US20120189640A1 - Methods and Compositions Related to Soluble Monoclonal Variable Lymphocyte Receptors of Defined Antigen Specificity - Google Patents

Methods and Compositions Related to Soluble Monoclonal Variable Lymphocyte Receptors of Defined Antigen Specificity Download PDF

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US20120189640A1
US20120189640A1 US12/375,804 US37580407A US2012189640A1 US 20120189640 A1 US20120189640 A1 US 20120189640A1 US 37580407 A US37580407 A US 37580407A US 2012189640 A1 US2012189640 A1 US 2012189640A1
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antigen specific
vlr
antigen
polypeptide
specific polypeptide
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Max D. Cooper
Brantley R. Herrin
Matthew N. Alder
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UAB Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1278—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Bacillus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/34—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood group antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • VLRs variable lymphocyte receptors
  • LRR leucine-rich repeat
  • the present application relates to antigen specific polypeptides and methods and compositions related thereto.
  • the present application further relates to methods of making soluble, monoclonal VLRs. These methods are commercially useful for the large scale production of VLRs.
  • VLRs made by these methods, including, by way of example and not limitation, VLRs specific for pathogens like anthrax, HIV, and influenza and specific for carbohydrates such as blood group determinants.
  • antibodies to VLRs and nucleic acids that encode VLRs. Methods of using VLRs, encoding nucleic acids, and antibodies to VLRs are also disclosed.
  • FIG. 1A is a Western blot of VLRs isolated from lamprey serum and VLRs isolated from culture medium. Blood was collected from lamprey larvae in the presence of EDTA as an anti-coagulant. Blood cells were pelleted by centrifugation at 1,000 g for 5 min, followed by removal of the plasma supernatant. The plasma was treated with the reducing agent 2-mercapto-ethanol (2-ME) or left untreated, then loaded onto SDS-PAGE gels (left panel). In the right panel of FIG. 1A , a cloned VLR cDNA was transfected into HEK-293T cells.
  • 2-ME 2-mercapto-ethanol
  • FIG. 1B is a model of a multivalent VLR.
  • FIG. 2 is a schematic of the method for making antigen-specific VLRs.
  • FIGS. 3A , 3 B and 3 C are Western blots of multimeric VLRs secreted from transfected HEK-293 cells.
  • FIG. 3A shows the results using detergent soluble lysates prepared from transfected HEK-293 cells treated with 2-mercaptoethanol before loading onto SDS-PAGE gels.
  • FIGS. 3B and 3C are Western blots of supernatants removed from VLR transfectants 48 hours after transfection, then loaded directly onto SDS-PAGE gels (B) or pre-treated with 2-mercaptonethanol (C). VLR expression was detected by Western blotting with anti-VLR mAb (4C4).
  • FIG. 4 is a bar graph identifying the specificity of binding of several VLRs.
  • Culture supernatants from VLR transfected HEK-293 cells were incubated in 96-well plates coated with the indicated antigens.
  • VLR binding was detected with anti-VLR mAb (4C4) followed by AP conjugated goat anti-mouse-Ig secondary antibody.
  • FIG. 5A is an alignment of B. anthracis (SEQ ID NO: 1) and B. cereus (SEQ ID NO:2) BclA-CTD amino acid sequence. Non-conserved residues are highlighted in white.
  • FIG. 5B is a sequence alignment showing the comparison of the variable region of VLR4 (SEQ ID NO:3), which binds BclA, and the variable region of VRL5 (SEQ ID NO:4), which does not bind BclA. White indicates amino acid differences, (*) denotes residues predicted to be positively selected and located on the inner surface of the VLR solenoid structure.
  • FIG. 6 shows a FACS histogram demonstrating that lamprey VLRs recognize a human blood group carbohydrate antigen. The results show that only plasma from lamprey immunized with human erythrocytes stained CHO cells transfected with the enzymes to produce the H antigen.
  • FIG. 7 is a sequence alignment of the full length VLR-4 (SEQ ID NO:5) and the full length VLR-5 (SEQ ID NO:6) denoting the various LRR domains.
  • FIG. 8A is a schematic showing a method for producing antigen specific monoclonal VLR-B antibodies.
  • FIG. 8A shows lamprey were immunized by intraperitoneal (I.P.) injection with antigen for four to eight weeks. After immunization, buffy-coat lymphocytes were isolated from peripheral blood and total RNA was prepared.
  • VLR-B cDNAs were isolated by PCR with primers specific for 5′ and 3′ constant regions and cloned into a mammalian expression vector to construct a library.
  • VLR-B cDNAs were transiently transfected into HEK-293T cells and transfectant supernatants were used to screen for antigen binding by ELISA or flow cytometry.
  • FIG. 8B shows time investment required for mouse monoclonal antibody (mAb) versus lamprey monoclonal VLR-B (mVLR-B) antibody production.
  • FIGS. 9A-E show production of monoclonal VLR-B antibodies specific for BclA of B. anthracis .
  • FIG. 9A plates were coated with recombinant BclA-CTD-GST or GST protein, then incubated with supernatant from VLR-B-transfected HEK-293T cells.
