type SIRPα, a SIRPα variant, or a fragment of wild-type SIRPα or a SIRPα variant. In some embodiments, the drug portion that binds to human CD47 comprises a SIRPα variant, which comprises one or more amino acid substitutions, insertions, deletions, N-terminal extensions, and / or C-terminal extensions compared to wild-type SIRPα. In some embodiments, the drug portion that binds to human CD47 comprises a fragment of a SIRPα variant, which comprises the extracellular domain of a SIRPα variant. In some embodiments, the drug portion that binds to human CD47 comprises wild-type SIRPγ, a SIRPγ variant, or a fragment of wild-type SIRPγ or a SIRPγ variant. In some embodiments, the drug portion that binds to human CD47 comprises a SIRPγ variant, which comprises one or more amino acid substitutions, insertions, deletions, N-terminal elongations, C-terminal elongations, or any combination thereof, compared to wild-type SIRPγ. In some embodiments, the drug portion that binds to human CD47 comprises a fragment of the SIRPγ variant, which comprises the extracellular domain of the SIRPγ variant. In some embodiments, the drug portion that binds to human CD47 comprises a SIRPβ variant or a fragment of the SIRPβ variant. In some embodiments, the drug portion that binds to human CD47 comprises a SIRPβ variant, which comprises one or more amino acid substitutions, insertions, deletions, N-terminal elongations, C-terminal elongations, or any combination thereof, compared to wild-type SIRPβ. In some embodiments, the drug portion that binds to human CD47 comprises a fragment of a SIRPβ variant, the fragment comprising the extracellular domain of the SIRPβ variant.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 thereof.

[0017] In some embodiments of any of the methods described herein, the serological assay is an ABO / Rh typing assay. In some embodiments, the serological assay is an immediate spin (IS) assay. In some embodiments, the serological assay is a direct antiglobulin (DAT) assay using a multispecific reagent to detect IgG and complement C3. In some embodiments, the serological assay is a direct antiglobulin (DAT) assay using a monospecific reagent to detect complement C3. In some embodiments, the serological assay is a PEG-enhanced serological assay. In some embodiments, the serological assay is an elution test performed after the DAT assay. In some embodiments, the serological assay is a test tube assay or a solid-phase erythrocyte assay (SPRCA).

[0018] CD47 polymers comprising at least two CD47 polypeptide monomers are provided herein. In some embodiments, the CD47 polymer comprises 2 to 100 CD47 polypeptide monomers. In some embodiments, the CD47 polymer comprises CD47 polypeptide monomers comprising drug-binding wild-type CD47 or a fragment thereof. In some embodiments, the CD47 polymer comprises drug-binding CD47 polypeptide monomers comprising wild-type human CD47, wild-type mouse CD47, wild-type rat CD47, wild-type rhesus monkey CD47, wild-type cynomolgus monkey CD47, or a fragment of any one of the above. In some embodiments, the CD47 polymer comprises CD47 polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the CD47 polymer comprises CD47 polypeptide monomers comprising CD47 variants comprising one or more amino acid substitutions, insertions, deletions, N-terminal elongations, or C-terminal elongations compared to drug-binding wild-type CD47 or a fragment thereof. In some embodiments, the CD47 multimer comprises a CD47 polypeptide monomer containing the amino acid sequence described in any one of SEQ ID NOs: 2-6. In some embodiments, the CD47 multimer comprises a CD47 polypeptide monomer containing a fusion polypeptide. In some embodiments, the fusion polypeptide contains a multimerizing domain. In some embodiments, the multimerizing domain contains an Fc monomer, a c-Jun leucine zipper domain, or a c-Fos leucine zipper domain. In some embodiments, the Fc monomer is a mouse Fc monomer. In some embodiments, the mouse Fc monomer contains the amino acid sequence described in any one of SEQ ID NOs: 81-83. In some embodiments, the fusion polypeptide CD47 polypeptide monomer contains the amino acid sequence described in any one of SEQ ID NOs: 84-86. In some embodiments, the CD47 multimer comprises a CD47 polypeptide monomer containing an epitope tag or ligand. In some embodiments, the epitope tag contains any one of SEQ ID NOs: 7-32 and 126, or the ligand contains biotin. In some embodiments, the CD47 polymer comprises a soluble CD47 polypeptide monomer.

[0019] In some embodiments, the CD47 multimer comprises at least two CD47 polypeptide monomers linked via peptide bonds. In some embodiments, the CD47 multimer comprises at least two CD47 polypeptide monomers linked via a linker peptide. In some embodiments, the linker peptide comprises any one of SEQ ID NOs: 85-109, 127-130, 140, and 141. In some embodiments, the linker peptide comprises one or more spacers. In some embodiments, the spacer comprises GS, GGS, or any one of SEQ ID NOs: 52-70. In some embodiments, the CD47 multimer comprises at least two CD47 multimers each comprising a plurality of CD47 polypeptide monomers bound to a solid support. In some embodiments, the solid support is a gold nanosphere, a gold nanoshell, a magnetic bead, a silica bead, a dextran polymer, a test tube, a slide, a gel column, or a microtiter well. In some embodiments, each of the at least two CD47 polypeptide monomers comprises an epitope tag or a ligand, and a capture agent that specifically binds to the epitope tag or ligand is immobilized on the solid support, and the CD47 polypeptide monomer is bound to the solid support by specific binding of the epitope tag or ligand by the capture agent. In some embodiments, the ligand is biotin and the capture agent is streptavidin. In some embodiments, at least one of the at least two CD47 polypeptide monomers comprises SEQ ID NO: 6. In some embodiments, the CD47 multimer comprises streptavidin or avidin bound to 2, 3, or 4 biotinylated CD47 polypeptide monomers. In some embodiments, at least one of the 2, 3, or 4 biotinylated CD47 polypeptide monomers comprises SEQ ID NO: 6. In some embodiments, the CD47 multimer is a homomultimer. In some embodiments, the CD47 multimer is a heteromultimer.

[0020] In some embodiments, a SIRP polymer is provided comprising at least two SIRP polypeptide monomers. In some embodiments, the SIRP polymer comprises 2 to 100 SIRP polypeptide monomers. In some embodiments, the SIRP polymer comprises a SIRP polypeptide monomer comprising wild-type SIRPα or a fragment thereof that can bind to human CD47. In some embodiments, the SIRP polymer comprises a SIRP polypeptide monomer comprising wild-type human SIRPα, wild-type mouse SIRPα, wild-type rat SIRPα, wild-type rhesus monkey SIRPα, wild-type cynomolgus monkey SIRPα, or a fragment of any one of the above that can bind to human CD47. In some embodiments, the SIRP polymer comprises a SIRP polypeptide monomer comprising a SIRPα variant comprising one or more amino acid substitutions, insertions, deletions, N-terminal elongation, or C-terminal elongation compared to wild-type SIRPα or a fragment thereof that can bind to CD47. In some embodiments, the SIRP polymer comprises a SIRP polypeptide monomer comprising wild-type SIRPγ or a fragment thereof that can bind to human CD47. In some embodiments, the SIRP multimer comprises a SIRP polypeptide monomer containing a fragment of wild-type human SIRPγ, wild-type mouse SIRPγ, wild-type rat SIRPγ, wild-type rhesus monkey SIRPγ, wild-type cynomolgus monkey SIRPγ, or any one of the above, which is bindable to human CD47. In some embodiments, the SIRP multimer comprises a SIRP polypeptide monomer containing a SIRPγ variant that includes one or more amino acid substitutions, insertions, deletions, N-terminal elongation, or C-terminal elongation compared to wild-type SIRPγ or a fragment thereof, which is bindable to CD47. In some embodiments, the SIRP multimer comprises a SIRP polypeptide monomer containing a SIRPβ variant that includes one or more amino acid substitutions, insertions, deletions, N-terminal elongation, or C-terminal elongation compared to wild-type SIRPβ or a fragment thereof, and the SIRPβ variant or a fragment thereof is bindable to CD47. In some embodiments, the SIRP multimer comprises a SIRP polypeptide monomer containing any one of the amino acid sequences of SEQ ID NOs. 33 to 45.In some embodiments, the SIRP multimer comprises SIRP polypeptide monomers comprising a fusion polypeptide. In some embodiments, the fusion polypeptide comprises a multimerization domain. In some embodiments, the multimerization domain comprises an Fc monomer, a c-Jun leucine zipper domain, or a c-Fos leucine zipper domain. In some embodiments, the Fc monomer is a mouse Fc monomer. In some embodiments, the mouse Fc monomer comprises the amino acid sequence set forth in any one of SEQ ID NOs: 81-83. In some embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 110. In some embodiments, the SIRP multimer comprises SIRP polypeptide monomers comprising an epitope tag or a ligand. In some embodiments, the epitope tag comprises any one of SEQ ID NOs: 7-32 and 126, or the ligand comprises biotin. In some embodiments, the SIRP multimer comprises soluble SIRP polypeptide monomers.

[0021] In some embodiments, the SIRP polymer comprises at least two SIRP polypeptide monomers linked via peptide bonds. In some embodiments, the SIRP polymer comprises at least two SIRP polypeptide monomers linked via a linker peptide. In some embodiments, the linker peptide comprises one of SEQ ID NOs: 85-109, 127-130, 140, and 141. In some embodiments, the linker peptide comprises one or more spacers. In some embodiments, the spacers comprise GS, GGS, or one of SEQ ID NOs: 52-70. In some embodiments, the SIRP polymer comprises at least two SIRP polypeptide monomers bound to a solid support. In some embodiments, the solid support is a gold nanosphere, gold nanoshell, magnetic beads, silica beads, dextran polymer, test tube, slide, gel column, or microtiter well. In some embodiments, each of at least two SIRP polypeptide monomers contains an epitope tag or ligand, and a scavenger that specifically binds to the epitope tag or ligand is immobilized on a solid support, and the SIRP polypeptide monomer is bound to the solid support by the specific binding of the epitope tag or ligand by the scavenger. In some embodiments, the ligand is biotin and the scavenger is streptavidin. In some embodiments, at least one of the at least two SIRP polypeptide monomers contains SEQ ID NO: 111. In some embodiments, the SIRP polymer contains streptavidin or avidin bound to two, three, or four biotinylated SIRP polypeptide monomers. In some embodiments, at least one of the two, three, or four biotinylated SIRP polypeptide monomers contains SEQ ID NO: 111. In some embodiments, the SIRP polymer is a homopolymer. In some embodiments, the SIRP polymer is a heteropolymer.

[0022] In some embodiments, anti-SIRP multimers comprising one or more anti-SIRP antibodies or drug-binding fragments thereof are provided herein. In some embodiments, the anti-SIRP multimer comprises 1 to 100 anti-SIRP antibodies or drug-binding fragments thereof. In some embodiments, the anti-SIRP multimer comprises wild-type SIRPα, SIRPα variants, SIRPβ variants, wild-type SIRPγ, SIRPγ variants, or anti-SIRP antibodies or drug-binding fragments bound to any two or more of the above. In some embodiments, the anti-SIRP multimer comprises anti-SIRP antibodies or drug-binding fragments comprising: (a) a heavy chain variable domain (V) comprising SEQ ID NO: 46 H ) and light chain variable domain (V) containing sequence number 47 L (b) Heavy chain variable domain containing sequence number 48 (V H ) and light chain variable domain (V) containing sequence number 49 L (c) Heavy chain variable domain containing sequence number 50 (V H ) and light chain variable domain (V) containing sequence number 51 L (d) Heavy chain variable domain containing sequence number 113 (V H ) and light chain variable domain (V) containing sequence number 114 L (e) Heavy chain variable domain containing sequence number 115 (V H ) and light chain variable domain (V) containing sequence number 116 L ); and / or (f) heavy chain variable domain (V) containing sequence number 133 H ) and light chain variable domain (V) containing sequence number 134 L). In some embodiments, the anti-SIRP multimer comprises a full-length anti-SIRP antibody. In some embodiments, the anti-SIRP antibody comprises a mouse Fc domain. In some embodiments, the mouse Fc domain comprises the amino acid sequence described in any one of SEQ ID NOs: 81-83. In some embodiments, the anti-SIRP antibody comprises: (a) a heavy chain containing SEQ ID NO: 117 and a light chain containing SEQ ID NO: 118; (b) a heavy chain containing SEQ ID NO: 119 and a light chain containing SEQ ID NO: 118; (c) a heavy chain containing SEQ ID NO: 120 and a light chain containing SEQ ID NO: 121; or (d) a heavy chain containing SEQ ID NO: 122 and a light chain containing SEQ ID NO: 121. In some embodiments, the drug-binding fragment of the anti-SIRP antibody is Fab, Fab', F(ab')2, Fab'-SH, Fv, diabody, one-arm antibody, scFv, scFv-Fc, single-domain antibody, or single-heavy-chain antibody. In some embodiments, the drug-binding fragment comprises F(ab')2, where F(ab')2 comprises SEQ ID NOs: 131 and 132. In some embodiments, the anti-SIRP antibody or its drug-binding fragment includes an epitope tag or ligand. In some embodiments, the epitope tag includes one of SEQ ID NOs. 7-32 and 126, or the ligand includes biotin. In some embodiments, the epitope tag includes HHHHHHGLNDIFEAQKIEWHE (SEQ ID NO: 135) or GSGSHHHHHGLNDIFEAQKIEWHE (SEQ ID NO: 126).

[0023] In some embodiments, the anti-SIRP multimer comprises one or more anti-SIRP antibodies or drug-binding fragments thereof bound to a solid support. In some embodiments, the solid support is a gold nanosphere, gold nanoshell, magnetic beads, silica beads, dextran polymer, test tube, slide, gel column, or microtiter well. In some embodiments, each of the one or more anti-SIRP antibodies or drug-binding fragments comprises an epitope tag or ligand, and a capture agent that specifically binds to the epitope tag or ligand is immobilized on the solid support, and the anti-SIRP antibody or drug-binding fragment is bound to the solid support by the specific binding of the epitope tag or ligand by the capture agent. In some embodiments, the ligand is biotin, and the capture agent is streptavidin. In some embodiments, the anti-SIRP multimer comprises two, three, or four biotinylated anti-SIRP antibodies or fragments thereof bound to streptavidin or avidin. In some embodiments, the anti-SIRP multimer comprises streptavidin or avidin bound to two, three, or four biotinylated F(ab')2 fragments, where two or more biotinylated F(ab')2 fragments include SEQ ID NOs. 131 and 132. In some embodiments, the anti-SIRP multimer is a homomultimer. In some embodiments, the anti-SIRP multimer is a heteromultimer.

[0024] All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are incorporated herein by reference in whole, as if each individual reference were specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawing]

[0025] [Figure 1A]This describes a serological assay in which a plasma sample obtained from a subject is mixed with reagent red blood cells (i.e., red blood cells known to express a specific cell surface antigen, or group of cell surface antigens ("RBCs")) to detect the presence of antibodies in a plasma sample that bind to RBC surface antigens. Alternatively, such a serological assay may 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.

[0026] [Figure 1B] This shows how the presence of a drug containing (i) an antibody Fc region and (ii) a portion that binds to human CD47 in a plasma sample interferes with the assay in Figure 1A.

[0027] [Figure 1C] This describes a serological assay in which a blood sample obtained from a subject is mixed with reagent plasma / antiserum (i.e., plasma or antiserum known to contain antibodies against specific RBC surface antigens or platelet surface antigens) to detect the presence of antigens on the target RBCs and / or platelets.

[0028] [Figure 1D] Figure 1C illustrates how the presence of a drug containing (i) an antibody Fc region and (ii) a portion that binds to human CD47 in a blood sample interferes with the assay.

[0029] [Figure 2] This paper describes a method for reducing interference in serological assays, which involves adding CD47 multimers to plasma samples obtained from drug-treated subjects. In summary, CD47 multimers bind to drugs in plasma samples, resulting in little to no drugs available to bind to CD47 on the surface of reagent RBCs or reagent platelets.

[0030] [Figure 3A]This paper describes a method for reducing interference in serological assays, which involves adding a CD47-binding SIRP multimer to reagent RBCs or reagent platelets. In summary, the SIRP multimer binds to CD47 on the surface of reagent RBCs or reagent platelets. The binding of the SIRP multimer to the reagent RBCs (or reagent platelets) blocks the binding of the drug to the reagent RBCs (or reagent platelets).

[0031] [Figure 3B] This paper describes a method to reduce interference in serological assays, including the addition of a CD47-binding SIRP multimer to plasma derived from drug-treated subjects. In summary, the SIRP multimer competes with the drug for binding to CD47 expressed on the surface of reagent RBCs or reagent platelets, minimizing the amount of drug-binding reagent RBCs or drug-binding reagent platelets in the assay (or removing drug-binding reagent RBCs and / or drug-binding reagent platelets from the assay).

[0032] [Figure 3C] This paper describes a method to reduce interference in serological assays, including the addition of a CD47-binding SIRP multimer to blood samples derived from drug-treated subjects. In summary, the SIRP multimer competes with the drug for binding to CD47 expressed on the surface of the subject's RBCs and / or platelets, minimizing (or removing) the amount of drug-bound RBCs and / or platelets in the assay.

[0033] [Figure 3D] This paper describes a method to reduce interference in serological assays, which involves adding a drug-binding SIRP multimer to plasma samples derived from drug-treated subjects. In summary, the SIRP multimer binds to the drug in the plasma sample, resulting in little to no drug available to bind to CD47 on the surface of reagent RBCs or reagent platelets.

[0034] [Figure 4]This report presents the results of experiments conducted in an IAT test tube assay to compare the degree to which CD47 polypeptide monomer, CD47 polymer B, anti-SIRP polymer C, and anti-SIRP polymer D inhibit interference by drug A.

[0035] [Figure 5] Further experimental results are provided for IAT test tube assays, which were conducted to compare the extent to which CD47 polypeptide monomer, CD47 polymer B, anti-SIRP polymer C, and anti-SIRP polymer D inhibit drug A interference.

[0036] [Figure 6] Further experimental results are provided for IAT test tube assays, which were conducted to compare the extent to which CD47 polypeptide monomer, CD47 polymer B, anti-SIRP polymer C, and anti-SIRP polymer D inhibit drug A interference.

[0037] [Figure 7] This paper presents the results of experiments conducted to compare the degree to which CD47 polypeptide monomer, CD47 polymer B, anti-SIRP polymer C, and anti-SIRP polymer D inhibit drug A interference in a solid-phase erythrocyte adhesion assay (SPRCA).

[0038] [Figure 8] This paper provides the results of experiments conducted to test whether anti-SIRP multimer C or anti-SIRP multimer F inhibits interference by drug A in a solid-phase erythrocyte adhesion assay (SPRCA). [Modes for carrying out the invention]

[0039] I. Methods to reduce interference from serological assays before blood transfusion. CD47 is a transmembrane protein that interacts with several molecules on immune cells, including thrombospondin 1 (TSP-1) and signal regulatory protein alpha (SIRPα). Upon binding to CD47, SIRPα initiates a signaling cascade that inhibits phagocytosis and prevents the immune system from removing healthy cells by phagocytic cells. However, many cancers overexpress CD47, evading phagocytic clearance. Therefore, drugs that target CD47 (e.g., anti-CD47 antibodies and fusion proteins containing the antibody Fc region and a CD47-binding portion) are of significant therapeutic interest. CD47 is also expressed on the surface of human red blood cells (RBCs) and platelets. Consequently, after administration of a drug containing (i) the antibody Fc region and (ii) a human CD47-binding portion to a target, interference in a given pre-transfusion serological assay may occur due to the presence of the drug in the target's plasma or the drug bound to the target's RBCs and / or platelets.

