WO2017132701A1 - Procédé de criblage d'anticorps - Google Patents

Procédé de criblage d'anticorps Download PDF

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Publication number
WO2017132701A1
WO2017132701A1 PCT/US2017/015797 US2017015797W WO2017132701A1 WO 2017132701 A1 WO2017132701 A1 WO 2017132701A1 US 2017015797 W US2017015797 W US 2017015797W WO 2017132701 A1 WO2017132701 A1 WO 2017132701A1
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protein
cell
region
cells
membrane
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PCT/US2017/015797
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English (en)
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Shuang Zhang
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Shuang Zhang
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Publication of WO2017132701A1 publication Critical patent/WO2017132701A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/12—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, Konjac gum, Locust bean gum or Guar gum
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
    • G01N2405/04—Phospholipids, i.e. phosphoglycerides

Definitions

  • the present invention relates to a method for screening plasma cells secreting antibodies for specific antigen using a fusion protein which is capable of attaching to cell membrane and binding to antibodies secreting from a plasma cell.
  • the terminally-differentiated plasma cell subset of B cells both the relatively stable population of long-lived plasma cells residing in the bone marrow and the short-lived plasma cells in the spleen and other secondary lymphoid organs, also represent an excellent source of high quality antibodies. Comparing to memory B cells, plasma cells is much more abundant and diversified, and has higher level of immunoglobulin mRNA. However, mainly due to the lack of surface-associated IgG and other markers, flow cytometry has not been used extensively to interrogate the plasma cell subset from these important niches. [0006] To exploit the high secretory capacity of plasma cells, a number of techniques have been developed that allow for the identification and isolation of antigen-specific cells.
  • Fischer in U.S. Pat. No. 4,264,766, discloses immunological reagents comprising discrete particles of a latex carrier to which a water-soluble polyhydroxy compound is covalently bound, having condensed thereto a known immunologically active material.
  • Means et al. in U.S. Pat. No. 4,808,530, disclose immobilizing biologically active proteins by reacting the protein with an imidoester or imidothioester having a hydrophobic moiety to form a hydrophobic amidine derivative of the protein, and then adsorbing the protein derivative to a hydrophobic surface.
  • Takkinen et al., in Protein Engineering 4:837-841 (1991) disclose preparing an active single-chain antibody containing a cellulase linker domain which is secreted by E. coli. These molecules were designed to allow incorporation of the single chain antibodies into lipid micelles.
  • the lipophilic antibody was genetically engineered and found to be active and capable of functioning in lipid micelles.
  • this protein did not provide an enhanced signal over unbound antibody.
  • the lipid-tagged antibody caries a single covalently bound glycerolipid anchor at the amino-terminal cysteinal residue which is separated from the variable region of the immunoglobulin heavy chain by a linker peptide. These antibodies are used for making immunoliposomes for fluoroimmunoassay.
  • Lipid modifications facilitate the attachment of soluble proteins to biological membranes, but they also enable protein-protein interactions and, in some cases, the shuttling of proteins between the plasma membrane and the cytosol or other membrane compartments.
  • These modifications which are found in all eukaryotic cells, fall into three major classes and are characterized by the type of lipid and the site of modification in the protein. They include N-myristylation, palmitoylation and prenylation.
  • the present invention provides a vector for expressing a capturing protein, which consists of two domains - one binds to an antibody and another attaches to cell membrane.
  • the antibody-binding domain is preferably derived from protein G, A, or a secondary antibody.
  • the membrane-attach domain is a GPI site or a site to which a lipid can be added in vitro, such as a sortase A recognition site (LPETG).
  • the invention provides a novel method for using a capturing protein to screen for cells secreting antibodies against specific antigens.
  • the method includes incubating cells with capturing proteins so that a capturing protein attaches itself to the membrane of a cell and grabs antibodies secreted by the cell. Then upon fluorescent-labeled specific antigens added, cells expressing antibodies that bind to the antigen will be sorted out by Fluorescence Activated Cell Sorting (FACS).