  • VLR-B binding was detected with anti-VLR-B mAb (4C4) and AP-conjugated goat anti-mouse polyclonal Ab.
  • FIG. 9B spores were adsorbed to poly-L-lysine-treated plates, then incubated with VLR-B transfectant supernatant. VLR-B binding was detected by ELISA as described in FIG.
  • FIG. 9C shows sequence alignment of BclA-CTD from B. anthracis (SEQ ID NO: 1) and B. cereusT (SEQ ID NO:2). Solvent exposed amino acid differences are shaded black, buried amino acid differences are shaded gray.
  • FIG. 9E shows surface representation of B. anthracis BclA-CTD tertiary structure. Differences in amino acid sequence between B. anthracis and B. cereus are shaded black.
  • FIGS. 10A-D show that recombinant VLR-B is assembled into disulfide-linked multimeric complexes.
  • TEA triethylamine
  • EtGlycol ethylene glycol
  • VLR4 was purified from stable transfectant supernatant by BclA-CTD affinity purification and eluted with triethylamine pH11.5. Purified VLR4 was separated by non-reducing 8% SDS-PAGE and detected with Gelcode blue staining.
  • FIG. 10C the relative migration of purified recombinant VLR4 and high molecular weight protein standards (Amersham Biosciences) in 5, 6, 7, 8, 10, and 12% native polyacrylamide gels were measured and used to construct Ferguson plots to estimate the molecular weight of multimeric VLR4.
  • monomers, dimers, and oligomers were detected by Western blotting VLR4-containing supernatant under partial reducing conditions.
  • FIGS. 11A-D show that the cysteine-rich C-terminus of VLR-B is required for oligomer assembly.
  • FIG. 11A supernatants from VLR4 wild-type (WT) and GPI-stop transfected HEK-293T cells were separated on a non-reducing 10% SDS-PAGE gel and Western blotted with anti-VLR mAb (4C4) followed by HRP-conjugated goat anti-mouse polyclonal Ab.
  • FIG. 11B VLR4 was purified from HEK-293T cell supernatant, separated by reducing SDS-PAGE, Gel-code blue stained, and excised by scalpel.
  • FIG. 11C is a schematic of VLR4 WT and GPI-stop constructs. GPI cleavage site is shown in italics and indicated by an arrow. Tryptic peptide identified by MS/MS is indicated by a black line above the sequence (SEQ ID NO:40).
  • FIG. 10D is a graph showing results of ELISA of VLR4 WT and GPI-stop binding to BclA-1-island coated plates.
  • FIGS. 12A-C show modulation of VLR5 avidity by site-directed mutagenesis of hypervariable amino acids on the concave surface.
  • FIG. 12A is a multiple sequence alignment of high avidity (vBA41 (SEQ ID NO:41), vBA191 (SEQ ID NO:42), and VLR4 (SEQ ID NO:43)) and low avidity (VLR5 (SEQ ID NO:44)) anti-Bcla-CTD VLR-B antibodies.
  • Hypervariable positions are in the boxes, VLR5 amino acids that differ from consensus residues utilized by high avidity VLR-B antibodies are shaded with a certain pattern if they reside in hypervariable positions. Sequence differences outside of hypervariable positions are shaded grey.
  • FIG. 12B is a model of the concave surface of VLR5. Discrepancies in amino acids utilized by VLR5 versus the consensus of the high avidity anti-BclA-CTD VLR-B antibodies are shaded with the same pattern as in A.
  • the pattern over the H at position 13 in FIG. 12A corresponds to the circles shaded with the same pattern in FIG. 12B .
  • the pattern over the Y at position 34 in FIG. 12A corresponds to the circles shaded with the same pattern in FIG. 12B .
  • the pattern over the T at position 37 in FIG. 12A corresponds to the circles shaded with the same pattern in FIG. 12B .
  • the pattern over the Q at position 80 in FIG. 12A corresponds to the circles shaded with the same pattern as in FIG. 12B .
  • the pattern over the S at position 82 in FIG. 12A corresponds to the circles shaded with the same pattern in FIG. 12B .
  • the pattern over the W at position 106 in FIG. 12A corresponds to the circles shaded with the same pattern in FIG. 12B .
  • FIG. 12C the relative avidity of VLR-B antibodies were measured by surface plasmon resonance (BiaCore 3000). BclA-1-island was covalently conjugated to a Biacore CM5 chip, then VLR transfectant supernatants, normalized for protein expression, were flowed over the chip. The chip was regenerated after each binding cycle with triethylamine pH 11.5.
  • FIG. 13 is a model of anti-H antigen monoclonal VLR-B (vRBC-36 (SEQ ID NO:20)) antigen binding site.
  • the vRBC-36 model was constructed by homology-based modeling to hagfish VLR-B (PDB ID: 206R) crystal structure data using SWISS-MODEL (http://swissmodel.expasy.org/). Hypervariable amino acid positions are highlighted dark grey.
  • the arrow denotes a depression on the concave surface that is the likely contact surface of the fucose sugar that distinguishes the H antigen from other carbohydrate moieties.