[0040] For example, Figure 1A shows a serological assay in which a plasma sample obtained from a subject is mixed with a reagent RBC or "reference RBC" (i.e., an RBC known to express a specific cell surface antigen or group of cell surface antigens), or a reagent platelet or "reference platelet" (i.e., a platelet known to express a specific cell surface antigen or group of cell surface antigens). This detects the presence of antibodies in the plasma sample that bind to cell surface antigens known to be expressed on the reagent RBC or reagent platelet. After the plasma sample and reagent RBC (or reagent platelet) are mixed, anti-human globulin (AHG) is added, and if the plasma sample contains antibodies that bind to the RBC surface antigen (or platelet surface antigen), aggregation of the reagent RBC (or reagent platelet) occurs (e.g., agglutination). However, if the plasma of the subject contains a drug that includes (i) an antibody Fc region and (ii) a portion that binds to human CD47, the assay may be interfered with, potentially leading to a false positive result. As shown in Figure 1B, after the target plasma and reagent RBCs (or reagent platelets) are mixed, the drug can bind to CD47 expressed on the surface of the reagent RBCs (or reagent platelets). When AHG is added to the mixture, aggregation of the reagent RBCs (or reagent platelets) occurs.

[0041] Figure 1C shows a serological assay for detecting the presence of antigens on target RBCs and / or platelets (a blood sample from the target is mixed with reagent plasma / antiserum (i.e., plasma or antiserum containing antibodies against known RBC surface antigens or known platelet surface antigens)). After mixing the reagent plasma / antiserum with the sample from the target, if the antigens recognized by the antibodies in the reagent plasma / antiserum are expressed on the target RBCs and / or platelets, agglutination will occur upon addition of AHG. If a drug containing (i) an antibody Fc region and (ii) a portion that binds to human CD47 is present in the sample containing the target RBCs and / or platelets, it may interfere with the assay and produce a false positive result. As shown in Figure 1D, after AHG is added to the mixture containing the target blood sample and reagent plasma / antiserum, drugs bound to CD47 on the target RBCs or platelets will cause agglutination.

[0042] The methods described below reduce (and in some embodiments eliminate) drug-induced interference, as shown in Figures 1B and 1D.

[0043] II. A method using drug-binding CD47 macromers to reduce interference from pre-transfusion serological assays. In some embodiments, the method comprises (a) adding a CD47 multimer bound to a drug (i.e., a portion of the drug containing a portion that binds to human CD47) to a plasma sample derived from a subject being treated with the drug, and (b) after step (a), performing a serological assay of the plasma sample using 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), wherein the drug contains (i) an antibody Fc region and (ii) a portion that binds to human CD47. Such embodiments are generally shown in Figure 2. As shown in Figure 2, the CD47 multimer binds to the drug in the plasma sample of the subject (e.g., the portion of the drug that binds to human CD47), blocking the drug from binding to the reagent RBCs and / or reagent platelets. There is little to no free drug bound to CD47 on the surface of the reagent RBCs and / or reagent platelets. Interference resulting from the binding of the drug to reagent RBCs and / or reagent platelets (shown in Figure 1B) is minimized (or, in some embodiments, eliminated), thereby preventing false-positive results in serological assays. In some embodiments, CD47 multimers are added to the plasma sample to obtain one of the anti-SIRP multimers in a molar excess of approximately 1x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, 10x, 10.5x, 11x, 11.5x, 12x, 12.5x, 13x, 13.5x, 14x, 14.5x, or 15x compared to the amount of drug in the plasma. In some embodiments, CD47 multimers are also added to reagent RBCs and / or reagent platelets before the serological assay is performed. In some embodiments, the CD47 macromer is added to the reagent RBC and / or reagent platelets (e.g., reagent RBC and / or reagent platelets only) before the serological assay is performed.

[0044] In some embodiments, the method is carried out in a solution in which, for example, the CD47 polymer is soluble. In some embodiments, the CD47 polymer is immobilized on a solid phase before the method is carried out via adsorption, covalent bonding, or non-covalent bonding to a matrix or surface. In some embodiments, the CD47 polymer is conjugable to a drug after immobilization on a solid phase or solid support. The solid phase or solid support used for immobilization can be any inert support, surface, or carrier that is essentially water-insoluble and useful for immunoassays, including, for example, supports in the form of surfaces, particles, porous matrices, cellulose polymer sponges (ImmunoCAP®, Phadia). Examples of supports commonly used include small sheets, Sephadex, polyvinyl chloride, plastic beads, gold beads, microparticles, assay plates, or test tubes made from polyethylene, polypropylene, polystyrene, etc. In some embodiments, the CD47 polymer is coated onto a microtiter plate, such as a multi-well microtiter plate, which can be used to analyze multiple samples simultaneously.

[0045] In some embodiments, the drug portion that binds to human CD47 includes wild-type SIRPα, a SIRPα variant, or a CD47-binding fragment of wild-type SIRPα or a SIRPα variant. In some embodiments, the drug portion that binds to human CD47 includes a SIRPα variant (or its CD47-binding fragment), which includes one or more amino acid substitutions, insertions, deletions, N-terminal extensions, and / or C-terminal extensions compared to wild-type SIRPα (or its CD47-binding fragment). In some embodiments, the drug portion that binds to human CD47 includes a fragment of a SIRPα variant, which includes the extracellular domain of a SIRPα variant. In some embodiments, the CD47 multimer is capable of binding to wild-type SIRPα, a SIRPα variant, or a fragment of wild-type SIRPα or a SIRPα variant.

[0046] In some embodiments, the drug portion that binds to human CD47 comprises wild-type SIRPγ, a SIRPγ variant, or a CD47-binding fragment of wild-type SIRPγ or a SIRPγ variant. In some embodiments, the drug portion that binds to human CD47 comprises a SIRPγ variant, which comprises one or more amino acid substitutions, insertions, deletions, N-terminal extensions, C-terminal extensions, or any combination thereof, compared to wild-type SIRPγ. In some embodiments, the drug portion that binds to human CD47 comprises a CD47-binding fragment of a SIRPγ variant, which comprises the extracellular domain of the SIRPγ variant. In some embodiments, the CD47 multimer is capable of binding to wild-type SIRPγ, a SIRPγ variant, or a fragment of wild-type SIRPγ or a SIRPγ variant.

[0047] In some embodiments, the drug portion that binds to human CD47 comprises a SIRPβ variant or a fragment of a SIRPβ variant that can bind to CD47 (e.g., human CD47). In some embodiments, the drug portion that binds to human CD47 comprises a SIRPβ variant, which comprises one or more amino acid substitutions, insertions, deletions, N-terminal extensions, C-terminal extensions, or any combination thereof, compared to wild-type SIRPβ. In some embodiments, the drug portion that binds to human CD47 comprises a fragment of a SIRPβ variant, which comprises the extracellular domain of the SIRPβ variant and is capable of binding to CD47 (e.g., human CD47). In some embodiments, a CD47 multimer is capable of binding to a SIRPβ variant or a CD47-binding fragment of a SIRPβ variant.

[0048] In some embodiments, the drug comprises an anti-CD47 antibody (or its CD47-binding fragment), and the CD47 polymer is capable of binding to the anti-CD47 antibody (or its CD47-binding fragment).

[0049] (a) CD47 polymer containing CD47 polypeptide monomer In some embodiments, the CD47 multimer comprises multiple CD47 polypeptide monomers. In some embodiments, the CD47 multimer comprises at least one CD47 polypeptide monomer from 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or up to 100 (including any range between these values). In some embodiments, the CD47 polypeptide monomer comprises the extracellular domain of wild-type CD47 ("WTCD47-ECD"), or a portion of WT CD47-ECD, which is capable of binding to a drug and blocking the drug from binding to reagent RBCs and / or reagent platelets. In some embodiments, the CD47 polypeptide monomer is a soluble polypeptide that does not contain the transmembrane domain of CD47 or any portion thereof. In some embodiments, the CD47 polypeptide monomer comprises a fusion polypeptide, e.g., a fusion polypeptide containing CD47 (or a fragment thereof). In some embodiments, the fusion polypeptide comprises a CD47 polypeptide monomer (or a fragment thereof) and, e.g., an antibody Fc domain, e.g., a mouse Fc domain. As discussed in further detail elsewhere in this specification, in some embodiments, the fusion polypeptide comprises a CD47 polypeptide monomer and a polymerizing domain. In some embodiments, the CD47 polypeptide monomer comprises human CD47, mouse CD47, rat CD47, rhesus monkey CD47, cynomolgus monkey CD47, or CD47 of any origin, capable of binding to a drug to block the drug from binding to reagent RBCs and / or reagent platelets. In some embodiments, the CD47 polypeptide monomer comprises a fragment of human CD47, mouse CD47, rat CD47, rhesus monkey CD47, cynomolgus monkey CD47, or CD47 of any origin. However, the fragment can bind to the drug, blocking the drug from binding to the reagent RBC and / or reagent platelet.In some embodiments, the CD47 polypeptide monomer comprises a variant of wild-type CD47 (or a fragment thereof, e.g., a variant of WT CD47-ECD or CD47 that does not contain the transmembrane domain or any part of CD47), provided that the variant is drug-binding. In some embodiments, the variant (or fragment thereof) comprises one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions compared to wild-type CD47 (e.g., wild-type human, rat, mouse, rhesus monkey, or cynomolgus monkey CD47). In some embodiments, one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions present in a variant (i.e., a "CD47 variant") alter the glycosylation pattern of the CD47 variant compared to wild-type CD47 (e.g., wild-type human, rat, mouse, rhesus monkey, or cynomolgus monkey CD47). In some embodiments, one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions present in a CD47 variant increase the affinity of the CD47 variant for a drug compared to wild-type CD47 (e.g., wild-type human, rat, mouse, rhesus monkey, or cynomolgus monkey CD47). In some embodiments, the affinity of a drug for the CD47 polypeptide monomer is greater than the affinity of the drug for human CD47.

[0050] In some embodiments, the CD47 polypeptide monomer comprises a CD47 variant containing one of the following amino acid sequences: QLLFNKTKSV EFTFSNDTVV IPCFVTNMEA QNTTEVYVKW KFKGRDIYTF DGALNKSTVP TDFSSAKIEV SQLLKGDASL KMDKSDAVSH TGNYTCEVTE LTREGETIIE LKYRVVS(Sequence ID 1) WQLPLLFNKT KSVEFTFGND TVVIPCFVTN MEAQNTTEVY VKWKFKGRDI YTFDGDKNKS TVPTDFSSAK IEVSQLLKGD ASLKMDKSDA VSHTGNYTCE VTELTREGET IIELKYRVVS(Sequence ID 2) WQPPLLFNKT KSVEFTFGND TVVIPCFVTN MEAQNTTEVY VKWKFKGRDI YTFDGQANKS TVPTDFSSAK IEVSQLLKGD ASLKMDKSDA VSHTGNYTCE VTELTREGET IIELKYRVVS(Sequence ID 3) WQPPLLFNKT KSVEFTFCND TVVIPCFVTN MEAQNTTEVY VKWKFKGRDI YTFDGQANKS TVPTDFSSAK IEVSQLLKGD ASLKMDKSDA VSHTGNYTCE VTELTREGET IIELKYRVVS(Sequence ID 4) WQPPLLFNKT KSVEFTCGND TVVIPCFVTN MEAQNTTEVY VKWKFKGRDI YTFDGQANKS TVPTDFSSAK IEVSQLLKGD ASLKMDKSDA VSHTGNYTCE VTELTREGET IIELKYRVVS(Sequence ID 5) QLLFNKTKSV EFTFSNDTVV IPCFVTNMEA QNTTEVYVKW KFKGRDIYTF DGALNKSTVP TDFSSAKIEV SQLLKGDASL KMDKSDAVSH TGNYTCEVTE LTREGETIIE LKYRVVSHHH HHHGLNDIFE AQKIEWHE(Sequence ID 6)

[0051] In some embodiments, the CD47 polymer comprises SEQ ID NO: 6. In some embodiments, the CD47 polymer is added to a plasma sample (e.g., a plasma sample obtained from a subject being treated with a drug) to obtain one of the following CD47 polymers in a molar excess of approximately 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times the amount of drug in the plasma.

[0052] Further details regarding exemplary CD47 polypeptide monomers (e.g., including CD47 variants) that can be polymerized and used in the methods described herein are provided in Ho et al. (2015) “'Velcro' Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein a(SIRPα) Antagonists That Enhance Antibody-Dependent Cellular Phagocytosis.” J Biol Chem. 290:12650-12663 and WO2016 / 179399, the contents of which are incorporated herein by reference in their entirety.

[0053] In some embodiments, the CD47 polypeptide monomer comprises a fusion polypeptide containing a polymerizing domain. Exemplary polymerizing domains include, but are not limited to, Fc monomers such as the mouse Fc monomer, the c-Jun leucine zipper domain, and the c-Fos leucine zipper domain. Each of these polymerizing domains is capable of forming a dimer. In some embodiments, the polymerization includes repeating units of GVGVP (SEQ ID NO: 71), VPGG (SEQ ID NO: 142), APGVGV (SEQ ID NO: 72), GAGAGS (SEQ ID NO: 73), GPGGG (SEQ ID NO: 74), GPGGX (wherein X is any amino acid (SEQ ID NO: 123)), GPGQQ (SEQ ID NO: 124), GPGGY (SEQ ID NO: 125), GGYGPGS (SEQ ID NO: 75), GAPGAPGSQGAPGLQ (SEQ ID NO: 76), GAPGTPGPQGLPGSP (SEQ ID NO: 77), AKLKLAEAKLELA (SEQ ID NO: 78), PPAKVPEVPEPKKPVPEEKVPVPKKPEA (SEQ ID NO: 79), and / or GGFGGMGGGX (wherein X is any amino acid (SEQ ID NO: 80)). For example, see: Tatham et al. (2000) Trends in Biochemical Sciences, 25, 567-571; Sanford and Kumar (2005) Current Opinion in Biotechnology, 16, 416-421; and Casal et al. (2014) Future Trends for Recombinant Protein-Based Polymers: The Case Study of Development and Application of Silk-Elastin-Like Polymers. In Kabasci (Ed.) Bio-Based Plastics: Materials and Applications (pp. 311-32) John Wiley & Sons, Ltd.

[0054] In some embodiments, the CD47 polypeptide monomer comprises a fusion polypeptide containing one of SEQ ID NOs: 1-6 and one of SEQ ID NOs: 81-83. The amino acid sequences of SEQ ID NOs: 81-83 are described below. SEQ ID NO: 81 contains the CH2 and CH3 domains of mouse IgG1. SEQ ID NO: 82 contains the CH2 and CH3 domains of mouse IgG1, the CH3 domain containing the N297A substitution, and the amino acid numbering follows the Kabat EU index. SEQ ID NO: 83 contains the CH2 and CH3 domains of mouse IgG2a. VPRDSGCKPC ICTVPEVSSV FIFPPKPKDV LTITLTPKVT CVVVDISKDD PEVQFSWFVD DVEVHTAQTQ PREEQFNSTF RSVSELPIMH QDWLNGKEFK CRVNSAAFPA PIEKTISKTK GRPKAPQVYT IPPPKEQMAK DKVSLTCMIT DFFPEDITVE WQWNGQPAEN YKNTQPIMDT DGSYFIYSKL NVQKSNWEAG NTFTCSVLHE GLHNHHTEKS LSHSPG (Sequence number 81) VPRDSGCKPC ICTVPEVSSV FIFPPKPKDV LTITLTPKVT CVVVDISKDD PEVQFSWFVD DVEVHTAQTQ PREEQFASTF RSVSELPIMH QDWLNGKEFK CRVNSAAFPA PIEKTISKTK GRPKAPQVYT IPPPKEQMAK DKVSLTCMIT DFFPEDITVE WQWNGQPAEN YKNTQPIMDT DGSYFIYSKL NVQKSNWEAG NTFTCSVLHE GLHNHHTEKS LSHSPG (SEQ ID NO: 82) EPRGPTIKPC PPCKCPAPNL LGGPSVFIFP PKIKDVLMIS LSPIVTCVVV DVSEDDPDVQ ISWFVNNVEV HTAQTQTHRE DYNSTLRVVS ALPIQHQDWM SGKEFKCKVN NKDLPAPIER TISKPKGSVR APQVYVLPPP EEEMTKKQVT LTCMVTDFMP EDIYVEWTNN GKTELNYKNT EPVLDSDGSY FMYSKLRVEK KNWVERNSYS CSVVHEGLHN HHTTKSFSRT PG (Sequence ID 83)

[0055] In some embodiments, the CD47 polypeptide monomer comprises a fusion polypeptide whose amino acid sequence is described in any one of the following SEQ ID NOs: 84-86. QLLFNKTKSV EFTFSNDTVV IPCFVTNMEA QNTTEVYVKW KFKGRDIYTF DGALNKSTVP TDFSSAKIEV SQLLKGDASL KMDKSDAVSH TGNYTCEVTE LTREGETIIE LKYRVVSVPR DSGCKPCICT VPEVSSVFIF PPKPKDVLTI TLTPKVTCVV VDISKDDPEV QFSWFVDDVE VHTAQTQPRE EQFNSTFRSV SELPIMHQDW LNGKEFKCRV NSAAFPAPIE KTISKTKGRP KAPQVYTIPP PKEQMAKDKV SLTCMITDFF PEDITVEWQW NGQPAENYKN TQPIMDTDGS YFIYSKLNVQ KSNWEAGNTF TCSVLHEGLH NHHTEKSLSH SPG(Sequence ID 84) QLLFNKTKSV EFTFSNDTVV IPCFVTNMEA QNTTEVYVKW KFKGRDIYTF DGALNKSTVP TDFSSAKIEV SQLLKGDASL KMDKSDAVSH TGNYTCEVTE LTREGETIIE LKYRVVSVPR DSGCKPCICT VPEVSSVFIF PPKPKDVLTI TLTPKVTCVV VDISKDDPEV QFSWFVDDVE VHTAQTQPRE EQFASTFRSV SELPIMHQDW LNGKEFKCRV NSAAFPAPIE KTISKTKGRP KAPQVYTIPP PKEQMAKDKV SLTCMITDFF PEDITVEWQW NGQPAENYKN TQPIMDTDGS YFIYSKLNVQ KSNWEAGNTF TCSVLHEGLH NHHTEKSLSH SPG(Sequence ID 85) QLLFNKTKSV EFTFSNDTVV IPCFVTNMEA QNTTEVYVKW KFKGRDIYTF DGALNKSTVP TDFSSAKIEV SQLLKGDASL KMDKSDAVSH TGNYTCEVTE LTREGETIIE LKYRVVSEPR GPTIKPSPPC KCPAPNLLGG PSVFIFPPKI KDVLMISLSP IVTCVVVDVS EDDPDVQISW FVNNVEVHTA QTQTHREDYN STLRVVSALP IQHQDWMSGK EFKCKVNNKD LPAPIERTIS KPKGSVRAPQ VYVLPPPEEE MTKKQVTLTC MVTDFMPEDI YVEWTNNGKT ELNYKNTEPV LDSDGSYFMY SKLRVEKKNW VERNSYSCSV VHEGLHNHHT TKSFSRTPG(Sequence ID 86)

[0056] In some embodiments, the CD47 polypeptide monomer (e.g., fusion polypeptide) includes an epitope tag (e.g., further includes). In some embodiments, the epitope tag facilitates the polymerization of the CD47 polypeptide monomer. In some embodiments, the tag facilitates the immobilization of the CD47 polypeptide monomer onto a solid support (e.g., beads, glass slides, etc.). Examples of epitope tags include, for example, HHHHHH (sequence number 7), GLNDIFEAQKIEWHE (sequence number 8), SRLEEELRRRLTE (sequence number 9), KRRWKKNFIAVSAANRFKKISSSGAL (sequence number 10), polyglutamic acid tags such as EEEEEE (sequence number 11), GAVVPYPDPLEPR (sequence number 12), DYKDDDDK (sequence number 13), YPYDVPDYA (sequence number 14), TKENPRSNQEESYDDNES (sequence number 15), TETSQVAPA (sequence number 16), KETAAAKFERQHMDS (sequence number 17), MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (sequence number 18), S Examples include, but are not limited to, LAELLNAGLGGS (SEQ ID NO: 19), TQDPSRVG (SEQ ID NO: 20), WSHPQFEK (SEQ ID NO: 21), MASMTGGQQMG (SEQ ID NO: 22), EVHTNQDPLD (SEQ ID NO: 23); GKPIPNPLLGLDST (SEQ ID NO: 24), YTDIEMNRLGK (SEQ ID NO: 25), DLYDDDDK (SEQ ID NO: 26), TDKDMTITFTNKKDAE (SEQ ID NO: 27), AHIVMVDAYKPTK (SEQ ID NO: 28), KLGDIEFIKVNK (SEQ ID NO: 29), KLGSIEFIKVNK (SEQ ID NO: 30), DIPATYEFTDGKHYITNEPIPPK (SEQ ID NO: 31), and DPIVMIDNDKPIT (SEQ ID NO: 32).