  • FACS Fluorescence Activated Cell Sorting
  • the invention provides methods for using a capturing protein to screen for plasma cells that produce antibodies specific against certain antigen.
  • the complex of a plasma cell, membrane bound capturing protein and antibodies secreted from the plasma cell will be sorted out with fluorescent-labeled specific antigens and fluorescent-labeled plasma cell surface markers, such as CD138, CD78, CD27, and/or CD319.
  • the invention provides methods for using a capturing protein to screen for plasmablasts that produce antibodies specific against certain antigen.
  • FIG. 1 is a representation of sG'-GPI gene, containing a signal peptide, a His-tag, an antibody- binding region, and a GPI anchor region (Human DAF);
  • FIG. 2 is a flowchart of expression and purification of sG'-GPI proteins
  • FIG. 3 is a flowchart of this invention for screening a cell secreting antibodies for specific antigen
  • FIG. 4 is a representation of pG'-LPETG gene, containing an antibody-binding region, a sortase A recognition sequence (LPETG), and a His-tag;
  • FIG. 5 is a flowchart of expression and purification of pG'-LPETG proteins, then preparation of pG'-lipid;
  • FIG. 6 is shows that pG'-lipids attach to cell membrane and are capable of binding antibodies.
  • Panel 1 with PI; panel 2, with PI and FITC-labeied antibody; panel 3, with PI and lipid modified pG' in Fig.4B; panel 4, with PI and lipid modified pG' in Fig.4C; panel 5, with PI, FITC-labeled HA and lipid modified pG' in Fig.4B; panel 6, with PI, FITC-labeled antibody, and lipid modified pG' in Fig.4B; panel 7, with PI, FITC-labeled antibody, and lipid modified pG' in Fig.4C.
  • FIG. 1 two versions of fusion proteins are presented, where both comprises two regions (i) a first region capable of anchoring the protein to cell membrane surface; and (ii) a second region capable of capturing antibodies secreted from the cells.
  • sG'-GPI is expressed and purified from mammalian cell culture
  • pG'-LPETG is expressed and purified from E.coli.
  • pG'- LPETG is preferred economically.
  • a short linker is inserted between pG' domain and LPETG site in Fig. 4C.
  • Step 1 incubating a sample of cells, such as plasma cells from bone marrow, with a fusion protein (Fig. 1 and Fig. 2).
  • the fusion protein comprises two regions (i) a first region capable of anchoring the protein to cell membrane surface; and (ii) a second region capable of capturing antibodies secreted from the cells.
  • Step 2 incubating the cells from Step 1 with fluorescent labeled antigen;
  • Step 3 a cell that is capable of recruiting fluorescent labeled antigen to its membrane surface from Step 2 is then sorted out by Fluorescence Activated Cell Sorting (FACS). The sorted cell is then subject to RT-PCR to rescue the heavy and light chains of the antibody.
  • FACS Fluorescence Activated Cell Sorting
  • the fusion protein was purified by affinity chromatography from the membranes of transfected 293 cells, following extraction with a detergent (either octylglucoside or CHAPS).
  • the fusion is recovered from host cell membranes by preparing cell membrane extracts in substantially the same fashion as mDAF or other anchored polypeptides heretofore have been isolated.
  • Other methods for obtaining preparations of membrane anchored polypeptides such as receptors also are known and are adaptable for use in recovering the fusions described herein.
  • the host cell membranes are separated from the cytoplasm, solubilized with nonionic detergent, and the fusion recovered by adsorption on immunoaffinity, substrate or ligand affinity columns.
  • a his-tag is attached to the N-terminal of the fusion to facilitate purification of the protein.
  • the fusions will be recovered as polypeptides containing the heterologous polypeptide with a C-terminally linked GPI-anchor glycophospholipid.
  • the fusion protein incorporating into cell membrane is readily accomplished by mixing a solution of the GPI-linked fusion protein or pG'-lipid with a plasma cell suspension and incubating until the insertion of the fusions into the cell membrane.
  • the fusion protein is able to capture antibodies when it attaches to cell membrane (Fig. 6). Dead cells were stained by Propidium iodide (PI). For each panel, the cells at lower section are alive. Comparing panels 1,2 with 3,4, it indicates that incubation with pG'-lipid has no or minimal effect on cell viability. Comparing panel 5 with 6,7, it indicates that membrane-bound pG'-lipid is able to capture antibodies, and pG'-lipid is quite evenly distributed among cells. Comparing panel 6 with 7, it indicates that addition of a linker between pG' domain and the lipid may affect the intensity of florescent on cells.