  • FIGS. 14A-D show the analysis of VLR-B antibodies produced after immunization with human blood group O erythrocytes.
  • FIG. 14A shows hemagglutinin responses of animals immunized with increasing numbers of human O erythrocytes. Blood samples were obtained before and 28 days after immunizations; immunization was on days 1 and 14.
  • FIG. 14B shows hemagglutination titers before and after plasma adsorption with beads coated with a monoclonal anti-VLR-B or a control antibody. Error bars indicate standard error of the mean.
  • FIG. 14A shows hemagglutinin responses of animals immunized with increasing numbers of human O erythrocytes. Blood samples were obtained before and 28 days after immunizations; immunization was on days 1 and 14.
  • FIG. 14B shows hemagglutination titers before and after plasma adsorption with beads coated with a monoclonal anti-VLR-B or
  • FIG. 14C shows flow cytometric analysis comparing H antigen reactivity of plasma from immunized lamprey versus an anti-H monoclonal mouse antibody; staining is shown for ⁇ 1,2-fucosyltransferase CHO cell transfectants expressing the H antigen. No reactivity was observed for non-transfected CHO cells.
  • FIG. 14D shows that depletion of H antigen-specific VLR antibodies by adsorption with H antigen-bearing CHO cells removes hemagglutinating activity from plasma. Depletion of H antigen-reactive VLRs has little effect on the VLR plasma level.
  • FIGS. 15A and B show recombinant VLR antibody specificity for the H antigen.
  • FIG. 15A CHO cells transfected with ⁇ 1,2-fucosyltransferase to produce the H antigen or vector alone transfected cells were stained with anti-H mAb or supernatant from HEK 293T cells transfected with VLR-B specific for the H antigen. Gray represents unstained cells and black with no fill represents cells stained with mAb or VLR antibodies.
  • FIG. 15B shows a Western blot of lamprey plasma before and after treatment with 2-mercaptoethanol to reduce disulfide bonds.
  • FIGS. 16A-C show VLR antibody response to immunization with B. anthracis exosporium. Plasma samples from immunized (black bars) and unimmunized (white bars) lamprey were assayed by ELISA.
  • FIG. 16A shows evaluation of antigen dose requirement. VLR antibody response to BclA before (x) and after two intraperitoneal immunizations with 1 ( ⁇ ), 0.1 ( ⁇ ) or 0.01 ( ⁇ ) ⁇ g of B. anthracis exosporium. Booster immunizations were given after two weeks and plasma samples were obtained at four weeks.
  • FIG. 16B shows that the VLR antibody response is directed toward the C terminal domain of the spore coat protein BclA (BslA-CTD).
  • FIG. 16C shows the specificity of VLR antibodies for B. anthracis spore coat protein BclA after two immunizations with anthrax exosporium (1 ⁇ g). Error bars indicate standard error of the
  • FIGS. 17A-D show tissue distribution of VLR+ lymphocytes.
  • FIG. 17A shows immunohistochemical analysis of VLR-B+ cells in different organs. Paraffin sections were stained with hematoxylin and eosin (top) or anti-VLR mAb using DAB as a chromogen (bottom).
  • FIG. 17B shows immunofluorescence identification of VLR-B+ lymphocytes within a large blood vessel of the gill region (corresponds with large blood vessel at gill base in top left panel of A).
  • FIG. 17C shows immunofluorescence analysis of VLR expression by lymphocytes from blood, kidney, and typhlosole. Histograms depict analysis of cells in the ‘lymphocyte gate’ isolated from different tissues.
  • FIG. 17A shows immunohistochemical analysis of VLR-B+ cells in different organs. Paraffin sections were stained with hematoxylin and eosin (top) or anti-VLR mAb using DAB as a chromogen
  • 17D shows transmission electron microscopy (EM) of VLR-B+ and VLR-B ⁇ cells sorted from ‘lymphocyte gate’ of blood sample: photomicrographs of resting VLR+ lymphocyte (top) and thrombocyte with characteristic nuclear cleft (bottom).
  • EM transmission electron microscopy
  • FIG. 18 is a graph showing the gene expression profile for VLR-B+ and VLR-B ⁇ lymphocyte populations. Quantitative PCR analysis of VLR-B+ and VLR-B-cells isolated by fluorescence activated cell sorting of cells in ‘lymphocyte gate’.
  • FIGS. 19A and 19B show lymphoblastoid response of VLR-B+lymphocytes in lamprey hyperimmunized with anthrax exosporium.
  • FIG. 19A shows flow cytometric analysis of forward and side light scatter characteristics of ungated blood leukocytes versus VLR-B+ cells. Blood samples were from animals 14 days after booster injection of a super-immunogenic dose of B. anthracis exosporium (>25 ⁇ g).
  • FIG. 19B shows cell surface expression of VLR-B. There was a decrease in VLR-B expression levels following hyper immunization with anthrax exosporium.
  • FIGS. 20A and 20B show analysis of the frequency of antigen binding VLR-B+ cells before and after immunization with B. anthracis exosporium.