[0057] In some embodiments, the CD47 polypeptide monomer contains a ligand (e.g., is bound to a ligand). In some embodiments, the ligand is biotin.

[0058] In some embodiments, the CD47 polypeptide monomer contains the amino acid sequence of SEQ ID NO: 6 (see above). SEQ ID NO: 6 contains, from N-terminus to C-terminus, the amino acid sequence of WT human CD47, a hexahistidine peptide (i.e., HHHHHH (SEQ ID NO: 7)), and a 15-amino acid tag GLNDIFEAQKIEWHE (SEQ ID NO: 8). GLNDIFEAQKIEWHE (SEQ ID NO: 8), also known as AVITAG®, is specifically biotinylated by the E. coli biotin ligase BirA. In some embodiments, the CD47 polypeptide monomer contains the amino acid sequence of SEQ ID NO: G, from N-terminus to C-terminus, the amino acid sequence of WT human CD47 and a hexahistidine peptide (i.e., HHHHHH (SEQ ID NO: 7)).

[0059] In some embodiments, the CD47 polymer is a homopolymer containing identical CD47 polypeptide monomers (e.g., 2 to 100 identical CD47 polypeptide monomers as described herein). In some embodiments, the CD47 polymer is a heteropolymer containing at least two different CD47 polypeptide monomers (e.g., CD47 polypeptide monomers as described herein). CD47 heteropolymers containing any combination of two or more different CD47 polypeptide monomers are intended.

[0060] In some embodiments, CD47 polypeptide monomers in a CD47 polymer are linked via peptide bonds, for example, to form a chain of CD47 polypeptide monomers. In some embodiments, CD47 polypeptide monomers in a CD47 polymer are linked via linker peptides. Exemplary linker peptides include, but are not limited to, the following: LSGX1RX2X3SX4DNH (SEQ ID NO: 127) (wherein each of X1 to X4 is any naturally occurring amino acid); X1SGSRKX2RVX3X4X5 (SEQ ID NO: 128) (wherein each of X1 to X5 is any naturally occurring amino acid); SGRXSA (SEQ ID NO: 129) (wherein X is any naturally occurring amino acid); LSGX1RX2X3SX4DNH (Sequence ID 130) (wherein X1 to X4 is any naturally occurring amino acid);RX1X2X3RKX4VX5X6GX7 (Sequence ID 137) (wherein X1 to X7 is any naturally occurring amino acid);RQARXVV (Sequence ID 138) (wherein X is any naturally occurring amino acid);RX1X2RKVX3G (Sequence ID 87) (wherein X1 to X3 is any naturally occurring amino acid);KRRKQGASRKA (Sequence ID: 88); LSGX1RX2X3SX4DNH (Sequence ID: 89) (wherein X1 to X4 are any naturally occurring amino acids); X1X2X3X4X5X6NX7X8X9 (Sequence ID: 90) (wherein X1 to X9 are any naturally occurring amino acids); AANXL (Sequence ID: 91) (wherein X is any naturally occurring amino acid); ATNXL (Sequence ID: 139) (wherein X is any naturally occurring amino acid); SIS QX1YQRSSX2X3 (SEQ ID NO: 92) (wherein X1 to X3 are any naturally occurring amino acids); SSKLQ (SEQ ID NO: 93); X1PX2X3LIX4X5X6 (SEQ ID NO: 94) (wherein X1 to X6 are any naturally occurring amino acids); GPAX1GLX2GX3 (SEQ ID NO: 95) (wherein X1 to X3 are any naturally occurring amino acids); GPLGIAGQ (SEQ ID NO: 96); PVGLIG (SEQ ID NO: 97);HPVGLLAR(SEQ ID NO: 98); X1X2X3VIATX4X5X6X7(SEQ ID NO: 99) (wherein X1 to X7 are any naturally occurring amino acids); X1YYVTAX2X3X4X5(SEQ ID NO: 100) (wherein X1 to X5 are any naturally occurring amino acids); PRFKIIGG(SEQ ID NO: 101); PRFRIIGG(SEQ ID NO: 102); SSRHRRALD(SEQ ID NO: 103); RKSSIIIRMRDVVL(SEQ ID NO: 104); SSSFDKGKYKGDDA(SEQ ID NO: 105); SSSFDKGKYKRGDDA(SEQ ID NO: 106); IEGR(SEQ ID NO: 141); IDGR(SEQ ID NO: 140); GGSIDGR(SEQ ID NO: 107); PLGLWA(SEQ ID NO: 108); and DVAQFVLT(SEQ ID NO: 109).

[0061] In some embodiments, CD47 polypeptide monomers in a CD47 polymer are linked via a linker peptide and at least one spacer. In some embodiments, the spacer contains 3 to 200 amino acids. In some embodiments, the spacer contains one or more glycine and / or serine residues. In certain embodiments, the spacer contains multiple or repeating motifs including GS, GGS, GGGGS (SEQ ID NO: 52), GGSG (SEQ ID NO: 53), or SGGG (SEQ ID NO: 54). In certain embodiments, the spacer contains multiple or repeating motifs including GSGS (SEQ ID NO: 55), GSGSGS (SEQ ID NO: 56), GSGSGSGS (SEQ ID NO: 57), GSGSGSGSGS (SEQ ID NO: 58), or GSGSGSGSGSGS (SEQ ID NO: 59). In some embodiments, the spacer contains multiple or repeating motifs including GGS, e.g., GGSGGS (SEQ ID NO: 60), GGSGGSGGS (SEQ ID NO: 61), and GGSGGSGGSGGS (SEQ ID NO: 136). In some embodiments, the spacer includes multiple or repeating motifs including GGSG (SEQ ID NO: 53), GGSGGGSG (SEQ ID NO: 62), or GGSGGGSGGGSG (SEQ ID NO: 63). In some embodiments, the spacer includes multiple repetitions of SEQ ID NO: 52, for example, 2 to 10 repetitions. In some embodiments, the spacer includes GENLYFQSGG (SEQ ID NO: 64), SACYCELS (SEQ ID NO: 65), RSIAT (SEQ ID NO: 66), RPACKIPNDLKQKVMNH (SEQ ID NO: 67), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 68), AAANSSIDLISVPVDSR (SEQ ID NO: 69), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 70).

[0062] In some embodiments, the CD47 polymer comprises a plurality of CD47 polypeptide monomers (e.g., 2 to 100 CD47 polypeptide monomers) bound to a solid support. In some embodiments, the CD47 polypeptide monomers are bound to the solid support via covalent or non-covalent capture. In some embodiments, the solid support is a gold nanosphere, gold nanoshell, magnetic beads, silica beads, dextran polymer, test tube, slide, gel column, or microtiter well. In some embodiments, the CD47 polymer comprises a plurality of CD47 polypeptide monomers (e.g., two or more CD47 polypeptide monomers) and a solid support, each of which contains an epitope tag or ligand (e.g., as described above), and the capture agent is immobilized on the solid support, and the CD47 polypeptide monomers are bound to the solid support via specific binding of the epitope tag or ligand by the capture agent immobilized on the solid support. In some embodiments, the ligand is biotin and the capture agent is streptavidin. In some embodiments, the CD47 polymer (e.g., homopolymer or heteropolymer) comprises streptavidin or avidin bound to two, three, or four biotinylated CD47 polypeptide monomers. In some embodiments, the biotinylated CD47 polypeptide monomer is produced by biotinylation of SEQ ID NO: 6, for example, as described in the examples.

[0063] (b) Method for preparing CD47 polymers (i) Recombination generation In some embodiments, the CD47 multimer (e.g., a homomultimer or heteromultimer comprising at least two CD47 polypeptide monomers linked via peptide bonds or linker peptides) is generated by recombinant host cells. The host cell refers to a vehicle containing the necessary cellular components, e.g., organelles, required to express the CD47 multimer described herein from the corresponding nucleic acid. The nucleic acid may be contained in a nucleic acid vector that can be introduced into the host cell by conventional methods known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.). The selection of the nucleic acid vector depends in part on the host cell to be used. Generally, the host cell is either of prokaryotic (e.g., bacteria) or eukaryotic (e.g., mammalian) origin.

[0064] (A) Nucleic acids, vectors, and host cells The polynucleotide sequence encoding the amino acid sequence of the CD47 multimer can be prepared by various methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. The polynucleotide molecule encoding the CD47 multimer can be obtained using standard methods, e.g., gene synthesis. In some embodiments, the polynucleotide molecule encoding the CD47 multimer can be mutated to contain specific substitutions using standard methods in the art, e.g., QUIKCHANGE® mutagenesis. The polynucleotide can be synthesized using a nucleotide synthesizer or PCR method. The polynucleotide sequence encoding the CD47 multimer can be inserted into a vector capable of replicating and expressing the polynucleotide in prokaryotic or eukaryotic host cells. Many vectors are available in the art. Each vector may contain various components that can be tuned and optimized for compatibility with specific host cells. For example, vector components may include, but are not limited to, an origin of replication, a selection marker gene, a promoter, a ribosome binding site, a signal sequence, a polynucleotide sequence encoding the CD47 multimer, and a transcription termination sequence. In some embodiments, the vector may include an internal ribosome entry site (IRES) that enables the expression of multiple CD47 multimers. Some examples of bacterial expression vectors include, but are not limited to, the pGEX series vectors (e.g., pGEX-2T, pGEX-3X, pGEX-4T, pGEX-5X, pGEX-6P), the pET series vectors (e.g., pET-21, pET-21a, pET-21b, pET-23, pET-24), the pACYC series vectors (e.g., pACYDuet-1), the pDEST series vectors (e.g., pDEST14, pDEST15, pDEST24, pDEST42), and pBR322 and its derivatives (e.g., see U.S. Patent No. 5,648,237). Some examples of mammalian expression vectors include, but are not limited to, pCDNA3, pCDNA4, pNICE, pSELECT, and pFLAG-CMV.Other types of nucleic acid vectors include viral vectors used to express proteins within host cells (for example, baculovirus vectors used to express proteins within insect host cells).

[0065] In some embodiments, E. coli cells are used as host cells. Examples of E. coli strains include, but are not limited to, E. coli 294 (ATCC® 31,446), E. coli λ 1776 (ATCC® 31,537), E. coli BL21 (DE3) (ATCC® BAA-1025), and E. coli RV308 (ATCC® 31,608). In some embodiments, mammalian cells are used as host cells. Examples of mammalian cell types include, but are not limited to, human fetal kidney (HEK) cells, Chinese hamster ovary (CHO) cells, HeLa cells, PC3 cells, Vero cells, and MC3T3 cells. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of protein products. Appropriate cell lines or host systems may be selected to ensure proper modification and processing of expressed proteins. The expression vectors described above can be introduced into suitable host cells using conventional methods in the art, such as transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. Once the vectors are introduced into host cells for protein synthesis, the host cells are cultured in conventional nutrient media that have been appropriately modified for promoter induction, transformant selection, or amplification of the gene encoding the desired sequence.

[0066] (B) Protein production, recovery, and purification Host cells used to produce CD47 multimers can be grown in culture media suitable for host cell culture, selected from those known in the art. Examples of suitable media for bacterial host cells include Luria broth (LB) and necessary adjuvants, e.g., selective agents, e.g., ampicillin. Examples of suitable media for mammalian host cells include minimal essential medium (MEM), Dulbecco's modified Eagle medium (DMEM), DMEM supplemented with fetal bovine serum (FBS), and RPMI-1640. Host cells are cultured at a suitable temperature, e.g., about 20°C to about 39°C, e.g., 25°C to about 37°C. The pH of the medium is generally about 6.8 to about 7.4, e.g., about 7.0, mainly depending on the host organism. When an inducible promoter is used in the expression vector, protein expression is induced under conditions suitable for promoter activation. In some embodiments, the recovery of CD47 multimers produced by host cells usually involves disruption of the host cells by means such as osmotic shock, sonication, or lysis. Once the cells are destroyed, the cell debris can be removed by centrifugation or filtration. In some embodiments, the CD47 multimer is secreted into the culture medium. The supernatant of such an expression system is generally first concentrated using a commercially available protein concentration filter, e.g., an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of exogenous contaminants. The CD47 multimer may be further purified, for example, by affinity resin chromatography. Standard protein purification methods known in the art can be used. The following procedure is an example of a preferred purification procedure: fractionation on an immunoaffinity or ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica or cation exchange resin, SOS-PAGE, and gel filtration.

[0067] (ii) Non-covalent capture to a solid support In some embodiments, CD47 polymers (e.g., CD47 homopolymers or CD47 heteropolymers) are produced by binding multiple CD47 polypeptide monomers (e.g., 2 to 100 CD47 polypeptide monomers) to a solid support via non-covalent capture. In some embodiments, the solid support is gold nanospheres, gold nanoshells, magnetic beads, silica beads, dextran polymer, test tubes, slides, gel columns, or microtiter wells. In some embodiments, the solid support includes, or is made from, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, each CD47 polypeptide monomer contains a ligand, and a capture agent is immobilized on the solid support. CD47 polymers are produced by binding CD47 polypeptide monomers to the solid support via specific binding of the ligand by the capture agent immobilized on the solid support. In some embodiments, the ligand is biotin, and the capture agent is streptavidin. CD47 polymers (e.g., homopolymers or heteropolymers) comprising at least two CD47 polypeptide monomers immobilized on a solid phase or solid support (e.g., the solid phase or solid support described herein) are also provided herein. In some embodiments, each CD47 polypeptide monomer is biotinylated, and the streptavidin molecule is immobilized on a solid support, with the CD47 polypeptide monomers bound to the solid support via specific binding of biotin to avidin.

[0068] In some embodiments, CD47 polymers are prepared by culturing host cells containing nucleic acids encoding the CD47 polypeptide monomer, including SEQ ID NO: 6, under appropriate conditions to induce expression of the CD47 polypeptide monomer, and then recovering the CD47 polypeptide monomer. The CD47 polypeptide monomer, containing the amino acid sequence of the biotin receptor peptide, is biotinylated using E. coli biotin ligase (BirA). Streptavidin and avidin are tetrameric biotin-binding glycoproteins. Each subunit of streptavidin and avidin exhibits high specificity and high affinity (K D = about 10 -15 ) can be bound to biotin. In some embodiments, CD47 polymers are produced by conjugating biotinylated CD47 polypeptide monomers to a solid support on which streptavidin or avidin molecules are immobilized. In some embodiments, CD47 polymers (e.g., homopolymers or heteropolymers) are provided, for example, comprising multiple CD47 polypeptide monomers (e.g., 2 to 100 CD47 polypeptide monomers) conjugated to a streptavidin or avidin conjugate solid support. Exemplary streptavidin or avidin conjugate solid supports include, but are not limited to, gold beads (e.g., available from Nanocs, Nanocomposix, and Cytodiagnostics), gold nanoshells (e.g., available from Nanocs, Nanocomposix, and Cytodiagnostics), dextran polymers (e.g., available from FinaBiosoltions), silica beads (e.g., available from BangsLabs, ThermoFisher, and VectorLabs), magnetic beads (e.g., available from CD Bioparticles, ThermoFisher, and SigmaAldrich), and Sepharose beads (e.g., available from BioVision and CellSignal). In some embodiments, CD47 polymers (e.g., homopolymers or heteropolymers) containing streptavidin or avidin conjugated to two, three, or four biotinylated CD47 polypeptide monomers are provided.

[0069] In some embodiments, CD47 polymers are produced, for example, by linking two or more CD47 polypeptide monomers together via a bifunctional protein coupling agent. Exemplary bifunctional protein coupling agents include, but are not limited to, N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (e.g., triene 2,6-diisocyanate), and bisactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0070] In some embodiments, CD47 polymers are produced, for example, by linking two or more CD47 polypeptide monomers to a solid support via a difunctional drug. Commonly used crosslinking agents include, but are not limited to, drugs such as 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, e.g., esters with 4-azidosalicylic acid, homodifunctional imide esters including disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl-propionate), difunctional maleimides, e.g., bis-N-maleimide-1,8-octane, and methyl-3-[(p-azidophenyl)-dithio]propioimidate.

[0071] Next, the CD47 macromers generated as described above are used in the manner provided herein to prevent interference by drugs that bind to CD47 in serological assays.

[0072] III. Method using CD47-binding SIRP multimers to reduce interference from pre-transfusion serological assays (a) Method using SIRP multimers that bind to human CD47 In some embodiments, the method comprises (a) adding a SIRP multimer that binds to human CD47 and does not contain an antibody Fc region that binds to anti-human globulin (AHG) to 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), and (b) after step (a), performing a serological assay of a plasma sample using the reagent RBCs and / or reagent platelets, wherein the plasma sample is derived from a subject that has been treated with a drug, and the drug comprises (i) an antibody Fc region and (ii) a portion that binds to human CD47. Such embodiments are generally shown in Figure 3A. As shown in Figure 3A, the SIRP multimer binds to CD47 expressed on the surface of the reagent RBC (and / or reagent platelet), blocking the drug from binding to the reagent RBC (and / or reagent platelet), thereby minimizing (or, in some embodiments, eliminating) the interference resulting from the binding of the drug to the reagent RBC and / or reagent platelet (shown in Figure 1B). In some embodiments, a CD47 multimer is added to a plasma sample to obtain one of the following SIRP multimers in a molar excess of approximately 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15 times compared to the amount of drug in the plasma sample derived from the subject. In some embodiments, the SIRP multimer is added to the plasma of the subject, as well as to reagent RBCs and / or reagent platelets, before the serological assay is performed. In some embodiments, a SIRP polymer is added to a plasma sample to obtain one of the anti-SIRP polymers in a molar excess of approximately 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15 times the amount of drug in the plasma.