  • His-tag is an amino acid motif in proteins that consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein.
  • Plasma cells also called plasma B cells, plasmocytes, plasmacytes, or effector B cells, are white blood cells that secrete large volumes of antibodies. They are transported by the blood plasma and the lymphatic system. Plasma cells originate in the bone marrow; B cells differentiate into plasma cells that produce antibody molecules closely modeled after the receptors of the precursor B cell. Once released into the blood and lymph, these antibody molecules bind to the target antigen (foreign substance) and initiate its neutralization or destruction.
  • target antigen foreign substance
  • Plasmablasts secrete more antibodies than B cells, but less than plasma cells. They divide rapidly and are still capable of internalizing antigens and presenting them to T cells. A cell may stay in this state for several days, and then either die or irrevocably differentiate into a mature, fully differentiated plasma cell.
  • Terminally differentiated plasma cells express relatively few surface antigens, and do not express common pan-B cell markers, such as CD19 and CD20. Instead, plasma cells are identified through flow cytometry by their additional expression of CD138, CD78, the lnterleukin-6 receptor and lack of expression of CD45.
  • CD27 is a good marker for plasma cells
  • naive B cells are CD27-
  • memory B-cells are CD27+
  • plasma cells are CD27++.
  • the surface antigen CD138 (syndecan-1) is expressed at high levels.
  • CD319 Another important surface antigen is CD319 (SLAMF7). This antigen is expressed at high levels on normal human plasma cells. It is also expressed on malignant plasma cells in multiple myeloma. Compared with CD138, which disappears rapidly ex vivo, the expression of CD319 is considerably more stable.
  • the fusion anchoring to cell membrane surface could be directly inserted into the lipid bilayer or indirectly bound to integral proteins by a combination of hydrophobic, electrostatic, and other non- covalent interactions.
  • the fusion protein is generally post- translationally modified in vivo, with addition of fatty acid or prenyl chains, or GPI (glycosylphosphatidylinositol), which may be anchored in the lipid bilayer.
  • GPI glycosethylphosphatidylinositol
  • lipid and/or glycan can in vitro be added to fusion protein chemically.
  • Glycosylphosphatidylinositol is a glycolipid that can be attached to the C-terminus of a protein during posttranslational modification. Proteins containing a GPI anchor play key roles in a wide variety of biological processes. It is composed of a phosphatidylinositol group linked through a carbohydrate-containing linker (glucosamine and mannose glycosidically bound to the inositol residue) and via an ethanolamine phosphate (EtIMP) bridge to the C-terminal amino acid of a mature protein. The two fatty acids within the hydrophobic phosphatidyl-inositol group anchor the protein to the cell membrane.
  • GPI anchor is a glycolipid that can be attached to the C-terminus of a protein during posttranslational modification. Proteins containing a GPI anchor play key roles in a wide variety of biological processes. It is composed of a phosphatidylinositol group linked through a carbohydrate-containing link
  • Glypiated (GPI-linked) proteins contain a signal sequence, thus directing them to the endoplasmic reticulum (ER).
  • the protein is co-translationally inserted in the ER membrane and is attached to the ER membrane by its hydrophobic C terminus; the majority of the protein extends into the ER lumen.
  • the hydrophobic C-terminal sequence is then cleaved off and replaced by the GPI-anchor.
  • the carboxyl terminal domain that specifies glycophospholipid membrane anchor attachment for mDAF (referred to as the GPI signal domain, wherein GPI is an abbreviation of glycophosphatidylinositol), or functionally equivalent domains from other proteins which also are anchored by glycophospholipids, are fused to proteins or multimers of such proteins which are heterologous to the source of the GPI signal domain, for example hormones, antigens (especially from infectious organisms), allergens, immunoglobulins, enzymes, receptors and the like.
  • the anchor fusions are used in combination with the recombinant cells which express them or are recovered and formulated into therapeutic compositions, used as diagnostic assay components, or employed in affinity purification procedures.
  • the fusions will contain the heterologous polypeptide fused at its C-terminus to the GPI signal domain, that specifies a processing event in the cell that results in cleavage and removal of the GPI signal domain, and covalent attachment of a GPI anchor to the new C-terminus of the protein.