  • FIG. 20A shows flow cytometric analysis of VLR-B+ cells in blood samples from na ⁇ ve and immunized animals co-stained with 4C4 anti-VLR monoclonal antibody and fluorescent-tagged spores.
  • FIG. 20B shows percentage of anthrax spore binding cells before and after (28 days) immunization with B. anthracis exosporium.
  • FIGS. 21A and 21B show characterization of VLR-B secreting cells induced by immunization with B. anthracis exosporium. Pooled cells were sorted from six 13 cm lamprey larvae 14 days after a booster immunization with 1 ⁇ g of exosporium.
  • FIG. 21A shows ELISPOT assay of VLR-B antibody secreting cells among VLR-B+ and VLR-B ⁇ populations of cells with different light scatter characteristics. Cells secreting VLR-B antibodies specific for the BclA anthrax coat protein were found in the subpopulation of relatively large VLR-B bearing cells.
  • FIG. 21B shows EM analysis of large VLR-B+ producing cells indicates their plasmacytoid morphology with expanded rough endoplasmic reticulum.
  • FIG. 22 shows comparison of na ⁇ ve and immunized lamprey following injection with non-mitogenic dose of anthrax exosporium.
  • Immunized lamprey were injected twice with 1 ⁇ g of anthrax exosporium. This dose of anthrax dose not induce lymphoblastoid transformation but still generates a specific immune response to BclA-CTD.
  • FIG. 23 shows that lamprey immunized with influenza virus produce VLRs specific for immunogen.
  • ELISA assay performed with 1:50 dilution of lamprey plasma.
  • FIG. 24 shows that lamprey immunized with HIV virus like particles (VLPs) produce VLR-B antibodies specific for HIV envelope protein gp120 subunit.
  • ELISA plates were coated with purified recombinant HIV gp120 overnight and then incubated with na ⁇ ve or HIV VLP immunized lamprey plasma.
  • Gp120 binding VLR-B antibodies were detected with anti-VLR-B mAb (4C4) and alkaline phosphatase-conjugated goat anti-mouse IgG polyclonal antibody.
  • V lymphocytes The adaptive immune system in jawless vertebrates is comprised of clonally diverse lymphocytes. They have been named V lymphocytes, because they express Variable Lymphocyte Receptors (VLRs) derived from the assembly of leucine-rich repeat (LRR) gene segments, rather than the immunoglobulin V, D, and J gene subunits utilized by jawed vertebrates.
  • VLRs Variable Lymphocyte Receptors
  • LRR leucine-rich repeat
  • Two VLR genes, VLR-A and VLR-B have been identified in lamprey and hagfish, the two extant representatives of the jawless vertebrates (agnathans).
  • the germline VLR genes are incomplete in that they have coding regions only for the invariant N-terminal and C-terminal sequences separated by intervening sequences, lacking canonical splice sites.
  • flanking LRR modular units are sequentially inserted into the incomplete VLR gene with a concomitant deletion of the intervening sequences via a gene conversion mechanism to generate a mature VLR gene.
  • the gene conversion process may be catalyzed by recently identified activation-induced deaminase/apolipoprotein B-editing catalytic protein (AID-APOBEC) family members with lymphocyte restricted expression.
  • AID-APOBEC activation-induced deaminase/apolipoprotein B-editing catalytic protein
  • VLR-B+ lymphocytes constitute a major component of the humoral arm of the lamprey adaptive immune system.
  • immunization of lamprey with particulate antigens such as, for example, B. anthracis exosporium or human red blood cells, induces the differentiation of plasmacytoid cells and their secretion of antigen-specific VLR-B antibodies.
  • Structural analysis of hagfish VLR-B lacking most of the stalk region confirmed the previous modeling prediction that the hypervariable amino acids are concentrated on the concave surface of the receptor to form a putative antigen binding site.
  • VLR-B antibodies function analogously to antibodies in jawed vertebrates, whereby antigen stimulation results in secretion of VLR-B as an effector molecule, which binds to antigen and promotes clearance of infection, presumably by neutralization, opsonization, and other mechanisms.
  • Monoclonal antibodies are valuable research and therapeutic tools that take advantage of the remarkable ability of the jawed vertebrate adaptive immune system to recognize almost any foreign molecule.
  • the tremendous repertoire of diversity of the agnathan adaptive immune system can be exploited to produce soluble VLR-B clones of known specificity, with similar properties to monoclonal antibodies.
  • Described herein is a method of producing soluble, recombinant monoclonal VLR-B antibodies of defined antigen specificity.
  • compositions including specific VLRs, multivalent VLRs, and antibodies to VLRs as well as methods of using the compositions.
  • the method of making a soluble, monoclonal antigen specific polypeptide comprises the steps of (1) isolating a cDNA clone encoding an antigen specific polypeptide, wherein the antigen specific polypeptide comprises an N-terminal leucine rich repeat (LRRNT), one or more leucine rich repeats (LRRs), a C-terminal leucine rich repeat (LRCCT), and a connecting peptide, wherein the connecting peptide comprises an alpha helix; (2) transfecting a cell with the cDNA clone in culture medium, wherein the cell proliferates; and (3) isolating the antigen specific polypeptide from the culture medium.