[0073] In some embodiments, the method comprises (a) adding a SIRP multimer that binds to human CD47 and does not contain an antibody Fc region that binds to anti-human globulin (AHG) to a plasma sample from a subject treated with the drug, and (b) performing a serological assay of the plasma sample after step (a) using reagent RBCs and / or reagent platelets, wherein the drug comprises (i) an antibody Fc region and (ii) a portion that binds to human CD47. As shown in Figure 3B, the SIRP multimer competes with the drug for binding to CD47 expressed on the surface of the reagent RBC (and / or reagent platelet), thereby minimizing (or, in some embodiments, eliminating) interference (shown in Figure 1B) resulting from the binding of the drug to the reagent RBC and / or reagent platelet.

[0074] In some embodiments, the method comprises (a) adding a SIRP multimer that binds to human CD47 but does not contain an antibody Fc region that binds to anti-human globulin (AHG) to a blood sample from a subject treated with the drug, and (b) performing a serological assay of the blood sample using reagent plasma / antiserum after step (a), wherein the drug contains (i) an antibody Fc region and (ii) a portion that binds to human CD47. As shown in Figure 3C, the SIRP multimer 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 (shown in Figure 1D) resulting from the binding of the drug to the subject's RBCs and / or platelets. In some embodiments, the SIRP multimer is added to the reagent plasma / antiserum and the blood sample from the subject before the serological assay is performed.

[0075] In some embodiments, the method is carried out in a solution in which the SIRP polymer is soluble, for example. In some embodiments, the SIRP polymer is immobilized on a solid phase before the method is carried out via adsorption, covalent bonding, or non-covalent bonding to a matrix or surface. In some embodiments, the SIRP polymer is bondable to CD47 after immobilization on a solid phase or solid support. The solid phase or solid support used for immobilization can be any inert support, surface, or carrier that is essentially water-insoluble and useful for immunoassays, including supports in the form of surfaces, particles, porous matrices, cellulose polymer sponges (ImmunoCAP®, Phadia), etc. Examples of supports commonly used include small sheets, Sephadex, polyvinyl chloride, plastic beads, gold beads, microparticles, assay plates, or test tubes made from polyethylene, polypropylene, polystyrene, etc. In some embodiments, the SIRP polymer is coated onto a microtiter plate, such as a multi-well microtiter plate, which can be used to analyze multiple samples simultaneously.

[0076] In some embodiments, the drug portion that binds to human CD47 comprises wild-type SIRPα, a SIRPα variant, or a CD47-binding fragment of wild-type SIRPα or a SIRPα variant. In some embodiments, the drug portion that binds to human CD47 comprises a SIRPα variant (or its CD47-binding fragment), which comprises one or more amino acid substitutions, insertions, deletions, N-terminal extensions, and / or C-terminal extensions compared to wild-type SIRPα (or its CD47-binding fragment). In some embodiments, the drug portion that binds to human CD47 comprises a fragment of a SIRPα variant, which comprises the extracellular domain of the SIRPα variant.

[0077] In some embodiments, the drug portion that binds to human CD47 comprises wild-type SIRPγ, a SIRPγ variant, or a CD47-binding fragment of wild-type SIRPγ or a SIRPγ variant. In some embodiments, the drug portion that binds to human CD47 comprises a SIRPγ variant, which comprises one or more amino acid substitutions, insertions, deletions, N-terminal elongations, C-terminal elongations, or any combination thereof, compared to wild-type SIRPγ. In some embodiments, the drug portion that binds to human CD47 comprises a CD47-binding fragment of a SIRPγ variant, which comprises the extracellular domain of the SIRPγ variant.

[0078] In some embodiments, the drug portion that binds to human CD47 comprises a SIRPβ variant or a fragment of a SIRPβ variant that can bind to CD47 (e.g., human CD47). In some embodiments, the drug portion that binds to human CD47 comprises a SIRPβ variant, which comprises one or more amino acid substitutions, insertions, deletions, N-terminal extensions, C-terminal extensions, or any combination thereof, compared to wild-type SIRPβ. In some embodiments, the drug portion that binds to human CD47 comprises a fragment of a SIRPβ variant, which comprises the extracellular domain of the SIRPβ variant and is capable of binding to CD47 (e.g., human CD47).

[0079] In some embodiments, the drug comprises an anti-CD47 antibody.

[0080] (b) SIRP polymer containing a SIRP polypeptide monomer bound to CD47 In some embodiments, the SIRP polymer that binds to human CD47 comprises a plurality of SIRP polypeptide monomers. In some embodiments, the SIRP polymer comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or up to 100 SIRP polypeptide monomers that can bind to CD47 (e.g., human CD47). In some embodiments, “SIRP polypeptide monomer” refers to a SIRPα polypeptide monomer, a SIRPγ polypeptide monomer, or a SIRPβ variant polypeptide monomer that can bind to CD47 (e.g., human CD47). In some embodiments, the SIRP polypeptide monomer does not contain an antibody Fc region. Exemplary SIRPα polypeptide monomers, SIRPγ polypeptide monomers, and SIRPβ variant polypeptide monomers are described in further detail below.

[0081] In some embodiments, the SIRP multimer comprises a SIRPα polypeptide monomer that binds to human CD47. In some embodiments, the SIRPα polypeptide monomer comprises a CD47-binding fragment of SIRPα (e.g., the extracellular domain of wild-type SIRPα ("WT SIRPα-ECD") or its D1 domain). In some embodiments, the CD47-binding fragment of SIRPα is a soluble fragment (e.g., a fragment of SIRPα that does not contain the transmembrane domain or any part thereof). In some embodiments, the SIRPα polypeptide monomer comprises human SIRPα, mouse SIRPα, rat SIRPα, rhesus monkey SIRPα, cynomolgus monkey SIRPα, or SIRPα of any origin, provided that SIRPα is binding to CD47 (e.g., human CD47 expressed on the surface of reagent RBCs and / or reagent platelets). In some embodiments, the SIRPα polypeptide monomer includes human SIRPα, mouse SIRPα, rat SIRPα, rhesus monkey SIRPα, cynomolgus monkey SIRPα, or a fragment of SIRPα of any origin, provided that the fragment is bindable to CD47 (e.g., human CD47 expressed on the surface of reagent RBCs and / or reagent platelets). In some embodiments, the SIRPα polypeptide monomer includes a SIRPα variant (or its fragment, e.g., a variant of WT SIRPα-ECD or its D1 domain) that is bindable to CD47 (e.g., human CD47). In some embodiments, the CD47-bound SIRPα variant (or its fragment) includes one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions compared to wild-type SIRPα. In some embodiments, one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions present in a SIRPα variant (or its fragment (which can bind to CD47)) alter the glycosylation pattern of the SIRPα variant compared to wild-type SIRPα.In some embodiments, one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions present in the SIRPα variant (or its fragment (CD47-binding)) increase the affinity of the SIRPα variant (or its fragment (CD47-binding)) to human CD47 compared to wild-type SIRPα.

[0082] In some embodiments, the SIRP multimer comprises a SIRPγ polypeptide monomer that binds to human CD47. In some embodiments, the SIRPγ polypeptide monomer comprises a CD47-binding fragment of SIRPγ (e.g., the extracellular domain of wild-type SIRPγ ("WT SIRPγ-ECD") or its D1 domain). In some embodiments, the CD47-binding fragment of SIRPγ is a soluble fragment (e.g., a fragment of SIRPγ that does not contain the transmembrane domain or any part thereof). In some embodiments, the SIRPγ polypeptide monomer comprises human SIRPγ, mouse SIRPγ, rat SIRPγ, rhesus monkey SIRPγ, cynomolgus monkey SIRPγ, or SIRPγ of any origin, provided that SIRPγ is binding to CD47 (e.g., human CD47 expressed on the surface of reagent RBCs and / or reagent platelets). In some embodiments, the SIRPγ polypeptide monomer includes human SIRPγ, mouse SIRPγ, rat SIRPγ, rhesus monkey SIRPγ, cynomolgus monkey SIRPγ, or a fragment of SIRPγ of any origin, provided that the fragment is bindable to CD47 (e.g., human CD47 expressed on the surface of reagent RBCs and / or reagent platelets). In some embodiments, the SIRPγ polypeptide monomer includes a SIRPγ variant (or its fragment, e.g., a variant of WT SIRPγ-ECD or its D1 domain) that is bindable to CD47 (e.g., human CD47 expressed on the surface of reagent RBCs and / or reagent platelets). In some embodiments, the SIRPγ variant (or its fragment (CD47-binding)) includes one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions compared to wild-type SIRPγ. In some embodiments, one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions present in the SIRPγ variant (or its fragment (which can bind to CD47)) alter the glycosylation pattern of the SIRPγ variant compared to wild-type SIRPγ.In some embodiments, one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions present in the SIRPγ variant (or its fragment (CD47-binding)) increase the affinity of the SIRPγ variant to human CD47 compared to wild-type SIRPγ.

[0083] In some embodiments, the SIRP multimer comprises a SIRPβ variant polypeptide monomer that binds to human CD47, or a fragment thereof that binds to human CD47. In some embodiments, the CD47-binding-capable fragment of the SIRPβ variant polypeptide monomer is a soluble fragment (e.g., a fragment of the SIRPβ variant polypeptide that does not contain the transmembrane domain or any portion thereof). In some embodiments, the SIRPβ variant polypeptide monomer comprises one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions compared to wild-type SIRPβ, which increases the affinity of the SIRPβ variant polypeptide monomer to human CD47 compared to wild-type SIRPβ. In some embodiments, the SIRPβ variant polypeptide monomer comprises a CD47-binding-capable fragment of the SIRPβ variant (e.g., the extracellular domain of the SIRPβ variant or its D1 domain). In some embodiments, the CD47-binding SIRPβ variant fragment is a soluble fragment (e.g., a SIRPβ variant fragment that does not contain a transmembrane domain or any portion thereof). In some embodiments, the SIRPβ variant polypeptide monomer includes a variant of wild-type human SIRPβ, a wild-type mouse SIRPβ variant, a wild-type rat SIRPβ variant, a wild-type rhesus monkey SIRPβ variant, a wild-type cynomolgus monkey SIRPβ variant, or a SIRPβ variant of any origin, provided that the SIRPβ variant is binding to CD47 (e.g., human CD47 expressed on the surface of reagent RBCs and / or reagent platelets). In some embodiments, the SIRPβ variant polypeptide monomer includes a variant of wild-type human SIRPβ, a variant of wild-type mouse SIRPβ, a variant of wild-type rat SIRPβ, a wild-type variant, a variant of wild-type rhesus macaque SIRPβ, a variant of wild-type cynomolgus macaque SIRPβ, or a fragment of a SIRPβ variant of any origin.However, the fragment is bindable to CD47 (e.g., human CD47 expressed on the surface of reagent RBCs and / or reagent platelets). In some embodiments, one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions present in the SIRPβ variant polypeptide monomer (or its fragment (which is CD47-binding)) alter the glycosylation pattern of the SIRPβ variant polypeptide monomer compared to wild-type SIRPβ.

[0084] In some embodiments, the SIRPα polypeptide monomer, SIRPβ variant polypeptide monomer, or SIRPγ polypeptide monomer contains one of the following amino acid sequences: SEQ ID NOs: 33-41. EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQRQGPFPRV TTVSDTTKRN NMDFSIRIGA ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPS(Sequence ID 33) EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRELIY NQREGPFPRV TTVSDTTKRN NMDFSIRIGA ITPADAGTYY CVKFRKGSPD DVEFKSGAGT ELSVRAKPS(Sequence ID 34) EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQREGPFPRV TTVSDTTKRN NMDFSIRIGA ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPS(Sequence ID 35) EDELQIIQPE KSVSVAAGES ATLRCAITSL FPVGPIQWFR GAGAGRVLIY NQRQGPFPRV TTVSETTKRN NLDFSISISN ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPS(Sequence ID 36) EEELQIIQPD KSISVAAGES ATLHCTITSL FPVGPIQWFR GAGPGRVLIY NQRQGPFPRV TTVSDTTKRN NMDFSIRISN ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPS(Sequence ID 37) EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGTPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 38) EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 39) EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGHFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 40) EEELQIIQPE KLLLVTVGKT ATLHCTITSH FPVGPIQWFR GVGPGRVLIY NQKDGHFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 41) EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPVLWFR GVGPGRVLIY NQRQGPFPRV TTVSDTTKRN NMDFSIRISS ITPADVGTYY CVKFRKGTPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 42) EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRELIY NAREGRFPRV TTVSDLTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 43) EEELQIIQPE KLLLVTVGKT ATLHCTITSL LPVGPIQWFR GVGPGRELIY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGTPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 44) EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQRQGPFPRV TTVSDTTKRN NMDFSIRIGN ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPS(Sequence ID 45)

[0085] Further details regarding exemplary SIRPα polypeptide monomers (e.g., SIRPα variants), SIRPβ variant polypeptide monomers, and SIRPγ polypeptide monomers (e.g., SIRPγ variants) that can be polymerized and used by the methods described herein can be found in WO2013 / 109752;US2015 / 0071905;USP9,944,911;WO2016 / 023040;WO2017 / 027422;US2017 / 01072 Provided in 70;USP10,259,859;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.

[0086] In some embodiments, the SIRP polypeptide monomer includes a fusion polypeptide comprising a polymerizing domain, for example, but not limited to, the exemplary polymerizing domain described above relative to the CD47 polypeptide monomer.

[0087] In some embodiments, the SIRP polypeptide monomer comprises a fusion polypeptide containing a multimerizing domain described in any one of SEQ ID NOs: 33-45 and any one of SEQ ID NOs: 81-83. In some embodiments, the SIRP polypeptide monomer comprises a fusion polypeptide containing a multimerizing domain described in any one of SEQ ID NOs: 33-45 and 81-83. The invention comprises a fusion polypeptide comprising a SIRP polypeptide monomer disclosed in any one of 014094122;US2015329616;US20180312563;WO2018176132;WO2018081898;WO2018081897;US20180141986A1; and EP3287470A1, as well as a polymerizing domain described in any one of SEQ ID NOs. 81-83. In some embodiments, the SIRP polypeptide monomer comprises a fusion polypeptide comprising the amino acid sequence described in SEQ ID NO. 110. EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQRQGPFPRV TTVSDTTKRN NMDFSIRIGN ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPSA KTTAPSVYPL APVCGDTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRG PTIKPCPPCK CPAPNLLGGP SVFIFPKIK DVLMISLSPI VTCVVVDVSE DDPDVQISWF VNNVEVHTA QTQTHREDYN STLRVVSALP IQHQDWMSGK EFKCKVNNKD LPAPIERTIS KPKGSVRAPQ VYVLPPPEEE MTKKQVTLTC MVTDFMPEDI YVEWTNNGKT ELNYKNTEPV LDSDGSYFMY SKLRVEKKNW VERNSYSCSV VHEGLHNHHT TKSFSRTPG(Sequence ID 110).

[0088] In some embodiments, the SIRP polypeptide monomer (e.g., fusion polypeptide) includes an epitope tag (e.g., further includes). In some embodiments, the epitope tag facilitates the polymerization of the SIRP polypeptide monomer. In some embodiments, the tag facilitates the immobilization of the SIRP polypeptide monomer onto a solid support (e.g., beads, glass surface, etc.). Exemplary epitope tags include, but are not limited to, SEQ ID NOs. 7-32 above for the CD47 polypeptide monomer.

[0089] In some embodiments, the SIRP polypeptide monomer contains a ligand (for example, one to which it is bound). In some embodiments, the ligand is biotin.

[0090] In some embodiments, the SIRP polypeptide monomer contains the amino acid sequence of SEQ ID NO: 111 (see below). SEQ ID NO: 111 contains, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 45, a hexahistidine peptide (i.e., HHHHHH (SEQ ID NO: 7)), and a 15-amino acid tag GLNDIFEAQKIEWHE (SEQ ID NO: 8). GLNDIFEAQKIEWHE (SEQ ID NO: 8), also known as AVITAG®, is specifically biotinylated by the E. coli biotin ligase BirA. In some embodiments, the CD47 polypeptide monomer contains the amino acid sequence of SEQ ID NO: 112 (see below), from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 45 and a hexahistidine peptide (i.e., HHHHHH (SEQ ID NO: 7)). EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQRQGPFPRV TTVSDTTKRN NMDFSIRIGN ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPSH HHHHHGLNDI FEAQKIEWHE(Sequence ID 111) EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQRQGPFPRV TTVSDTTKRN NMDFSIRIGN ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPSH HHHHH(Sequence ID 112)

[0091] In some embodiments, the SIRP polymer bound to human CD47 is a homopolymer comprising the same SIRP polypeptide monomer (e.g., the same SIRPα polypeptide monomer, the same SIRPβ variant polypeptide monomer, or the same SIRPγ polypeptide monomer, e.g., the SIRP polypeptide monomer described herein). In some embodiments, the SIRP polymer bound to human CD47 is a heteropolymer comprising at least two different SIRPα polypeptide monomers, two different SIRPβ variant polypeptide monomers, two different SIRPγ polypeptide monomers, or any combination thereof. A SIRP heteropolymer comprising any combination of two or more different SIRPα polypeptide monomers, SIRPβ variant polypeptide monomers, and / or SIRPγ polypeptide monomers (e.g., the SIRP polypeptide monomer described herein) is intended.

[0092] In some embodiments, SIRP polypeptide monomers in a SIRP polymer are linked, for example, via peptide bonds or linker peptides to form a chain of SIRP polypeptide monomers. In some embodiments, SIRP polypeptide monomers in a SIRP polymer are linked via linker peptides. Exemplary linker peptides include, but are not limited to, those described in SEQ ID NOs: 85-109, 127-130, 140, and 141, as well as other linkers used in CD47 polymers (see above). In some embodiments, SIRP polypeptide monomers in a SIRP polymer are linked via peptide linkers and at least one spacer. Exemplary peptide spacers include, but are not limited to, those described in SEQ ID NOs: 52-70, and other spacers used in CD47 polymers (see above).

[0093] In some embodiments, the SIRP polymer bound to human CD47 comprises multiple SIRP polypeptide monomers (e.g., 2 to 100 SIRP polypeptide monomers) bound to a solid support. In some embodiments, the SIRP polypeptide monomers are bound to the solid support via covalent or non-covalent capture. In some embodiments, the solid support is gold nanospheres, gold nanoshells, magnetic beads, silica beads, dextran polymer, test tubes, slides, gel columns, or microtiter wells. In some embodiments, the solid support comprises, or is made from, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, a SIRP multimer bound to human CD47 comprises a plurality of SIRP polypeptide monomers (e.g., two or more SIRP polypeptide monomers) and a solid support, each of which comprises an epitope tag or ligand (e.g., as described above), and a scavenger is immobilized on the solid support, with the SIRP polypeptide monomers bound to the solid support via specific binding of the epitope tag or ligand by the scavenger immobilized on the solid support. In some embodiments, the ligand is biotin and the scavenger is streptavidin. In some embodiments, the SIRP multimer (e.g., a homomultimer or a heteromultimer) comprises streptavidin or avidin bound to two, three, or four biotinylated SIRP polypeptide monomers.