  • the last about 30-50 residues of DAF contain a signal (the "GPI signal") that directs a processing event in cells in which the last about 28 residues are proteolytically removed and replaced with a hydrophobic glycolipid (GPI) that acts as a membrane anchor.
  • GPI hydrophobic glycolipid
  • immunoglobulin-binding bacterial proteins include Protein A, Protein G, Protein A/G, and Protein L.
  • Antibodies for instance secondary antibody, could also be raised against the Fc region of an antibody.
  • Protein A is a 42 kDa surface protein originally found in the cell wall of the bacterium Staphylococcus aureus. It is composed of five homologous Ig-binding domains (E, D, A, B and C) that fold into a three-helix bundle. Each domain is able to bind proteins from many mammalian species, most notably IgGs. It binds the heavy chain within the Fc region of most immunoglobulins and also within the Fab region in the case of the human VH3 family.
  • Protein G is an immunoglobulin-binding protein expressed in group C and G Streptococcal bacteria much like Protein A but with differing binding specificities. It is a 65-kDa (G148 protein G) and a 58 kDa (C40 protein G) cell surface protein that has found application in purifying antibodies through its binding to the Fab and Fc region.
  • Protein A/G is a genetically engineered fusion protein that combines the IgG binding profiles of both Protein A and Protein G.
  • Protein A/G contains 6x His-tag on the N-terminus, five Ig-binding regions of protein A fusion with three Ig-binding region of protein G. The 6x His-tag on the N- terminus can be used for affinity purification or for protein A/G detection using an anti-His-tag antibody.
  • Non-specific binding regions, including cell wall binding regions, albumin binding regions and other non-specific binding regions have been eliminated from this fusion protein A/G, to ensure the maximum specific IgG binding.
  • Protein L isolated from bacterial species Peptostreptococcus magnus binds antibodies through light chain interactions. Since no part of the heavy chain is involved in the binding interaction, Protein L binds a wider range of antibody classes than Protein A or G. Protein L binds to representatives of all antibody classes, including IgG, IgM, IgA, IgE and IgD. Single chain variable fragments (scFv) and Fab fragments also bind to Protein L. Protein L binding is restricted to those antibodies that contain certain kappa light chains.
  • VKI human VKI, VKI II and VKIV subtypes but does not bind the VKII subtype. Binding of mouse immunoglobulins is restricted to those having VKI light chains.
  • equivalent is used herein as denoting a nucleotide sequence or a polypeptide or protein which performs substantially as the nucleotide sequence, polypeptide, or protein identified here to produce molecules with substantially the same antigenic or immunogenic activity in essentially the same kind of hosts.
  • Equivalency of amino acid sequences can be measured in terms of homology and equivalency of function. Within this definition of “equivalents” are subfragments which have equivalent activity.
  • Soluble sortase A bearing an N-terminal His6-tag was expressed and purified from E. coli as described in Ton-That, H.et al. PNAS USA 96: 12424-12429 (1999).
  • a DNA fragment bearing an N-terminal His6-tag sortase A was cloned into pET21a expression vector.
  • the result expression plasmid was then transformed into BL21(DE3) E.coli cells and. selected on an Amp+ plate. IPTG induction was carried out for 2.5 hours and cell pellets were suspended in lysis buffer (20mM Tris- HCI pH8.0, 250mM NaCI, ImM DTT).
  • pG'-LPETG proteins in Fig. 4B and 4C were expressed and purified from E.coli in a way same as the one in Example 1.
  • GGGYC-PEG-Lipid was prepared as described in Tomita et al., Biotechnol. Bioeng. 110:
  • a maleimidyl PEG (4000)-lipid was purchased from Ponsure Biotechnology (Shanghai, China).
  • a pentapeptide with the sequence GGGYC was synthesized and purified by reversed-phase high performance liquid chromatography (RP-HPLC) by Bio Basic Canada Inc. (Ontario, Canada).
  • the GGGYC peptide was modified with the lipid in a MOPS buffer (20 mM, pH 7.0) including 5% DMSO.
  • n in pG'- LPET(G) n -lipid is three.
  • Myeloma cells were maintained in Dulbecco's Modified Eagles Medium (DMEM) with 10% FBS.
  • DMEM Dulbecco's Modified Eagles Medium
  • lxlO 6 celis were resuspended in 1ml of lxPBS or DMEM (without FBS) and incubated with 2 ⁇ g of pG'-LPET(G) n -lipid for 45 min at 37°C. Cells were washed with lxPBS for 3 times. Cells were then incubated with 2 ⁇ g/ml either FITC-labeled antibodies or FITC-labeled HA1 antigens in lxPBS (with 1% BSA) at RT for 45 min. After that, cells were washed with lxPBS for 3 times. Right before FACS analysis, 2 ⁇ of propidium iodide (PI) were added. In this case, the n in pG'-LPET(G) n -lipid is three.