  • LRRNT N-terminal leucine rich repeat
  • LRRs leucine rich repeats
  • LRCCT C-terminal leucine rich repeat
  • the method of making the antigen specific protein comprises (1) administering to a lamprey or hagfish a target antigen (e.g., a target carbohydrate, a target protein, a target pathogen, a target glycoprotein, a target lipid, a target glycolipid, etc.); (2) isolating an antigen specific protein-encoding RNA from lymphocytes of the lamprey or hagfish; (3) amplifying antigen specific protein encoding cDNA from the isolated RNA; (4) cloning the cDNA into an expression vector; (5) expressing the expression vector in a bacterium transformed with the expression vector; (6) isolating a cDNA clone; (7) transfecting a cultured cell with a the isolated cDNA clone; (8) screening the culture supernatant for an ability to bind the target antigen, and (9) isolating the antigen specific protein from the supernatant that binds the target antigen.
  • a target antigen e.g.
  • the antigen can be administered in an amount sufficient to produce antigen-specific VLRs.
  • 0.01, 0.1, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75 or 100 ⁇ g or any amount in between 0.01 and 100 ⁇ g or more of antigen can be administered to the lamprey or hagfish.
  • the isolated cDNA clone does not encode a sequence that prevents the formation of soluble VLRs.
  • approximately 50% of VLR clones contain: KNWIVQHASIVN-(P/L)-X-(S/Y/N/H)-GGVDNVK (SEQ ID NO:7) or KNWIVQHASIVN-(P/L)-XX-(S/Y/N/H)-GGVDNVK (SEQ ID NO: 8), where (P/L) means either P or L in that position, X means any amino acid and (S/Y/N/H) means either S, Y, N or H in that position.
  • VLRs without SEQ ID NOs:7 or 8 are only membrane bound.
  • SEQ ID NOs:7 or 8 can be mutated to prevent or reduce membrane anchoring in any cDNA clone that contains this sequence by methods known to those of ordinary skill in the art.
  • soluble, monoclonal antigen specific polypeptides made by the methods described herein.
  • a solube VLR contains SEQ ID NOs:7 or 8 or contains a mutation in SEQ ID NOs:7 or 8 that reduces or prevents membrane anchoring.
  • a soluble VLR optionally lacks the transmembrane domain, the GPI anchor, the hydrophobic tail, the stalk region, or any combination of these regions.
  • VLR variable lymphocyte receptor or VLR is an antigen specific polypeptide having certain structural characteristics and functions.
  • VLRs comprise 1-12 leucine rich repeats and have been shown to function in adaptive immunity. More particularly VLRs comprise an N-terminal leucine rich repeat (LRRNT), one or more leucine rich repeats (LRRs) (referred to herein as the internal LRRs), a C-terminal leucine rich repeat (LRRCT), and a connecting peptide, wherein the connecting peptide comprises an alpha helix.
  • LRRNT N-terminal leucine rich repeat
  • LRRs leucine rich repeats
  • LRRs leucine rich repeats
  • LRRCT C-terminal leucine rich repeat
  • the length of the VLR can comprise as few as about 130 amino acids or as many as about 225 amino acids.
  • the connecting peptide of the VLR is located on the N-terminal side of the LRRCT, and more specifically located between an internal LRR and the LRRCT.
  • the connecting peptide can be linked to an internal LRR and the LRRCT.
  • VLRs comprising a LRRNT, one or more internal LRRs, a connecting peptide, and a LRRCT, in that order.
  • VLRs wherein the internal LRR region between the LRRNT and the LRRCT comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 leucine rich repeats, with LRR 1 located adjacent to or closest to the LRRNT.
  • LRRs 1, 2, 3, 4, 5, 6, 7, 8, or 9 are considered to run from the LRRNT to the LLRCT, consecutively.
  • VLRs comprising a LRRNT, 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, or 1-9 LRRs, a connecting peptide, and a LRRCT, in that order.
  • LRRs are short sequence motifs typically involved in protein-protein interactions, wherein the LRRs comprise multiple leucine residues.
  • LRRs contain leucine or other aliphatic residues, for example, at positions 2 , 5 , 7 , 12 , 16 , 21 , and 24 .
  • the leucine or other aliphatic residues can occur at other positions in addition to or in the place of residues at positions 2 , 5 , 7 , 12 , 16 , 21 , and 24 .
  • a leucine can occur at position 3 rather than position 2 .
  • the LRR motifs form ⁇ -sheet structures.
  • a disclosed polypeptide comprising a LRRNT, 5 separate LRRs, a LRRCT, and a connecting peptide would comprise 7 ⁇ -sheet structures and the alpha helix of the connecting peptide.
  • each internal LRR can vary from the other internal LRRs in the VLR as well as from the LRRNT and LRRCT.
  • VLRs can comprise a LRRNT, 1 to 9 LRRs, a connecting peptide, and a LRRCT, wherein the first internal LRR is LRR1, and wherein LRR1 comprises less than about 20 amino acids.
  • VLRs wherein LRR1 comprises about 18 amino acids.