[0094] (c) Method for producing SIRP polymers. In some embodiments, SIRP multimers (e.g., homo-multimers or hetero-multimers containing at least two SIRP polypeptide monomers linked via peptide bonds or linker peptides) are generated by recombinant host cells. Any method herein that generates CD47 multimers using recombinant techniques is similarly applicable to the generation of SIRP multimers containing SIRP polypeptide monomers.

[0095] In some embodiments, SIRP polymers (e.g., homo-polymers or hetero-polymers) are produced by binding multiple SIRP polypeptide monomers (e.g., 2 to 100 SIRP polypeptide monomers) to a solid support via non-covalent capture. In some embodiments, the solid support is a gold nanosphere, gold nanoshell, magnetic beads, silica beads, dextran polymer, test tube, slide, gel column, or microtiter well. In some embodiments, each SIRP polypeptide monomer contains a ligand, and a capture agent is immobilized on the solid support. The SIRP polymer is produced by binding the SIRP polypeptide monomers to the solid support via specific binding of the ligand by the capture agent immobilized on the solid support. In some embodiments, the ligand is biotin, and the capture agent is streptavidin.

[0096] Any method described elsewhere in this specification for conjugating CD47 polypeptide monomers to a solid support to produce CD47 polymers is similarly applicable to conjugating SIRP polypeptide monomers to a solid support to produce SIRP polymers. For example, in some embodiments, SIRP polymers are prepared by culturing host cells containing nucleic acids encoding a SIRP polypeptide monomer, including SEQ ID NO: 111, under appropriate conditions to induce expression of the SIRP polypeptide monomer, and then recovering the SIRP polypeptide monomer. The SIRP polypeptide monomer, containing the amino acid sequence of the biotin receptor peptide, is biotinylated using E. coli biotin ligase (BirA). In some embodiments, SIRP polymers are produced by conjugating the biotinylated SIRP polypeptide monomer to a solid support on which streptavidin or avidin molecules are immobilized. In some embodiments, a SIRP polymer (e.g., a homo-polymer or hetero-polymer) is provided, comprising, for example, multiple SIRP polypeptide monomers (e.g., 2 to 100 SIRP polypeptide monomers) conjugated to a streptavidin or avidin conjugate solid support. Exemplary streptavidin or avidin conjugate solid supports are described in further detail elsewhere in this specification. In some embodiments, a SIRP polymer (e.g., a homo-polymer or hetero-polymer) is provided, comprising streptavidin or avidin conjugated to 2, 3, or 4 biotinylated SIRP polypeptide monomers.

[0097] In some embodiments, SIRP polymers are produced by using a bifunctional crosslinking agent (such as one described elsewhere in this specification) to link one or more SIRP monomer polypeptides to, for example, a solid support, or to each other.

[0098] Next, the SIRP multimer thus produced is used in a manner provided herein to prevent interference by drugs that bind to CD47 in a serological assay.

[0099] IV. Methods for using anti-SIRP multimers to reduce interference in pre-transfusion serological assays (a) A method using an anti-SIRP polymer that binds to the drug. In some embodiments, the method comprises (a) adding an anti-SIRP multimer that binds to a drug (i.e., a portion of the drug that includes a portion that binds to human CD47) to a plasma sample derived from a subject treated with the drug, and (b) after step (a), performing a serological assay of the plasma sample using 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), wherein the drug includes (i) an antibody Fc region and (ii) a portion that binds to human CD47. Such embodiments are generally shown in Figure 3D. As shown in Figure 3D, the anti-SIRP multimer binds to the drug in the plasma sample of the subject (e.g., the portion of the drug that binds to human CD47), blocking the drug from binding to the reagent RBCs and / or reagent platelets. There are few or no drugs that can bind to CD47 on the surface of reagent RBCs and / or reagent platelets. Interference resulting from the binding of the drug to reagent RBCs and / or reagent platelets (shown in Figure 1B) is minimized (or, in some embodiments, eliminated), thereby preventing false-positive results in serological assays. In some embodiments, an anti-SIRP multimer is added to a plasma sample to obtain one of the following anti-SIRP multimers in molar excess of approximately 1x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, 10x, 10.5x, 11x, 11.5x, 12x, 12.5x, 13x, 13.5x, 14x, 14.5x, or 15x compared to the amount of drug in the plasma. In some embodiments, the anti-SIRP multimer that binds to the drug is also added to the reagent RBCs and / or reagent platelets before the serological assay is performed. In some embodiments, the anti-SIRP multimer is added to the reagent RBCs and / or reagent platelets (e.g., reagent RBCs and / or reagent platelets only) before the serological assay is performed.

[0100] In some embodiments, the method is carried out in a solution in which, for example, the anti-SIRP polymer bound to the drug is soluble. In some embodiments, the anti-SIRP polymer bound to the drug is immobilized on a solid phase before the method is carried out, via adsorption, covalent bonding, or non-covalent bonding to a matrix or surface. In some embodiments, the anti-SIRP polymer bound to the drug is capable of binding to the drug after immobilization on a solid phase or solid support. The solid phase or solid support used for immobilization can be any inert support, surface, or carrier that is essentially water-insoluble and useful for immunoassays, and this includes supports in the form of, for example, surfaces, particles, porous matrices, cellulose polymer sponges (ImmunoCAP®, Phadia). Examples of supports commonly used include small sheets, Sephadex, polyvinyl chloride, plastic beads, gold beads, microparticles, assay plates, or test tubes made from polyethylene, polypropylene, polystyrene, etc. In some embodiments, the anti-SIRP polymer bound to the drug is coated on a microtiter plate, such as a multi-well microtiter plate, which can be used to analyze multiple samples simultaneously.

[0101] In some embodiments, the drug portion that binds to human CD47 comprises wild-type SIRPα, a SIRPα variant, or a CD47-binding fragment of wild-type SIRPα or a SIRPα variant. In some embodiments, the drug portion that binds to human CD47 comprises a SIRPα variant (or its CD47-binding fragment), which comprises one or more amino acid substitutions, insertions, deletions, N-terminal extensions, and / or C-terminal extensions compared to wild-type SIRPα (or its CD47-binding fragment). In some embodiments, the drug portion that binds to human CD47 comprises a fragment of a SIRPα variant, which comprises the extracellular domain of the SIRPα variant.

[0102] In some embodiments, the drug portion that binds to human CD47 comprises wild-type SIRPγ, a SIRPγ variant, or a CD47-binding fragment of wild-type SIRPγ or a SIRPγ variant. In some embodiments, the drug portion that binds to human CD47 comprises a SIRPγ variant, which comprises one or more amino acid substitutions, insertions, deletions, N-terminal elongations, C-terminal elongations, or any combination thereof, compared to wild-type SIRPγ. In some embodiments, the drug portion that binds to human CD47 comprises a CD47-binding fragment of a SIRPγ variant, which comprises the extracellular domain of the SIRPγ variant.

[0103] In some embodiments, the drug portion that binds to human CD47 comprises a SIRPβ variant or a fragment of a SIRPβ variant that can bind to CD47 (e.g., human CD47). In some embodiments, the drug portion that binds to human CD47 comprises a SIRPβ variant, which comprises one or more amino acid substitutions, insertions, deletions, N-terminal extensions, C-terminal extensions, or any combination thereof, compared to wild-type SIRPβ. In some embodiments, the drug portion that binds to human CD47 comprises a fragment of a SIRPβ variant, which comprises the extracellular domain of the SIRPβ variant and is capable of binding to CD47 (e.g., human CD47).

[0104] (b) Anti-SIRP multimer In some embodiments, the anti-SIRP multimer that binds to a drug comprises an anti-SIRP antibody (or its drug-binding fragment) capable of binding to the SIRPα, SIRPα variant, SIRPβ variant, SIRPγ, or SIRPγ variant portion of the drug. In some embodiments, the anti-SIRP multimer that binds to a drug comprises one or more anti-SIRP antibodies (or its drug-binding fragment) capable of binding to the SIRPα, SIRPα variant, SIRPβ variant, SIRPγ, or SIRPγ variant portion of the drug. In some embodiments, the anti-SIRP multimer comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or up to 100 anti-SIRP antibodies (or its drug-binding fragment). In some embodiments, the anti-SIRP multimer comprises a bivalent anti-SIRP antibody (for example, that is). In some embodiments, “anti-SIRP antibody” refers to an antibody or its drug-binding fragment (e.g., its antigen-binding fragment) that specifically binds to SIRPα, SIRPα variants, SIRPγ, SIRPγ variants, or SIRPβ variants. The extracellular domains of SIRPα, SIRPβ, and SIRPγ have high homology. Therefore, in some embodiments, “anti-SIRP antibody” refers to an antibody or its drug-binding fragment that can cross-react with one or more of SIRPα, SIRPα variants, SIRPγ, SIRPγ variants, and / or SIRPβ variants.

[0105] In some embodiments, the anti-SIRP multimer comprises a full-length anti-SIRP antibody. In some embodiments, the anti-SIRP antibody comprises an Fc region (or portion thereof) that does not bind to the anti-human globulin reagent (AHG). (Further details regarding serological assays and reagents used in such assays are provided elsewhere in this specification.) In some embodiments, the anti-SIRP antibody comprises a mouse Fc region (or portion thereof). In some embodiments, the mouse Fc region comprises the amino acid sequence described in any one of SEQ ID NOs: 81-83. In some embodiments, the drug-binding fragment (e.g., antigen-binding fragment) of the anti-SIRP antibody is, for example, but not limited to, Fab, Fab', F(ab')2, Fab'-SH, Fv, diabody, one-arm antibody, scFv, scFv-Fc, single-domain antibody, single-heavy-chain antibody, etc. In some embodiments, the anti-SIRP antibody (or its drug-binding fragment) is an ADA (anti-drug antibody) or NAb (neutralizing antibody) that binds to a drug (i.e., a portion of the drug including SIRPα, a SIRPα variant, a SIRPβ variant, SIRPγ, or a SIRPγ variant). In some embodiments, the affinity of the drug to the anti-SIRP antibody is greater than the affinity of the drug to human CD47. In some embodiments, the anti-SIRP antibody (or its drug-binding fragment) includes a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 46 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 47. In some embodiments, the anti-SIRP antibody (or its drug-binding fragment) includes a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 48 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 49. In some embodiments, the anti-SIRP antibody (or its drug-binding fragment) includes a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 50 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 51. In some embodiments, the anti-SIRP antibody (or its drug-binding fragment) comprises a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 113 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 114.In some embodiments, the anti-SIRP antibody (or its drug-binding fragment) comprises a heavy-chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 115 and a light-chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 116. In some embodiments, the anti-SIRP antibody (or its drug-binding fragment) comprises a heavy-chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 133 and a light-chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 134. In some embodiments, the anti-SIRP antibody comprises a mouse Fc domain containing the amino acid sequence described in any one of SEQ ID NOs: 81-83. In some embodiments, the drug-binding fragment of the anti-SIRP antibody includes, for example, Fab, Fab', F(ab')2, Fab'-SH, Fv, diabody, one-arm antibody, scFv, scFv-Fc, single-domain antibody, single-heavy-chain antibody, etc. In some embodiments, the drug-binding fragment of the anti-SIRP antibody includes Fab or F(ab')2 containing SEQ ID NOs: 131 and 132. DVQLVESGGG VVRPGESLRL SCAASGFSFS SYAMNWVRQA PGEGLEWVSR INSGGGGTDY AESVKGRFTI SRDNSENTLY LQMNSLRAED TAVYYCAKQY DWNSFFDYWG LGALVTVSS(Sequence ID 46) ETVLTQSPAT LSVSPGERAT LSCRASQTVG SKLAWHQQKP GQAPRLLIYD ATNRATGISD RFSGSGSGTD FTLTISSLQT EDSAVYYCQQ YYYWPPYRFG GGTKVEIK(Sequence ID 47) DVQLVESGGG VVRPGESLRL SCEASGFTFS SNAMSWVRQA PGKGLEWVAG ISSGSDTYYG DSVKGRLTIS RDNSKNILYL QMNSLTAEDT AVYYCARETW NHLFDYWGQG TLVTVSS(Sequence ID 48) SYELTQPPSV SVSPGQTARI TCSGGSYSSY YYAWYQQKPG QAPVTLIYSD DKRPSNIPER FSGSSSGTTV TLTISGVQAE DEADYYCGGY DQSSYTNPFG GGTKLTVL(Sequence ID 49) DVQLVESGGG VVRPGESLRL SCAASGFTFS SYDMNWVRQA PGEGLEWVSL ISGSGEIIYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKEN NRYRFFDDWG QGTLVTVSS(Sequence ID 50) ETVLTQSPGT LTLSPGERAT LTCRASQSVY TYLAWYQEKP GQAPRLLIYG ASSRATGIPD RFSGSGSGTE FTLTISSLQS EDFAVYYCQQ YYDRPPLTFG GGTKVEIK(Sequence ID 51) DVQLVESGGG VVRPGESLRL SCAASGFTFS SYAMSWVRQA PGKGLEWLAG ISAGGSDTYY IDSVKGRFTI SRDNPKNSLY LQMSSLTAED TAVYYCARET WNHLFDYWGL GTLVTVSS (Sequence ID 113) ALTQPASVSA NPGETVKITC SGGDYYSTYY AWYQQKSPGS APVTVIHSDD KRPSDIPSRF SGSASGSAAT LIITGVRVED EAVYYCGGYD GRTYINTFGA GTTLTVL (Sequence ID 114) DVQLVESGGG VVRPGESLRL SCAASGFTFS SNAMSWVRQA PGKGLEWLAG ISAGGSDTYY PASVKGRFTI SRDNSKNTLY LQMNTLTAED TAVYYCARET WNHLFDYWGL GTLVTVSS (Sequence ID 115) ALTQPASVSA NPGETVKIAC SGGDYYSYYY GWYQQKAPGS ALVTVIYSDD KRPSDIPSRF SGSASGSTAT LTITGVRAED EAVYYCGGYD YSTYANAFGA GTTLTVL(Sequence ID 116) DVQLVESGGG VVRPGESLRL SCEASGFTFS SNAMSWVRQA PGKGLEWVAG ISSGSDTYYG DSVKGRLTIS RDNSKNILYL QMNSLTAEDT AVYYCARETW NHLFDYWGLG TLVTVS(Sequence ID 133) ALTQPASVSA SPGETVEITC SGGSDSSYYY GWYQQKSPGS APVTVIYSDN KRPSNIPSRF SGSASGSTAT LTITGVRVED EAVYYCGGYD YSTYTNPFGA GTTLTVL(Sequence ID 134) DVQLVESGGG VVRPGESLRL SCEASGFTFS SNAMSWVRQA PGKGLEWVAG ISSGSDTYYG DSVKGRLTIS RDNSKNILYL QMNSLTAEDT AVYYCARETW NHLFDYWGLG TLVTVSSAKT TAPSVYPLAP VCGDTTGSSV TLGCLVKGYF PEPVTLTWNS GSLSSGVHTF PAVLQSDLYT LSSSVTVTSS TWPSQSITCN VAHPASSTKV DKKIEPRGPT IKPCPPCKCP GSGSHHHHHH GLNDIFEAQK IEWHE (Sequence ID 131) ALTQPASVSA SPGETVEITC SGGSDSSYYY GWYQQKSPGS APVTVIYSDN KRPSNIPSRF SGSASGSTAT LTITGVRVED EAVYYCGGYD YSTYTNPFGA GTTLTVLRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC (Sequence ID 132)

[0106] In some embodiments, the anti-SIRP antibody comprises a heavy chain containing SEQ ID NO: 117 and a light chain containing SEQ ID NO: 118. In some embodiments, the anti-SIRP antibody comprises a heavy chain containing SEQ ID NO: 119 and a light chain containing SEQ ID NO: 118. In some embodiments, the anti-SIRP antibody comprises a heavy chain containing SEQ ID NO: 120 and a light chain containing SEQ ID NO: 121. In some embodiments, the anti-SIRP antibody comprises a heavy chain containing SEQ ID NO: 122 and a light chain containing SEQ ID NO: 121. The amino acid sequences of SEQ ID NOs. 117-122 are provided below. DVQLVESGGG VVRPGESLRL SCAASGFTFS SNAMSWVRQA PGKGLEWLAG ISAGGSDTYY PASVKGRFTI SRDNSKNTLY LQMNTLTAED TAVYYCARET WNHLFDYWGL GTLVTVSSAK TTPPSVYPLA PGSAAQTNSM VTLGCLVKGY FPEPVTVTWN SGSLSSGVHT FPAVLQSDLY TLSSSVTVPS STWPSETVTC NVAHPASSTK VDKKIVPRDC GCKPCICTVP EVSSVFIFPP KPKDVLTITL TPKVTCVVVD ISKDDPEVQF SWFVDDVEVH TAQTQPREEQ FASTFRSVSE LPIMHQDWLN GKEFKCRVNS AAFPAPIEKT ISKTKGRPKA PQVYTIPPPK EQMAKDKVSL TCMITDFFPE DITVEWQWNG QPAENYKNTQ PIMDTDGSYF IYSKLNVQKS NWEAGNTFTC SVLHEGLHNH HTEKSLSHSP G (Sequence ID 117) ALTQPASVSA NPGETVKIAC SGGDYYSYYY GWYQQKAPGS ALVTVIYSDD KRPSDIPSRF SGSASGSTAT LTITGVRAED EAVYYCGGYD YSTYANAFGA GTTLTVLGQP KSSPSVTLFP PSSEELETNK ATLVCTITDF YPGVVTVDWK VDGTPVTQGM ETTQPSKQSN NKYMASSYLT LTARAWERHS SYSCQVTHEG HTVEKSLSRA DCS (Sequence ID 118) DVQLVESGGG VVRPGESLRL SCAASGFTFS SNAMSWVRQA PGKGLEWLAG ISAGGSDTYY PASVKGRFTI SRDNSKNTLY LQMNTLTAED TAVYYCARET WNHLFDYWGL GTLVTVSSAK TTAPSVYPLA PVCGDTTGSS VTLGCLVKGY FPEPVTLTWN SGSLSSGVHT FPAVLQSDLY TLSSSVTVTS STWPSQSITC NVAHPASSTK VDKKIEPRGP TIKCPPCKC PAPNLLGGPS VFIFPPKIKD VLMISLSPIV TCVVVDVSED DPDVQISWFV NNVEVHTAQT QTHREDYNST LRVVSALPIQ HQDWMSGKEF KCKVNNKDLP APIERTISKP KGSVRAPQVY VLPPPEEEMT KKQVTLTCMV TDFMPEDIYV EWTNNGKTEL NYKNTEPVLD SDGSYFMYSK LRVEKKNWVE RNSYSCSVVH EGLHNHHTTK SFSRTPG(Sequence ID 119) DVQLVESGGG VVRPGESLRL SCAASGFTFS SYAMSWVRQA PGKGLEWLAG ISAGGSDTYY IDSVKGRFTI SRDNPKNSLY LQMSSLTAED TAVYYCARET WNHLFDYWGL GTLVTVSSAK TTPPSVYPLA PGSAAQTNSM VTLGCLVKGY FPEPVTVTWN SGSLSSGVHT FPAVLQSDLY TLSSSVTVPS STWPSETVTC NVAHPASSTK VDKKIVPRDC GCKPCICTVP EVSSVFIFPP KPKDVLTITL TPKVTCVVVD ISKDDPEVQF SWFVDDVEVH TAQTQPREEQ FASTFRSVSE LPIMHQDWLN GKEFKCRVNS AAFPAPIEKT ISKTKGRPKA PQVYTIPPPK EQMAKDKVSL TCMITDFFPE DITVEWQWNG QPAENYKNTQ PIMDTDGSYF IYSKLNVQKS NWEAGNTFTC SVLHEGLHNH HTEKSLSHS PG (Sequence ID 120) ALTQPASVSA NPGETVKITC SGGDYYSTYY AWYQQKSPGS APVTVIHSDD KRPSDIPSRF SGSASGSAAT LIITGVRVED EAVYYCGGYD GRTYINTFGA GTTLTVLGQP KSSPSVTLFP PSSEELETNK ATLVCTITDF YPGVVTVDWK VDGTPVTQGM ETTQPSKQSN NKYMASSYLT LTARAWERHS SYSCQVTHEG HTVEKSLSRA DCS (Sequence ID 121) DVQLVESGGG VVRPGESLRL SCAASGFTFS SYAMSWVRQA PGKGLEWLAG ISAGGSDTYY IDSVKGRFTI SRDNPKNSLY LQMSSLTAED TAVYYCARET WNHLFDYWGL GTLVTVSSAK TTAPSVYPLA PVCGDTTGSS VTLGCLVKGY FPEPVTLTWN SGSLSSGVHT FPAVLQSDLY TLSSSVTVTS STWPSQSITC NVAHPASSTK VDKKIEPRGP TIKCPPCKC PAPNLLGGPS VFIFPPKIKD VLMISLSPIV TCVVVDVSED DPDVQISWFV NNVEVHTAQT QTHREDYNST LRVVSALPIQ HQDWMSGKEF KCKVNNKDLP APIERTISKP KGSVRAPQVY VLPPPEEEMT KKQVTLTCMV TDFMPEDIYV EWTNNGKTEL NYKNTEPVLD SDGSYFMYSK LRVEKKNWVE RNSYSCSVVH EGLHNHHTTK SFSRTPG(Sequence ID 122)

[0107] In some embodiments, the anti-SIRP multimer comprises an anti-SIRP antibody comprising VH containing SEQ ID NO: 115 and VL containing SEQ ID NO: 116. In some embodiments, the anti-SIRP multimer (e.g., anti-SIRP antibody) is added to a plasma sample (e.g., a plasma sample obtained from a subject being treated with a drug) to obtain an anti-SIRP multimer in a molar excess of one of the following: approximately 1x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, or 10x compared to the amount of drug in the plasma.