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Abstract

La présente invention concerne une nouvelle protéine hybride, pouvant se lier à la membrane d'une cellule et capturer des anticorps sécrétés par une telle cellule, et des procédés d'utilisation d'une telle protéine hybride pour cribler des cellules sécrétant des anticorps pour un antigène spécifique.
PCT/US2017/015797 2016-01-31 2017-01-31 Procédé de criblage d'anticorps WO2017132701A1 (fr)

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US62/289,316 2016-01-31

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220025326A1 (en) * 2020-07-27 2022-01-27 Namocell Inc. Kits and method of modifying, detecting, and sorting antibody-producing cells
CN118006552A (zh) * 2024-04-09 2024-05-10 暨南大学 一种抗原特异性浆细胞的筛选方法及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6919183B2 (en) * 2001-01-16 2005-07-19 Regeneron Pharmaceuticals, Inc. Isolating cells expressing secreted proteins
US20100098719A1 (en) * 2006-09-18 2010-04-22 Peter Van Endert Fusion Proteins Comprising Two or More IgG Binding Domains of Streptococcal Protein G

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6919183B2 (en) * 2001-01-16 2005-07-19 Regeneron Pharmaceuticals, Inc. Isolating cells expressing secreted proteins
US20100098719A1 (en) * 2006-09-18 2010-04-22 Peter Van Endert Fusion Proteins Comprising Two or More IgG Binding Domains of Streptococcal Protein G

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220025326A1 (en) * 2020-07-27 2022-01-27 Namocell Inc. Kits and method of modifying, detecting, and sorting antibody-producing cells
WO2022026357A1 (fr) * 2020-07-27 2022-02-03 Namocell Inc. Kits et procédé de modification, de détection et de tri de cellules productrices d'anticorps
CN118006552A (zh) * 2024-04-09 2024-05-10 暨南大学 一种抗原特异性浆细胞的筛选方法及其应用

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