  • the VLR further comprises LRRs 2 to 9, wherein LRRs 2 to 9 are less than about 25 amino acids each.
  • LRRs 1 to 9 can be the same or different from each other in a given VLR both in length and in specific amino acid sequence.
  • the terminal LRRs are typically longer than each internal LRR.
  • the LRRNT and LRRCT comprise invariant regions (regions that have little variation relative to the rest of the polypeptide as compared to similar variable lymphocyte receptors).
  • the variable regions provide the receptors with specificity, but the invariant regions and general structural similarities across receptors help maintain the protective immunity functions.
  • the VLR can comprise an LRRNT, wherein the LRRNT comprises less than about 40 amino acids.
  • the LRRNT optionally comprises the amino acid sequence CPSQCSC (SEQ ID NO:9), CPSRCSC (SEQ ID NO: 10), CPAQCSC (SEQ ID NO: 11), CPSQCLC (SEQ ID NO: 12), CPSQCPC (SEQ ID NO: 13), NGATCKK (SEQ ID NO: 14), or NEALCKK (SEQ ID NO: 15) in the presence or absence of one or more conservative amino acid substitutions.
  • VLRs comprising a LRRCT, wherein the LRRCT is less than about 60 amino acids, and optionally from 40 to 60 amino acids in length.
  • the LRRCT comprises the amino acid sequence TNTPVRAVTEASTSPSKCP (SEQ ID NO:16), SGKPVRSIICP (SEQ ID NO: 17), SSKAVLDVTEEEAAEDCV (SEQ ID NO: 18), or QSKAVLEITEKDAASDCV (SEQ ID NO: 19) in the presence or absence of conservative amino acid substitutions.
  • the connecting peptides of VLRs are short peptides less than 15 amino acids in length and comprise an alpha helix.
  • connecting peptides of 10, 11, 12, 13, 14, and 15 amino acids in length comprising an alpha helix.
  • the connecting peptide serves to link structural components of the VLR, including to the LRRCT.
  • the VLRs described herein selectively bind an antigen or an agent, much as an antibody selectively binds an antigen or agent.
  • selectively binding or specifically binding is meant that the VLR binds one agent or antigen to the partial or complete exclusion of other antigens or agents.
  • binding is meant a detectable binding at least about 1.5 times the background of the assay method. For selective or specific binding such a detectable binding can be detected for a given antigen or agent but not for a control antigen or agent.
  • VLRs may be naturally occurring or non-naturally occurring. Fragments or variants of VLRs are described below wherein the fragment or variant retains the ability of the VLR to selectively bind an antigen or agent. Thus, VLR, like the term antibody, includes various versions having various specificities. VLRs are tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their therapeutic, diagnostic or other purification activities are tested according to known testing methods. For example, ELISA, dot blot, Western blot analysis, and other testing methods can be used to test activity and/or specificity.
  • VLRs can be detected by direct labeling of the VLR or by using a secondary VLR or an antibody that binds VLRs, analogous to a secondary antibody, and wherein the antibody or secondary VLR are labeled directly or indirectly.
  • Antibodies to VLR and labels are described in more detail below.
  • a multivalent protein comprising multiple antigen specific polypeptides, such as VLRs wherein each antigen specific polypeptide comprises a N-terminal leucine rich repeat (LRRNT), one or more leucine rich repeats (LRRs), a C-terminal leucine rich repeat (LRCCT), and connecting peptide, wherein the connecting peptide comprises an alpha helix.
  • LRRNT N-terminal leucine rich repeat
  • LRRs leucine rich repeats
  • LRCCT C-terminal leucine rich repeat
  • connecting peptide comprises an alpha helix.
  • LRR-1 refers to the first LRR following the LRRNT.
  • LRRV refers to LRR Variable, which is an LRR that follows the LRR-1 but comes before the LRRCT.
  • LRRV e refers to LRR Variable end, which is the last LRR that comes before the LRRCT. However, if the VLR contains an LRRNT, one LRR and the LRRCT. The LRR between the LRRNT and LRRCT is designated LRR-1.
  • a schematic of a multivalent VLR is shown in FIG. 1 .
  • the multivalent protein comprises two to twelve (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) antigen specific polypeptides.
  • the multivalent protein binds a target protein, a target carbohydrate, target glycoprotein, target proteoglycan, or a target pathogen.
  • Multivalent proteins optionally are designed to bind a variety of target proteins, carbohydrates, glycoprotens, proteoglycans, pathogens, or any combination thereof.
  • a divalent protein can comprise a first and second antigen specific polypeptide, wherein the first antigen specific polypeptide selectively binds a first protein, carbohydrate, glycoprotein, proteoglycan, or pathogen and wherein the second antigen specific polypeptide selectively binds a second protein, carbohydrate, glycoprotein, proteoglycan, or pathogen.
  • a trivalent protein comprises a first, second, and third antigen specific polypeptide wherein each binds a different target; a tetravalent comprises a first, second, third and fourth antigen specific polypeptide wherein each binds a different target.
  • a divalent protein comprises a first antigen specific polypeptide that binds the H blood group determinant and a second antigen specific polypeptide that binds the A or B group determinant.