[0108] In some embodiments, the anti-SIRP multimer comprises an anti-SIRP antibody comprising a heavy chain containing SEQ ID NO: 119 and a light chain containing SEQ ID NO: 118. In some embodiments, the anti-SIRP multimer (e.g., anti-SIRP antibody) is added to a plasma sample (e.g., a plasma sample obtained from a subject being treated with a drug) to obtain an anti-SIRP multimer in a molar excess of one of the following: approximately 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times compared to the amount of drug in the plasma.

[0109] In some embodiments, the anti-SIRP multimer comprises an anti-SIRP antibody comprising a heavy chain containing SEQ ID NO: 117 and a light chain containing SEQ ID NO: 118. In some embodiments, the anti-SIRP multimer (e.g., anti-SIRP antibody) is added to a plasma sample (e.g., a plasma sample obtained from a subject being treated with a drug) to obtain an anti-SIRP multimer in a molar excess of one of the following: approximately 1x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, or 10x compared to the amount of drug in the plasma.

[0110] In some embodiments, the anti-SIRP multimer comprises an anti-SIRP antibody comprising a heavy chain containing SEQ ID NO: 120 and a light chain containing SEQ ID NO: 121. In some embodiments, the anti-SIRP multimer (e.g., anti-SIRP antibody) is added to a plasma sample (e.g., a plasma sample obtained from a subject being treated with a drug) to obtain an anti-SIRP multimer in a molar excess of one of the following: approximately 1x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, or 10x compared to the amount of drug in the plasma.

[0111] Other exemplary anti-SIRP antibodies (e.g., anti-SIRPα antibodies, anti-SIRPβ antibodies, and / or anti-SIRPγ antibodies that cross-react with SIRPα) or their drug-binding fragments that may be contained in the SIRP polymer used in the methods described herein are known in the art. Further details regarding such antibodies are provided, for example, in WO2018 / 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. In some embodiments, the anti-SIRP antibody disclosed in one of the above references comprises a mouse Fc domain (or a portion thereof). In some embodiments, the mouse Fc domain comprises the amino acid sequence described in any one of SEQ ID NOs. 81-83.

[0112] In some embodiments, the anti-SIRP multimer is a homopolymer containing the same anti-SIRP antibody (e.g., the same anti-SIRPα antibody, anti-SIRPα variant antibody, anti-SIRPβ variant antibody, anti-SIRPγ antibody, anti-SIRPγ variant antibody, or an antibody capable of cross-reacting with one or more of SIRPα, SIRPα variant, SIRPβ variant, SIRPγ, and / or SIRPγ variant) or a drug-binding fragment thereof. In some embodiments, the anti-SIRP multimer is a homopolymer containing a monospecific bivalent anti-SIRP antibody. In some embodiments, the SIRP multimer is a heteropolymer containing at least two different anti-SIRP antibodies or drug-binding fragments in any combination. In some embodiments, the anti-SIRP polymer is a heteropolymer containing a multispecific anti-SIRP antibody (i.e., a multispecific anti-SIRP antibody comprising a first VH / VL pair and a second VH / VL pair (where the first and second VH / VL pairs have different amino acid sequences, and the first and second VH / VL pairs each bind to the CD47 binding site of the drug)).

[0113] In some embodiments, the anti-SIRP antibody or its drug-binding fragment includes a polymerizing domain, for example, but not limited to, the exemplary polymerizing domain for the CD47 polypeptide monomer and the SIRP polypeptide monomer described above.

[0114] In some embodiments, the anti-SIRP antibody or its drug-binding fragment includes an epitope tag (for example, further includes). In some embodiments, the epitope tag facilitates the polymerization of the anti-SIRP antibody (or its drug-binding fragment). In some embodiments, the tag facilitates the immobilization of the anti-SIRP antibody (or its drug-binding fragment) onto a solid support (e.g., beads, glass surface, etc.). Exemplary epitope tags include, but are not limited to, SEQ ID NOs. 7-32, as described elsewhere in this specification.

[0115] In some embodiments, the anti-SIRP antibody (or its drug-binding fragment) includes a ligand (e.g., bound to it). In some embodiments, the ligand is biotin. In some embodiments, the anti-SIRP antibody (or its drug-binding fragment, e.g., Fab or F(ab')2) includes HHHHHHGLNDIFEAQKIEWHE (SEQ ID NO: 135) or GSGSHHHHHGLNDIFEAQKIEWHE (SEQ ID NO: 126). GLNDIFEAQKIEWHE (SEQ ID NO: 8), also known as AVITAG®, is specifically biotinylated by the E. coli biotin ligase BirA. In some embodiments, Fab or F(ab')2 includes SEQ ID NOs: 131 and 132. In some embodiments, an anti-SIRP polymer (e.g., Fab or F(ab')2 containing SEQ ID NOs. 131 and 132) is added to a plasma sample (e.g., a plasma sample obtained from a subject undergoing drug treatment) to obtain one of the anti-SIRP polymers in a molar excess of approximately 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times the amount of drug in the plasma.

[0116] In some embodiments, the anti-SIRP antibody (or its drug-binding fragment) in the anti-SIRP multimer is linked, for example, via peptide bonds, to form a linkage chain of anti-SIRP antibodies (or its drug-binding fragment). In some embodiments, the anti-SIRP antibody (or its drug-binding fragment) in the SIRP multimer is linked via a linker peptide. Exemplary linker peptides include, but are not limited to, those described in SEQ ID NOs: 85-109, 127-130, IEGR (SEQ ID NO: 141), IDGR (SEQ ID NO: 140), and other linkers used with the CD47 multimer and / or SIRP multimer (see above). In some embodiments, the anti-SIRP antibody (or its drug-binding fragment) in the anti-SIRP multimer is linked via a linker peptide and at least one spacer. Exemplary peptide spacers include, but are not limited to, those described in SEQ ID NOs: 52-70, and other spacers used with the CD47 multimer and / or SIRP multimer (see above).

[0117] In some embodiments, the anti-SIRP polymer comprises at least one (e.g., 1 to 100) anti-SIRP antibody (or its drug-binding fragment) bound to a solid support. In some embodiments, the anti-SIRP antibody, or the anti-SIRP antibody or its drug-binding fragment, is bound to the solid support via covalent or non-covalent capture. In some embodiments, the solid support is gold nanospheres, gold nanoshells, magnetic beads, silica beads, dextran polymer, test tubes, slides, gel columns, or microtiter wells. In some embodiments, the solid support comprises or is made from, for example, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, the anti-SIRP multimer comprises an anti-SIRP antibody, or a plurality of anti-SIRP antibodies (or their drug-binding fragments), and a solid support, wherein the anti-SIRP antibody, or the anti-SIRP antibody or its drug-binding fragment, comprises an epitope tag or ligand (e.g., as described above), and the capture agent is immobilized on the solid support, and the anti-SIRP antibody, or the anti-SIRP antibody or its drug-binding fragment, is bound to the solid support via specific binding of the epitope tag or ligand by the capture agent immobilized on the solid support. In some embodiments, the anti-SIRP antibody, or the anti-SIRP antibody or its drug-binding fragment, comprises SEQ ID NO: 111. In some embodiments, the ligand is biotin, and the capture agent is streptavidin. In some embodiments, the anti-SIRP multimer (e.g., homopolymer or heteropolymer) comprises streptavidin or avidin bound to two, three, or four biotinylated anti-SIRP antibodies (or their drug-binding fragments). In some embodiments, the anti-SIRP antibody (or its drug-binding fragment) comprises SEQ ID NO: 111. In some embodiments, the anti-SIRP multimer comprises streptavidin or avidin bound to two, three, or four F(ab')2 fragments. In some embodiments, two or more F(ab')2 fragments include SEQ ID NOs. 131 and SEQ ID NOs. 132.

[0118] (c) Method for producing anti-SIRP polymers In some embodiments, anti-SIRP multimers (e.g., homopolymers or heteropolymers, containing, for example, two or more anti-SIRP antibodies (or their drug-binding fragments) linked via peptide bonds or linker peptides) are generated by recombinant host cells. Any method herein for generating CD47 multimers or SIRP multimers using recombinant techniques is similarly applicable to the generation of anti-SIRP multimers containing anti-SIRP antibodies (or their antigen-binding fragments).

[0119] In some embodiments, anti-SIRP multimers (e.g., homo-multimers or hetero-multimers) are produced by conjugating one or more (e.g., 1 to 100) anti-SIRP antibodies (or their drug-binding fragments) to a solid support via non-covalent capture. In some embodiments, the solid support is a gold nanosphere, gold nanoshell, magnetic beads, silica beads, dextran polymer, test tube, slide, gel column, or microtiter well. In some embodiments, the anti-SIRP antibody, or anti-SIRP antibody (or its drug-binding fragment), each contains a ligand, and a capture agent is immobilized on the solid support. The anti-SIRP multimer is produced by conjugating the anti-SIRP antibody, or anti-SIRP antibody (or its drug-binding fragment) to the solid support via specific binding of the ligand by the capture agent immobilized on the solid support. In some embodiments, the ligand is biotin, and the capture agent is streptavidin. In some embodiments, the anti-SIRP antibody(s) (or their drug-binding fragments) include SEQ ID NO: 111 or SEQ ID NO: 126.

[0120] Any method described elsewhere herein for generating CD47 multimers by conjugating CD47 polypeptide monomers to a solid support can similarly be applied to generating anti-SIRP multimers by conjugating anti-SIRP antibodies (or their drug-conjugated fragments) to a solid support. For example, in some embodiments, anti-SIRP multimers are prepared by culturing host cells containing nucleic acids encoding an anti-SIRP antibody (or its drug-conjugated fragment), including SEQ ID NO: 111 or SEQ ID NO: 126, under appropriate conditions to induce expression of the anti-SIRP antibody (or its drug-conjugated fragment), and then recovering the anti-SIRP antibody (or its drug-conjugated fragment). Anti-SIRP antibodies (or their drug-conjugated fragments) containing biotin receptor peptide amino acid sequences are biotinylated using E. coli biotin ligase (BirA). In some embodiments, anti-SIRP multimers are produced by conjugating a biotinylated anti-SIRP antibody(s) (or its drug-conjugated fragment) to a solid support on which streptavidin or avidin molecules are immobilized. In some embodiments, anti-SIRP multimers (e.g., homopolymers or heteropolymers) are provided, for example, comprising one or more (e.g., 1 to 100) anti-SIRP antibodies (or their drug-binding fragments) conjugated to a streptavidin or avidin conjugate solid support. Exemplary streptavidin or avidin conjugate solid supports are described in further detail elsewhere in this specification. In some embodiments, anti-SIRP multimers (e.g., homopolymers or heteropolymers) are provided, comprising streptavidin or avidin conjugated to 2, 3, or 4 biotinylated anti-SIRP antibodies (or their drug-binding fragments).

[0121] In some embodiments, anti-SIRP polymers are produced by linking one or more anti-SIRP antibodies (or their drug-binding fragments) to, for example, a solid support, or to, for example, one another, using a bifunctional crosslinking agent (such as those described elsewhere in this specification).

[0122] Next, the anti-SIRP multimer thus produced is used in the manner provided herein to prevent interference by drugs that bind to CD47 in serological assays.

[0123] V. Exemplary Drugs The methods provided herein reduce (or, in some embodiments, eliminate) interference in serological assays. This is caused by the presence of a drug in a sample containing plasma or RBC / platelets obtained from a subject being treated with the drug, which includes (i) an antibody Fc region and (ii) a portion that binds to human CD47. In some embodiments, the drug includes an IgG Fc region, e.g., a human IgG Fc region, e.g., IgG1, IgG2, or IgG4 Fc region. In some embodiments, the drug includes a modified Fc region (e.g., a modified IgG Fc region), which includes one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions compared 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, WO2017 / 027422;US2017 / 0107270; and USP10,259,859, the contents of which are incorporated herein by reference in their entirety.

[0124] In some embodiments, the portion that binds to human CD47 is wild-type SIRPα lacking a transmembrane domain (e.g., the extracellular domain of any wild-type SIRPα capable of binding to human CD47). In some embodiments, the portion that binds to human CD47 is a SIRPα variant that is capable of binding to human CD47 and lacks a transmembrane domain. In some embodiments, the SIRPα variant includes one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions compared to the extracellular domain of wild-type SIRPα. In some embodiments, the SIRPα variant is a SIRPα-d1 domain variant. In some embodiments, the affinity of the SIRPα variant to human CD47 is higher than the affinity of wild-type SIRPα to human CD47.

[0125] In some embodiments, the portion that binds to human CD47 is wild-type SIRPγ lacking a transmembrane domain (e.g., the extracellular domain of any wild-type SIRPγ capable of binding to human CD47). In some embodiments, the portion that binds to human CD47 is a SIRPγ variant that is capable of binding to human CD47 and lacks a transmembrane domain. In some embodiments, the SIRPγ variant includes one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions compared to the extracellular domain of wild-type SIRPγ. In some embodiments, the SIRPγ variant is a SIRPγ-d1 domain variant. In some embodiments, the affinity of the SIRPγ variant to human CD47 is higher than the affinity of wild-type SIRPγ to human CD47.

[0126] 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 SIRPβ variant includes one or more amino acid substitutions, deletions, insertions, N-terminal additions, and / or C-terminal additions compared to the extracellular domain of wild-type SIRPβ. In some embodiments, the SIRPβ variant is a SIRPβ-d1 domain variant.

[0127] Exemplary SIRPα, SIRPβ, and SIRPγ variants are known in the art and are referred to in WO2013 / 109752;US2015 / 0071905;USP9,944,911;WO2016 / 023040;WO2017 / 027422;US2017 / 0107270;USP10,259,859;US9845345;WO20161872 26;US20180155405;WO2017177333;WO2014094122;US2015329616;US20180312563;WO2018176132;WO2018081898;WO2018081897;US20180141986A1; and EP3287470A1, the contents of which are incorporated herein by reference in their entirety.

[0128] In some embodiments of any of the above methods, 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. Further details regarding these and other therapeutic anti-CD47 antibodies can be found in WO2018175790A1;US20180142019;US20180171014;US20180057592;US20170283498,US9,518,116;US9,518,117;US20150274826;US201601377 33;US9,221,908;US20140161799;US20160137734;WO2015191861;WO2014093678;WO20 14123580;WO2013119714;US9,045,541;WO2016109415;WO2018183182;WO2018009499; WO2017196793;US9663575;US20140140989;WO2018237168;US20180037652;US201900 23784;WO2018095428;EP3411071;WO2019042285;WO2016081423;WO2011076781;WO201 It is available under 2172521;WO2014087248;US20140303354;WO2016156537;US20160289727;US20190062428;US20180201677;US9352037;US20170044258;US9650441; and US20180105591.

[0129] VI. Exemplary Serological Assays for Pre-Transfusion Testing Pre-transfusion testing is performed to confirm that a blood product intended for transfusion is compatible with the blood of the subject (i.e., the transfusion recipient). Pre-transfusion testing includes serological assays used to confirm ABO compatibility between donor and recipient blood, as well as those used to detect clinically important RBC / platelet alloantibodies that react with antigens on donor RBCs and / or donor platelets (see Technical Manual, 18th ed, AABB, Bethesda, MD, 2014). Other exemplary blood group antigens for which serological assays are performed to determine donor / recipient transfusion compatibility include, but are not limited to, Kell blood group antigen, Duffy blood group antigen, Knops blood group antigen, Cartwright blood group antigen, Scianna blood group antigen, Indian blood group antigen, Rhesus blood group antigen, Dombrock blood group antigen, Landsteiner-Wiener blood group antigen, and VEL blood group antigen. The methods provided herein reduce or prevent drug interference in many serological assays known in the art (e.g., interference by drugs containing (i) an antibody Fc region and (ii) a portion that binds to human CD47). Exemplary serological assays in which the methods can be used include, but are not limited to, those described in further detail below.

[0130] Typically, serological assays are performed using samples containing unhemolyzed blood, plasma (e.g., plasma samples anticoagulated in EDTA), coagulated blood, or serum from subjects requiring transfusion (e.g., subjects treated with drugs containing (i) an antibody Fc region and (ii) a portion that binds to human CD47). Generally, the subject's ABO and Rh types are determined first. Next, antibody screening is used to detect clinically significant, unexpected non-ABO blood group antibodies that may be present in the subject's plasma. If the screening test reveals the presence of such antibodies, the specificity of those antibodies is determined using an antibody identification panel. After the specificity of the antibodies is identified, appropriate ABO and Rh type donor units are screened for the corresponding antigens. Units that are negative for those antigens are cross-matched with subjects requiring transfusion to ensure compatibility.