  • the multivalent protein and/or the antigen specific polypeptides are soluble.
  • antigen specific polypeptides that bind a target carbohydrate including, for example, a blood group determinant.
  • the blood group determinant includes, for example, the A determinant, the B determinant, or the H determinant.
  • an antigen specific polypeptide that specifically binds the H determinant is provided.
  • the antigen specific polypeptide that specifically binds the H determinant comprises the amino acid sequence of SEQ ID NO:20.
  • nucleic acids that can encode the antigen specific polypeptide of SEQ ID NO:20 including, for example, SEQ ID NO:32.
  • Other examples include nucleic acids that encode SEQ ID NO:20 with one or more conservative amino acid substitutions.
  • Antigen specific polypeptides that bind carbohydrates have many uses in identifying, quantifying, isolating, and imaging the target carbohydrate.
  • a method of typing blood comprising contacting a blood sample with the antigen specific polypeptide that selectively binds a blood group determinant, wherein the antigen specific polypeptide is detectably labeled (directly or indirectly).
  • the labeled antigen specific polypeptide bound to one or more cells in the blood sample is detected.
  • the presence or absence of the label indicates the blood type.
  • the presence of label in a blood sample indicates an O blood type.
  • the presence of label when an A determinant-specific polypeptide is used indicates either A or AB blood type.
  • the presence of label when a B determinant-specific polypeptide is used indicates either B or AB blood type.
  • One or more antigen specific polypeptides can be used with the same blood sample.
  • different labels can be attached to each antigen specific polypeptide if they have a different specificity. Accordingly, an FITC label could be linked directly or indirectly to the VLR that selectively binds an H determinant, whereas fluorescent labels that fluoresce at different wavelengths can be linked directly or indirectly to a VLR that selectively bind an A or B determinant.
  • a method of typing blood comprising contacting a blood sample with a first antigen specific polypeptide, wherein the first antigen specific polypeptide is detectably labeled with a first label and wherein the first antigen specific polypeptide is specific for a first blood determinant; contacting the blood sample with a second antigen specific polypeptide, wherein the second antigen specific polypeptide is detectably labeled with a second label and wherein the second antigen specific polypeptide is specific for a second blood determinant; and detecting labeled first and second antigen specific polypeptides bound to one or more cell in the blood sample, the presence or absence of the first and second labels indicating the blood type.
  • VLRs that selectively binds an agent, such as a pathogenic agent, wherein the pathogen is a bacterium, and more particularly wherein the bacterium is Bacillus anthracis .
  • an antigen specific polypeptide wherein the binding polypeptide specifically binds a Bacillus anthracis cell surface polypeptide, such as BclA.
  • the antigen binding polypeptide has the amino acid sequence of SEQ ID NO: 5 (see FIG. 7 ) or SEQ ID NOs:22, 47, 49, 51, 53, 55, 57, 59 or 61.
  • nucleic acids that encode SEQ ID NOs:5, 22, 47, 49, 51, 53, 55, 57, 59 or 61, including, for example, SEQ ID NOs:21, 23, 46, 48, 50, 52, 54, 56, 58 and 60, respectively.
  • Pathogens include any known pathogens such as, for example, bacteria and viruses.
  • a method of detecting the presence of Bacillus anthracis in a sample comprising contacting the sample with the antigen specific polypeptide that binds Bacillus anthracis , wherein the antigen specific polypeptide is detectably labeled.
  • the labeled antigen specific polypeptide bound to the sample is detected and the presence of the label indicates the presence of Bacillus anthracis in the sample.
  • a method of reducing the pathogenicity of Bacillus anthracis in a subject comprising administering to the subject the antigen specific polypeptide that binds Bacillus anthracis.
  • a method of detecting the presence of a virus in a sample comprising contacting the sample with the antigen specific polypeptide that binds the virus wherein the antigen specific polypeptide is detectably labeled.
  • the labeled antigen specific polypeptide bound to the sample is detected and the presence of the label indicates the presence of virus in the sample.
  • a method of reducing the pathogenicity of a virus in a subject comprising administering to the subject the antigen specific polypeptide that binds the virus.
  • the virus can be, for example, HIV or influenza.
  • the antigen specific polypeptide can bind to, for example, HIV envelope protein gp120.
  • a method of removing a pathogen from a subject's blood sample or other biological fluid is also provided.
  • the method comprises contacting the sample with an antigen specific polypeptide that selectively binds the pathogen.
  • a method of reducing the amount of a pathogen in a subject's blood comprising contacting a portion of the subject's blood with an antigen specific polypeptide that selectively binds the pathogen.
  • the blood to be contacted is removed and then returned to the subject.
  • the antigen is bound to a solid support.
  • Also provided herein are methods of making antigen specific proteins having a selected antigen specificity and compositions useful in these methods comprising administering to a lamprey or hagfish one or more target antigens (e.g., a target carbohydrate, a target protein, a target pathogen, a target glycoprotein, a target lipid, a target glycolipid, a target cell and any combination thereof including, for example, two carbohydrates, one carbohydrate and one protein, etc.).