[0131] Serological assays can be performed in test tubes, on slides, on gel columns, or in microtiter well plates, and hemolysis and agglutination are signals indicating a positive (incompatible) test result. Agglutination, a reaction reflecting the binding of adjacent RBCs coated with antibody, can be scored visually and / or microscopically on a scale of 0 to 4+ in the most commonly used test tube method. A score of 0 indicates no reactivity and is characterized by smooth, easily dispersed cells. A score of 4+ indicates strong reactivity and is characterized by a single solid agglutination that does not easily disperse. Scores of 1+, 2+, or 3+ indicate intermediate levels of reactivity, characterized by gradually increasing agglutination size as the score increases. A similar principle of agglutination scoring can be applied when serological assays are performed using a gel column (gel card) containing anti-IgG antibodies in the column or a microtiter well plate (solid phase) containing bound erythrocyte antigens. Various methods are currently available for detecting antibody-RBC antigen interactions with varying sensitivities. In some embodiments, serological assays are performed manually. In some embodiments, serological assays are performed via automated machines.

[0132] For example, immediate spin (IS) (also known as "immediate spin cross-matching") is an assay that requires, for example, mixing reagent plasma / antiserum (i.e., plasma containing antibodies against known RBC and / or platelet surface antigens) with the target blood cells, immediately centrifugating the mixture at room temperature or 37°C for about 15-30 seconds, and visually inspecting the test tube for direct agglutination. Direct agglutination indicates a strong interaction between the antibody and the RBC surface antigen in the plasma. Alternatively, the target 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 direct agglutination can be visually evaluated.

[0133] Anti-human globulin (AHG) is used to detect antibody-bound red blood cell cells (RBCs) that do not directly agglutinate. AHG is a secondary anti-human globulin antibody produced in a different species. AHG reagents are either specific to a single class of human Ig (e.g., IgG) or multispecific, i.e., capable of binding to multiple human Ig classes (e.g., IgG, IgM, IgA) and complement. AHG serum can be used in direct antiglobulin tests (DAT) and / or indirect antiglobulin tests (IAT). The DAT demonstrates in vivo sensitization of red blood cells and is performed by directly testing washed patient red blood cell samples with AHG. The IAT shows the in vitro reaction between red blood cells and antibodies. In the IAT, serum (or plasma) is incubated with red blood cells, and then washed to remove unbound globulin. The presence of agglutination upon addition of AHG indicates that the antibody is bound to a specific red blood cell antigen. Some methods involve the addition of an enhancement reagent (enhancement), such as physiological saline, albumin, low ionic strength saline (LISS), or polyethylene glycol (PEG), followed by incubation of the sample at 37°C for 10–60 minutes before the AHG test. In some embodiments, the assay is a test tube assay. In some embodiments, the assay is a solid-phase erythrocyte adhesion assay (SPRCA).

[0134] ABO typing involves testing the recipient's red blood cells for the presence of A and B antigens using anti-A and anti-B antiserum (forward typing). Testing the recipient's plasma for the presence of anti-A and anti-B antigens using known A and B red blood cells (reverse typing) is also part of the prescribed ABO blood group testing.

[0135] 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 immediate spin (IS).

[0136] Alloantibodies against antigens not present in an individual's red blood cells can develop in individuals exposed to exogenous red blood cell antigens through pregnancy or blood transfusion. To detect antibodies against non-A or B type antigens, a sample of the patient's plasma or serum is tested against selected commercially available type O red blood cells expressing most clinically significant antigens other than A and B.

[0137] If antibody screening is positive, further serological testing is performed using a commercially available expanded panel of type O reagent RBCs to identify clinically significant antibodies. Next, once antibody specificity is determined, donor units must be screened for the corresponding antigens to select units lacking those antigens.

[0138] Antigenic typing (phenotyping) of recipient red blood cells can also be performed to determine which red blood cell antibodies an individual is most likely to produce. A serological assay for RBC phenotyping involves mixing recipient cells with a commercially available reagent antiserum containing specific antibodies.

[0139] IATs with and without enhancement (e.g., saline, LISS, PEG) are used for antibody detection and identification.

[0140] "Cross-matching" refers to a method for confirming compatibility between a patient's blood (plasma) and donor red blood cells. Cross-matching primarily means detecting and preventing ABO type incompatibility. Serological cross-matching assays (either IS cross-matching or AHG phase cross-matching) involve directly mixing donor red blood cells with recipient plasma, as well as scoring for hemolysis and agglutination after an immediate spin test or AHG test.

[0141] In some embodiments, serological assays are performed using a gel card. The principle of the gel card test is based on the gel method described in Lapierre, et al. (1990) “The gel test: a new way to detect red cells antigen-antibody reactions.” Transfusion, 30:109-113 for the detection of hemagglutination reactions. The plastic card consists of multiple microtubes. Each microtube contains an incubator chamber at the top of the column. Each microtube in the card is pre-filled with a buffered gel solution containing a specific antibody against an RBC or platelet surface antigen. Agglutination occurs when the target RBC or platelet reacts with the corresponding antibody present in the gel solution. Alternatively, the microtube is filled with the target serum, and agglutination occurs when the reagent RBC or reagent platelet reacts with an antibody present in the patient's serum or plasma. The gel column acts as a filter to trap agglutinated red blood cells as they pass through the gel column during centrifugation of the card. The gel column separates agglutinated red blood cells from non-agglutinated red blood cells based on size. Agglutinated red blood cells are captured at the top of the gel column or along the gel column, while non-aggregated red blood cells reach the bottom of the microtube and form a pellet.

[0142] In some embodiments, the serological assay is a solid-phase assay (e.g., a polymer immobilized on a solid support to mitigate / reduce interference from a drug conjugated to CD47 as described herein, a target RBC, a reagent RBC, or an antibody against a known RBC and / or platelet surface antigen). In some embodiments, the serological assay is performed manually. In some embodiments, the serological assay is performed using an automated system. In some embodiments, the automated system is a multiplexed high-throughput system that enables simultaneous analysis of multiple RBC, platelet, serum, or plasma samples obtained from different subjects. In some embodiments, the automated system is a multiplexed high-throughput system that enables simultaneous analysis of various serological assays using an RBC, platelet, serum, or plasma sample obtained from a single subject. Typically, automated blood analysis systems are microprocessor-controlled instruments. Exemplary automated blood analysis systems include, for example, Immucor's NEO® system (i.e., a solid-phase platform) and Diagast's QWALYS® 3 system. Qwalys® is a fully automated erythrocyte magnetization (EMT) system for ABO / D typing, Rh phenotyping, K typing, and antibody screening (ABS).

[0143] This specification is considered sufficient to enable those skilled in the art to carry out the invention. In addition to those shown and described herein, various modifications of the invention are apparent to those skilled in the art from the above description and are within the scope of the appended claims. All publications, patents, and patent applications referenced herein are incorporated herein by reference in their entirety for all purposes.

[0144] Each embodiment described herein may be combined with any other embodiment unless expressly indicated otherwise. In particular, any feature or embodiment shown as preferred or advantageous may be combined with any other feature or embodiment shown as preferred or advantageous unless expressly indicated otherwise. [Examples]

[0145] The following examples are provided to those skilled in the art to provide a complete disclosure and explanation of the methods of preparation and use of the present invention and are not intended to limit the scope of what the inventors consider to be the invention, nor are they intended to represent all or only the experiments performed. While efforts have been made to ensure the accuracy of the numerical values ​​used (e.g., quantity, temperature, etc.), some experimental errors and deviations should be taken into consideration. Unless otherwise indicated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0146] Example 1: Preparation of an exemplary CD47 polymer Preparation of CD47 polymer B CD47 polypeptide monomers containing Sequence ID No. 6 (see below) were transiently expressed in Expi293F cells, recovered, purified using Ni SEPHAROSE® 6 FAST FLOW chromatography resin, and then purified by size exclusion chromatography. QLLFNKTKSV EFTFSNDTVV IPCFVTNMEA QNTTEVYVKW KFKGRDIYTF DGALNKSTVP TDFSSAKIEV SQLLKGDASL KMDKSDAVSH TGNYTCEVTE LTREGETIIE LKYRVVSHHH HHHGLNDIFE AQKIEWHE(Sequence ID 6)

[0147] Purified CD47 polypeptide monomers were biotinylated in vitro using the BirA biotinylation kit available from Avidity LLC. The in vitro biotinylation reaction was then purified by size exclusion chromatography to separate the biotinylated CD47 polypeptide monomers from any excess biotin present in the mixture.

[0148] Next, biotinylated CD47 polypeptide monomers were incubated with streptavidin protein at 4°C overnight with gentle stirring in a molar ratio of biotinylated CD47 polypeptide monomer to streptavidin of 4.5:1. The reaction mixture was then purified by size exclusion chromatography to separate the CD47 polymer (i.e., the tetramerized biotinylated CD47 polypeptide monomer bound to streptavidin) from other species in the mixture. The CD47 polymer (i.e., the tetramer) was confirmed by size exclusion chromatography against a gel filtration standard.

[0149] Example 2: Preparation of an exemplary anti-SIRP polymer Formation of anti-SIRP octamer (anti-SIRP polymer C) Anti-SIRP F(ab')2 containing the VH-CH1 sequence containing SEQ ID NO: 131 and the VL-CL sequence containing SEQ ID NO: 132 was prepared as follows. The C-terminus of SEQ ID NO: 131 contains SEQ ID NO: 126, which contains the hexahistidine peptide HHHHHH (SEQ ID NO: 7) and the 15-amino acid tag GLNDIFEAQKIEWHE (SEQ ID NO: 8). GLNDIFEAQKIEWHE (SEQ ID NO: 8) is also known as AVITAG®. SEQ ID NO: 8 is specifically biotinylated with the E. coli biotin ligase BirA. DVQLVESGGG VVRPGESLRL SCEASGFTFS SNAMSWVRQA PGKGLEWVAG ISSGSDTYYG DSVKGRLTIS RDNSKNILYL QMNSLTAEDT AVYYCARETW NHLFDYWGLG TLVTVSSAKT TAPSVYPLAP VCGDTTGSSV TLGCLVKGYF PEPVTLTWNS GSLSSGVHTF PAVLQSDLYT LSSSVTVTSS TWPSQSITCN VAHPASSTKV DKKIEPRGPT IKPCPPCKCP GSGSHHHHHH GLNDIFEAQK IEWHE (Sequence ID 131) ALTQPASVSA SPGETVEITC SGGSDSSYYY GWYQQKSPGS APVTVIYSDN KRPSNIPSRF SGSASGSTAT LTITGVRVED EAVYYCGGYD YSTYTNPFGA GTTLTVLRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC (Sequence ID 132)

[0150] Sequence IDs 131 and 132 were transiently expressed in Expi293 cells. The transfection supernatant was purified using Ni SEPHAROSE® 6 FAST FLOW chromatography resin and polished by size exclusion chromatography. Purified anti-SIRP(Fab')2 was biotinylated in vitro using the BirA biotinylation kit available from Avidity LLC. The in vitro biotinylation reaction mixture was then purified by size exclusion chromatography to separate biotinylated anti-SIRP F(ab')2 from excess biotin present in the mixture.

[0151] Next, biotinylated anti-SIRP F(ab')2 was incubated with streptavidin protein overnight at 4°C with gentle stirring in a molar ratio of biotinylated anti-SIRP F(ab')2:streptavidin of 4.5:1. The reaction mixture was then purified by size exclusion chromatography to separate the anti-SIRP F(ab')2 multimer (i.e., tetramerized biotinylated anti-SIRP F(ab')2 bound to streptavidin) from other species in the mixture. The anti-SIRP F(ab')2 multimer (i.e., octamer) was confirmed by size exclusion chromatography against gel filtration standards.

[0152] Production of anti-SIRP polymer D Anti-SIRP antibodies containing SEQ ID NO: 115 (VH), SEQ ID NO: 116 (VL), and a mouse Fc domain were transiently expressed in Expi293 cells. (SEQ ID NOs: 115 and 116 are provided below.) Anti-SIRP antibodies were purified from cell culture supernatant via MabSelect LX and dialyzed in 1× phosphate-buffered saline. DVQLVESGGG VVRPGESLRL SCAASGFTFS SNAMSWVRQA PGKGLEWLAG ISAGGSDTYY PASVKGRFTI SRDNSKNTLY LQMNTLTAED TAVYYCARET WNHLFDYWGL GTLVTVSS (Sequence ID 115) ALTQPASVSA NPGETVKIAC SGGDYYSYYY GWYQQKAPGS ALVTVIYSDD KRPSDIPSRF SGSASGSTAT LTITGVRAED EAVYYCGGYD YSTYANAFGA GTTLTVL(Sequence ID 116)

[0153] Example 3: Mitigation of drug interference in a predetermined serological test using CD47 polymer B, anti-SIRP polymer C, or anti-SIRP polymer D, including (i) the antibody Fc region and (ii) the portion that binds to human CD47. Experiments were conducted to evaluate whether CD47 multimer B, anti-SIRP multimer C, or anti-SIRP multimer D mitigate the interference of drug A in in vitro antiglobulin tests (IATs). Drug A is an exemplary CD47-binding drug containing 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, and drug A has been previously shown to interfere with certain serological assays (see Kim et al. (2020) Transfusion. 1-9).

[0154] To prepare donor red blood cells (RBCs) and donor plasma, HemeQC whole blood samples (commercially available from Bio-Rad) from donors of various blood types were rotated at room temperature at 1000xg for 10 minutes to separate the plasma from the red blood cells. The pelletized red blood cells were then resuspended in MLB2 LISS (low ionic strength solution) at a concentration of 3.4%.

[0155] To perform the assay, 100 μL of drug A at 130 nM (10 μg / mL) was co-incubated at room temperature (RT) for 10 minutes at 1:2 dilution, starting at 20 times the molar concentration of drug A, in each donor plasma with CD47 multimer B, anti-SIRP multimer C, anti-SIRP multimer D, or CD47 polypeptide monomer (SEQ ID NO: 6). Next, 100 μL of each titration condition was added to capture wells containing 50 μL of 3.4% RBC from each donor and incubated at 37°C for 45 minutes. The wells were then washed and rotated three times at 400 × g for 5 minutes at room temperature using PBS. Two drops of AHG anti-IgG (i.e., anti-human globulin IgG) were added to each well and centrifuged at 800 × g for 30 seconds. Before acquiring images, the reaction mixture was gently agitated with the pelleted cells. The capture wells containing PBS served as negative controls in the assay, while the wells containing only drug A served as positive controls.

[0156] Figure 4 shows the results of IAT using a test tube assay with donor-derived blood samples with blood type / plasma type AsubB RhD+. Figure 5 shows the results of IAT using a test tube assay with donor-derived blood samples with anti-Fy RBC / plasma type O RhD+ R1r DCcee. Figure 6 shows the results of IAT using a test tube assay with donor-derived blood samples with anti-D blood type / plasma type A1 RhD-rr ccee. Different molar titrations (20x, 10x, 5x, 2.5x, 1.25x, 0.63x, 0.31x) (i.e., comparison with drug A) of CD47 polymer B, anti-SIRP polymer C, anti-SIRP polymer D, and CD47 monomer were tested.

[0157] In IAT assays using the CD47 polypeptide monomer, agglutination was observed across all molar titrations (see Figures 4-6). In contrast, no agglutination was observed in IAT assays with at least 5 × molar ratio of CD47 polymer B, 2.5 × molar ratio of anti-SIRP polymer C, and 2.5 × molar ratio of anti-SIRP polymer D. CD47 polymer B, anti-SIRP polymer C, and anti-SIRP polymer D demonstrated the ability to prevent drug A from interfering with blood typing when using the AHG anti-IgG reagent in a test tube assay.

[0158] Subsequent experiments will be conducted to evaluate whether CD47 multimer B, anti-SIRP multimer C, or anti-SIRP multimer D mitigates drug A interference in the solid-phase erythrocyte adhesion assay (SPRCA).

[0159] In summary, 50 μL of 130 nM (10 μg / mL) drug A was co-incubated in PBS at room temperature (RT) for 10 minutes with CD47 multimer B, anti-SIRP multimer C, or anti-SIRP multimer D, starting at a molar concentration of 20 × of drug A, in a 1:2 dilution. Next, 50 μL of each titration condition was added to Capture R wells and coated with erythrocyte membranes prepared from pool suspensions of equal proportions of erythrocytes from two type O donors. Then, two drops of Capture-R LISS reagent were added to each well, and the wells were incubated at 37°C for 45 minutes. Next, the wells were washed six times with PBS. After washing, one drop of Capture-R Ready Indicator Red cells was added to each well. The wells were rotated at 450 x g for 1 minute, and then images were acquired.

[0160] Agglutination was observed in the SPRCA assay using the CD47 polypeptide monomer (see Figure 7). In contrast, no interference by drug A was observed in the SPRCA assay with at least 0.31x molar ratio of CD47 polymer B, 0.31x molar ratio of anti-SIRP polymer C, or 0.63x molar ratio of anti-SIRP polymer D. CD47 polymer B, anti-SIRP polymer C, and anti-SIRP polymer D demonstrated the ability to prevent drug A from interfering with blood typing when using the AHG anti-IgG reagent in the solid-phase erythrocyte adhesion assay.

[0161] Example 4: Preparation of exemplary anti-SIRP polymers E and F Anti-SIRP polymer E contains a heavy chain containing SEQ ID NO: 117 and a light chain containing SEQ ID NO: 118. Anti-SIRP polymer F contains a heavy chain containing SEQ ID NO: 120 and a light chain containing SEQ ID NO: 121. DVQLVESGGG VVRPGESLRL SCAASGFTFS SNAMSWVRQA PGKGLEWLAG ISAGGSDTYY PASVKGRFTI SRDNSKNTLY LQMNTLTAED TAVYYCARET WNHLFDYWGL GTLVTVSSAK TTPPSVYPLA PGSAAQTNSM VTLGCLVKGY FPEPVTVTWN SGSLSSGVHT FPAVLQSDLY TLSSSVTVPS STWPSETVTC NVAHPASSTK VDKKIVPRDC GCKPCICTVP EVSSVFIFPP KPKDVLTITL TPKVTCVVVD ISKDDPEVQF SWFVDDVEVH TAQTQPREEQ FASTFRSVSE LPIMHQDWLN GKEFKCRVNS AAFPAPIEKT ISKTKGRPKA PQVYTIPPPK EQMAKDKVSL TCMITDFFPE DITVEWQWNG QPAENYKNTQ PIMDTDGSYF IYSKLNVQKS NWEAGNTFTC SVLHEGLHNH HTEKSLSHSP G (Sequence ID 117) ALTQPASVSA NPGETVKIAC SGGDYYSYYY GWYQQKAPGS ALVTVIYSDD KRPSDIPSRF SGSASGSTAT LTITGVRAED EAVYYCGGYD YSTYANAFGA GTTLTVLGQP KSSPSVTLFP PSSEELETNK ATLVCTITDF YPGVVTVDWK VDGTPVTQGM ETTQPSKQSN NKYMASSYLT LTARAWERHS SYSCQVTHEG HTVEKSLSRA DCS (Sequence ID 118) DVQLVESGGG VVRPGESLRL SCAASGFTFS SYAMSWVRQA PGKGLEWLAG ISAGGSDTYY IDSVKGRFTI SRDNPKNSLY LQMSSLTAED TAVYYCARET WNHLFDYWGL GTLVTVSSAK TTPPSVYPLA PGSAAQTNSM VTLGCLVKGY FPEPVTVTWN SGSLSSGVHT FPAVLQSDLY TLSSSVTVPS STWPSETVTC NVAHPASSTK VDKKIVPRDC GCKPCICTVP EVSSVFIFPP KPKDVLTITL TPKVTCVVVD ISKDDPEVQF SWFVDDVEVH TAQTQPREEQ FASTFRSVSE LPIMHQDWLN GKEFKCRVNS AAFPAPIEKT ISKTKGRPKA PQVYTIPPPK EQMAKDKVSL TCMITDFFPE DITVEWQWNG QPAENYKNTQ PIMDTDGSYF IYSKLNVQKS NWEAGNTFTC SVLHEGLHNH HTEKSLSHS PG (Sequence ID 120) ALTQPASVSA NPGETVKITC SGGDYYSTYY AWYQQKSPGS APVTVIHSDD KRPSDIPSRF SGSASGSAAT LIITGVRVED EAVYYCGGYD GRTYINTFGA GTTLTVLGQP KSSPSVTLFP PSSEELETNK ATLVCTITDF YPGVVTVDWK VDGTPVTQGM ETTQPSKQSN NKYMASSYLT LTARAWERHS SYSCQVTHEG HTVEKSLSRA DCS (Sequence ID 121)

[0162] To prepare anti-SIRP multimer E, SEQ ID NOs. 117 and 118 were expressed in 293FS cells. The multimers were purified by standard protein A affinity chromatography according to the manufacturer's recommended protocol (MabSelect LX, Cytiva) and dialyzed in 1× phosphate-buffered saline. Anti-SIRP multimer F was prepared by expressing SEQ ID NOs. 120 and 121 in 293F cells and purifying the multimer described for anti-SIRP multimer E.

[0163] Example 5: Reduction of drug interference in routine serological tests using anti-SIRP multimer E or anti-SIRP multimer F, including (i) the antibody Fc region and (ii) the portion that binds to human CD47. Experiments were conducted to compare the extent to which anti-SIRP multimer E and anti-SIRP multimer F could reduce the interference of drug A in the solid-phase erythrocyte adhesion assay (SPRCA). The SPRCA assay was performed as described in Example 3. As shown in Figure 8, both anti-SIRP multimer E and anti-SIRP multimer F could similarly reduce the interference of drug A by adding at least 1.25 times the molar ratio compared to drug A.

[0164] Example 6: Preparation of an exemplary anti-SIRP polymer G Anti-SIRP multimer G contains a heavy chain containing SEQ ID NO: 119 and a light chain containing SEQ ID NO: 118. DVQLVESGGG VVRPGESLRL SCAASGFTFS SNAMSWVRQA PGKGLEWLAG ISAGGSDTYY PASVKGRFTI SRDNSKNTLY LQMNTLTAED TAVYYCARET WNHLFDYWGL GTLVTVSSAK TTAPSVYPLA PVCGDTTGSS VTLGCLVKGY FPEPVTLTWN SGSLSSGVHT FPAVLQSDLY TLSSSVTVTS STWPSQSITC NVAHPASSTK VDKKIEPRGP TIKCPPCKC PAPNLLGGPS VFIFPPKIKD VLMISLSPIV TCVVVDVSED DPDVQISWFV NNVEVHTAQT QTHREDYNST LRVVSALPIQ HQDWMSGKEF KCKVNNKDLP APIERTISKP KGSVRAPQVY VLPPPEEEMT KKQVTLTCMV TDFMPEDIYV EWTNNGKTEL NYKNTEPVLD SDGSYFMYSK LRVEKKNWVE RNSYSCSVVH EGLHNHHTTK SFSRTPG(Sequence ID 119) ALTQPASVSA NPGETVKIAC SGGDYYSYYY GWYQQKAPGS ALVTVIYSDD KRPSDIPSRF SGSASGSTAT LTITGVRAED EAVYYCGGYD YSTYANAFGA GTTLTVLGQP KSSPSVTLFP PSSEELETNK ATLVCTITDF YPGVVTVDWK VDGTPVTQGM ETTQPSKQSN NKYMASSYLT LTARAWERHS SYSCQVTHEG HTVEKSLSRA DCS (Sequence ID 118)

[0165] To prepare anti-SIRP multimer G, sequence numbers 118 and 119 were expressed recombinantly in host cells. The multimers were purified using standard methods.

[0166] Example 7: Drug A interferes with blood typing assays performed using various platforms. Based on previous results regarding the potential interference of drug A with RBC antibody screening (see, e.g., Kim et al. (2020) Transfusion. 1-9; doi:10.1111 / trf.16009), drug A was spiked into normal pooled plasma confirmed to be free of antibodies binding to erythrocyte surface antigens. Plasma samples from patients used in this study were collected as part of routine hospital care. Final concentrations of drug A were 0.1, 1, 10, 100, 1000, and 2000 μg / mL, and this was tested using Bio-Rad antibody screening cells I and II in gel card format (Table 1), using Immucor's automated high-throughput NEO® system (Table 2) in solid phase, and using Diagast's automated high-throughput Qwalys3 system (Table 3) for antibody screening cells I, II, and III. As shown in Tables 1-3, agglutination reactivity ranged from 2+ to 4+ (on a scale of 0-4+) and was observed across all drug A concentrations tested. This suggests that the binding of drug A to RBCs and the interaction of the Fc portion of drug A with the AHG reagent interfere with the assay using the above format. Reagents were used according to the manufacturer's protocol. Bio-Rad reagent information includes antibody screening cells I and II: ID-DiaCell I-II, rr phenotype derived from ID cells: ID-DiaPanel: Gel card: ID card. The gel card assay was performed according to the manufacturer's protocol. In summary, 50 μL of 0.8% RBC suspension and 25 μL of plasma were used, incubated at 37°C for 15 minutes, and centrifuged for 10 minutes in a Bio-Rad ID-Centrifuge. The plasma was pooled from patients confirmed to be type AB and did not contain alloantibodies. [Table 1]

[0167] Reagents for the NEO® solid-phase platform, for normal use with the NEO® system, were supplied by Immucor. The reagents included Capture-R Ready-Screen (I and II). Reagent erythrocytes (in the erythrocyte interstitial morphology) were conjugated to the test wells during preparation. The plasma volume per well was 25 μL. [Table 2]

[0168] The specified reagents used in the QWALYS3 platform (Erythrocyte Magnetization Technology (EMT)) were supplied by Diagast. One reagent was Hemascreen I-II-III (1% magnetized red blood cells). The volumes of red blood cells and plasma per well were 15 and 15 μL, respectively. [Table 3]

[0169] The data in Table 1 show that drug A caused 2+ to 3+ agglutination in the gel card test (Bio-Rad). Similar agglutination was observed in the gel card test using D-, i.e., RBCs with significantly reduced CD47 expression, and in the gel card test using rr, i.e., RBCs with high CD47 expression. The data in Tables 2 and 3 show that even at very low concentrations, drug A caused 4+ agglutination in the high-throughput solid-phase test (NEO®, QWALYS®).

[0170] Example 8: Reduction of drug interference in routine serological tests using anti-SIRP multimer G, including (i) the antibody Fc region and (ii) the portion that binds to human CD47. Experiments were conducted to evaluate the extent to which anti-SIRP multimer G mitigates drug A interference in the Gelcard assay. The assay was performed as described in Example 7. In one series of tests, multimer G was added to plasma samples to obtain final concentrations of 0.1, 1, 10, 100, 1000, or 2000 μg / mL. The Gelcard serological assay was performed. As shown in Table 4, when drug A was present in the sample at a concentration of 2000 μg / mL, a 6-fold molar excess of anti-SIRP multimer G mitigated drug A interference. When drug A was present in the sample at a concentration of 1000 μg / mL, a 4-fold molar excess of anti-SIRP multimer G mitigated drug A interference. When drug A was present in the sample at concentrations of 0.1–100 μg / mL, a 3-fold molar excess of anti-SIRP multimer G mitigated drug A interference. [Table 4]

[0171] Next, drug A was added to plasma samples containing anti-Jka antibody or anti-E antibody (i.e., known alloantibodies) to obtain final concentrations of drug A of 0.1, 1, 10, 100, 1000, or 2000 μg / mL. The Gel Gard assay was performed. As shown in Table 5, drug A present in plasma at a concentration of 2000 μg / mL interfered with the detection of anti-Jka and anti-E alloantibodies. Multimer G neutralized the interference of drug A without interfering with the detection of anti-Jka and anti-E alloantibodies. [Table 5]

[0172] The data in Tables 4 and 5 support the conclusion that anti-SIRP multimer G mitigates interference caused by drug A present in plasma. Such mitigation enables the detection of allogeneic antibodies in plasma by the Gelcard assay.

[0173] Example 9: Serological assay interference caused by drugs containing (i) an antibody Fc region and (ii) a portion that binds to human CD47 can be mitigated by using multimer G across various platforms. Further tests were conducted to evaluate the extent to which drug A interferes with serological assays in test tube and gel card formats. Plasma samples (AB inactive plasma) were spiked with drug A to obtain final concentrations of 31.25, 125, 500, or 2000 μg / ml. In test tube tests, the presence of drug A in plasma at concentrations of 500 μg / ml and 2000 μg / ml resulted in a strong positive direct antiglobulin (DAT). Drug A at the tested concentrations of 31.25 μg / ml and 2000 μg / ml did not interfere with ABO typing (forward and reverse typing) or RhD typing. Positive DATs were observed to interfere with RhD typing for weak D in AHG. Drug A at all tested concentrations (31.25 μg / ml, 125 μg / ml, 500 μg / ml, and 2000 μg / ml) did not interfere with antibody screening during the initial spin (where conventional ABO incompatibility was being tested), but caused strong agglutination with all cells in the indirect antiglobulin test (IAT) using PEG and both AHG reagents tested (Immucor and Ortho). In the gel card test, drug A caused strong positive agglutination with all cells tested at all concentrations of drug A (i.e., 31.25 μg / ml, 125 μg / ml, 500 μg / ml, and 2000 μg / ml). (Data not shown).

[0174] When multimer G (100 mg / ml) was added to plasma in a 1:10 (v / v, multimer G:plasma) ratio and incubated at room temperature for 15 minutes, interference from drug A present in plasma at a concentration of 500 μg / ml was eliminated. See Table 6A. When multimer G (100 mg / ml) was added to plasma in a 1:10 (v / v, multimer G:plasma) ratio and incubated at room temperature or 37°C for up to 60 minutes, interference from drug A was partially mitigated ("vw+" or "very weak+", falling within the 0-1+ range), and antibody screening remained positive ("w1+" or "weak 1+", falling within the 0-1+ range) when present in plasma at a concentration of 2000 μg / ml. See Table 6B. Reactivity in antibody screening was significantly reduced, but testing by PEG-IAT and LISS-IAT remained weakly positive. See Table 6C. [Table 6] [Table 7] [Table 8]

[0175] The use of polymer G did not affect the reactivity of allogeneic anti-D as measured by LISS, PEGIAT, or gel card tests. See Table 7. The use of polymer G did not affect the reactivity of allogeneic anti-K as measured by LISS, PEGIAT, or gel card tests. See Table 8. [Table 9] [Table 10]

[0176] As shown in Table 9A, drug A causes aggregation in the LISSIAT, PEGIAT, and gel card test formats. [Table 11]

[0177] The following results are shown in Table 9B: Multimer G (150 mg / ml) was added to plasma in a ratio of 1:10 (v / v, multimer G:plasma). After a 30-minute incubation, when present in plasma at 750 μg / ml or 1000 μg / mL, interference by drug A was completely mitigated by multimer G in the LISS IAT test. After a 30-minute incubation, when present in plasma at 1250 μg / ml, 1500 μg / ml, or 2000 μg / mL, interference by drug A was substantially eliminated by multimer G in the LISS IAT test (micro-reactive). After a 30-minute incubation, interference from drug A at all tested concentrations (750 μg / mL, 1000 μg / mL, 1250 μg / mL, 1500 μg / mL, and 2000 μg / mL) was significantly reduced by multimer G in the PEG IAT test (1+ or micro+). After a 30-minute incubation, when drug A was present in plasma at 750 μg / mL, 1000 μg / mL, or 1250 μg / mL, interference from drug A was completely mitigated by multimer G in the IgG gel test. After a 30-minute incubation, when drug A was present in plasma at 1500 μg / mL or 2000 μg / mL, interference from drug A was significantly reduced by multimer G in the IgG gel test (1+ or + / -). [Table 12]

[0178] The following results are shown in Table 9C. Multimer G (150 mg / ml) was added to plasma in a ratio of 1:10 (v / v, multimer G:plasma). After 60 minutes of incubation, when present in plasma at concentrations of 750 μg / ml, 1000 μg / mL, or 1250 μg / mL, interference from drug A was completely mitigated by multimer G in the LISS IAT test. After 60 minutes of incubation, when present in plasma at a concentration of 1500 μg / mL, interference from drug A was virtually eliminated by multimer G in the LISS IAT test (micro-reactive). Mitigation of drug A interference by multimer G was not evaluated in the LISS IAT test for this experiment when present in plasma at a concentration of 2000 μg / mL. After 60 minutes of incubation, interference by drug A was substantially eliminated by multimer G in the PEG IAT test when present in plasma at concentrations of 750 μg / mL, 1000 μg / mL, 1250 μg / mL, or 1500 μg / mL (micro-reactive). The mitigation of drug A interference by multimer G was not assessed in the PEG IAT test for this experiment when present at 2000 μg / mL in plasma. After 60 minutes of incubation, interference by drug A was completely mitigated by multimer G when present in plasma at concentrations of 750 μg / mL or 1000 μg / mL, and significantly reduced by multimer G in the IgG gel test when present at 1250 μg / mL or 1500 μg / mL (weak + or + / -). The mitigation of drug A interference by multimer G was not assessed in the IgG gel test when present at 2000 μg / mL in plasma. [Table 13]

Claims

1. A method for reducing drug interference in a serological assay using reagent red blood cells (RBCs) or reagent platelets, wherein the method is: (a) Adding an anti-SIRP polymer that binds to the drug and blocks the drug from binding to the reagent RBC or the reagent platelet to a plasma sample from a subject treated with the drug, and (b) After step (a), perform a serological assay of the plasma sample using the reagent RBC or the reagent platelet. Includes, The drug comprises (i) a human antibody Fc region or a variant thereof and (ii) a portion that binds to human CD47, The method wherein the anti-SIRP multimer comprises two or more anti-SIRP antibodies or their drug-binding fragments.

2. The method according to claim 1, wherein the anti-SIRP polymer comprises 2 to 100 anti-SIRP antibodies or drug-binding fragments thereof.

3. The method according to claim 1 or 2, wherein the anti-SIRP macromer comprises a wild-type SIRPα, a SIRPα variant, a SIRPβ variant, a wild-type SIRPγ, a SIRPγ variant, or an anti-SIRP antibody or its drug-binding fragment that binds to two or more of these.

4. The aforementioned anti-SIRP polymer, (a) Heavy chain variable domain containing Sequence ID No. 113 (V Ｈ ) and light chain variable domain (V) containing sequence number 114 Ｌ ), (b) Heavy chain variable domain containing Sequence ID No. 115 (V Ｈ ) and light chain variable domain (V) containing sequence number 116 Ｌ ), and / or (c) Heavy chain variable domain containing Sequence ID No. 133 (V Ｈ ) and light chain variable domain (V) containing sequence number 134 Ｌ ), The method according to any one of claims 1 to 3, comprising an anti-SIRP antibody or a drug-binding fragment thereof.

5. The method according to any one of claims 1 to 4, wherein the anti-SIRP polymer comprises a full-length anti-SIRP antibody.

6. The method according to claim 5, wherein the anti-SIRP antibody comprises a mouse Fc domain.

7. The method according to claim 6, wherein the mouse Fc domain comprises the amino acid sequence described in any one of SEQ ID NOs: 81 to 83.

8. The aforementioned anti-SIRP antibody (a) A heavy chain containing SEQ ID NO: 117 and a light chain containing SEQ ID NO: 118, or (b) Heavy chain containing SEQ ID NO: 120 and light chain containing SEQ ID NO: 121 The method according to any one of claims 5 to 7, including the method described in any one of claims 5 to 7.

9. The drug-binding fragment of the anti-SIRP antibody is Fab, Fab', F(ab') ２ The method according to any one of claims 1 to 4, wherein the antibody is Fab'-SH, Fv, diabody, one-arm antibody, scFv, scFv-Fc, single-domain antibody, or single-heavy-chain antibody.

10. where the drug-binding fragment comprises F(ab') ２ and the F(ab') ２ comprises SEQ ID NOs: 131 and 132, the method according to claim 9

11. The method according to any one of claims 1 to 10, wherein the anti-SIRP antibody or its drug-binding fragment comprises an epitope tag or ligand.

12. The method according to claim 11, wherein the epitope tag comprises any one of sequence numbers 7, 8, and 126, or the ligand comprises biotin.

13. The method according to claim 11, wherein the epitope tag includes GSGSHHHHHGLNDIFEAQKIEWHE (Sequence ID 126).

14. The method according to any one of claims 1 to 13, wherein two or more anti-SIRP antibodies or drug-binding fragments thereof are bound to a solid support.

15. The method according to claim 14, wherein the solid support is a gold nanosphere, a gold nanoshell, a magnetic bead, a silica bead, a dextran polymer, a test tube, a slide, a gel column, or a microtiter well.

16. The method according to claim 14 or 15, wherein each of the two or more anti-SIRP antibodies or drug-binding fragments thereof comprises an epitope tag or ligand, a capture agent that specifically binds to the epitope tag or ligand is immobilized on the solid support, and the anti-SIRP antibody or drug-binding fragment thereof is bound to the solid support by the specific binding of the epitope tag or ligand by the capture agent.

17. The method according to claim 16, wherein the ligand is biotin and the scavenger is streptavidin.

18. The method according to any one of claims 1 to 13, wherein the anti-SIRP macromer comprises streptavidin or avidin bound to two, three, or four biotinylated anti-SIRP antibodies or fragments thereof.

19. The anti-SIRP polymer contains 2, 3, or 4 biotinylated F(ab') ２ The streptavidin or avidin bound to the fragment, and the biotinylated F(ab') ２ The method according to claim 18, wherein two or more fragments include sequence numbers 131 and 132.

20. The method according to any one of claims 1 to 19, wherein the anti-SIRP polymer is a homopolymer.

21. The method according to any one of claims 1 to 19, wherein the anti-SIRP polymer is a heteropolymer.

22. The method according to any one of claims 1 to 21, wherein 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, and the SIRPα variant has any amino acid sequence from SEQ ID NOs: 33 to 41.

23. The method according to claim 22, wherein the portion of the drug that binds to human CD47 comprises a fragment of the SIRPα variant, and the fragment comprises the extracellular domain of the SIRPα variant.

24. The method according to any one of claims 1 to 23, wherein the antibody Fc region of the drug is a human IgG Fc region.

25. The method according to claim 24, wherein the human IgG Fc region is a human IgG1, IgG2, or IgG4Fc region.

26. The serological assay described above, (a) ABO / Rh typing assay; or (b) Direct antiglobulin (DAT) assay using multispecific reagents for detecting IgG and complement C3; The method according to any one of claims 1 to 25.

27. The method according to any one of claims 1 to 26, wherein the serological assay is a PEG-enhanced serological assay.

28. The method according to claim 26, wherein the serological assay is an elution test performed after the DAT assay.

29. The serological assay described above, (a) Test tube assay; (b) Solid-phase erythrocyte assay (SPRCA); (c) Gel card assay; or (d) Solid-phase assay The method according to any one of claims 1 to 25.

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