  • a target antigens e.g., a target carbohydrate, a target protein, a target pathogen, a target glycoprotein, a target lipid, a target glycolipid, a target cell and any combination thereof including, for example, two carbohydrates, one carbohydrate and one protein, etc.
  • an antigen specific protein that binds a blood group determinant comprising administering to a lamprey or hagfish the blood group determinant; isolating an antigen specific protein-encoding RNA from lymphocytes of the lamprey or hagfish; amplifying antigen specific protein encoding cDNA from the isolated RNA; cloning the cDNA into an expression vector; expressing the expression vector in a bacterium transformed with the expression vector; isolating a cDNA clone; transfecting a cultured cell with a the cDNA clone; screening the culture supernatant for an ability to bind the blood group determinant, and isolating the antigen specific protein from the supernatant that binds the blood determinant.
  • the erythrocyte itself, for example type O human erythrocytes, can be administered to the lamprey or hagfish to generate antigen specific proteins.
  • VLRs that specifically bind a pathogen like Bacillus anthracis can be made by administering to a lamprey or hagfish a cell surface Bacillus anthracis polypeptide isolating an antigen specific protein-encoding RNA from lymphocytes of the lamprey or hagfish; amplifying antigen specific protein encoding cDNA from the isolated RNA; cloning the cDNA into an expression vector; expressing the expression vector in a bacterium transformed with the expression vector; isolating a cDNA clone; transfecting a cultured cell with a the cDNA clone; screening the culture supernatant for an ability to bind the cell surface Bacillus anthracis polypeptide, and isolating the antigen specific protein from the supernatant that binds the cell surface Bacillus anthracis polypeptide.
  • the pathogen itself for example the Bacillus anthracis
  • VLRs that specifically bind a pathogen such as, for example, a virus, like HIV or influenza
  • a pathogen such as, for example, a virus, like HIV or influenza
  • a viral antigen includes the virus, a virus-like particle, a fragment of the virus, a polypeptide expressed by the virus or any other portion or part of the virus that stimulates an antigenic response in the lamp
  • the methods of making the antigen specific polypeptides, as well as fragments and variants thereof, include making a stable cell line that expresses the nucleic acid that encodes the antigen specific polypeptide or fragment or variant thereof.
  • Stable cell lines can be produced by a variety of methods. For example, stable cell lines can be produced by transfecting cells with expression vectors that co-express a VLR cDNA and a selectable marker, such as a gene that encodes for resistance to antibiotics. In the case of antibiotic selection, cells that stably integrate the expression vector into their genome will be resistant to antibiotics selection and survive, while other cells will die upon treatment with the antibiotic.
  • Sub-clones may be established of cells that exhibit the highest levels of VLR secretion by such methods as limiting dilution cloning.
  • methods of making the antigen specific polypeptides by culturing cells of the stable cell line under conditions that allow the cells to express the antigen specific polypeptide and isolating the antigen specific polypeptide from the cells or culture medium.
  • VLR producing lymphocytes VLR producing lymphocytes, VLR cells and VLR lymphocytes are used synonymously.
  • an isolated population of VLR-B+ lymphocytes are provided.
  • VLR-B+ lymphocytes express VLR-B transcripts and not VLR-A transcripts.
  • VLR-B+ lymphocytes express TCR-like, CD-4-like and/or TNFR14.
  • VLR-A+ cells express VLR-A transcripts and not VLR-B transcripts.
  • the isolated population of VLR-A+ cells express CD45 and/or GATA.
  • the isolated populations of cells can be obtained using routine experimentation, for example, by flow cytometry or using VLR-B or VLR-A specific antibodies.
  • a isolated population of antigen specific VLR-B+ cells are also provided.
  • antigen specific VLR-B+ cells refers to cells that express an antigen specific polypeptide. Such cells can be produced, for example, by immunizing a lamprey or hagfish with antigen and isolating the VLR-B+ cells by flow cytometry or using VLR-B specific antibodies, such as those provided herein, for example, 4C4 or 6C3.
  • VLR-A+ cells can be similarly isolated.
  • nucleic acids including, for example, isolated nucleic acids and including RNA and DNA
  • Nucleic acids that can encode the VLRs or regions thereof as well as variants and fragments of disclosed VLRs are disclosed herein.
  • Nucleic acids that can encode VLRs include, but are not limited to, SEQ ID NOs:21, 23, 45, 46, 48, 50, 52, 54, 56, 58, 60 and 32.
  • Nucleic acids that can encode LRRNTs include, but are not limited to,
  • SEQ ID NO:28 (TGTCCCTCGCAGTGTCCGTGT).
  • Nucleic acids that can encode LRRCTs include, but are not limited to SEQ ID NO:29 (ACCAATACCCCCGTCCGTGCGGTCACCGAGGCCAGCACTAGCCCCTCGAA ATGCCCA). Examples of nucleic acids include all degenerate sequences related to a specific polypeptide sequence and variants and derivatives thereof. The nucleic acids provided herein include complements of the encoding sequence.
  • Nucleic acids are provided that encode any one of SEQ ID NOs:5, 6, 20, 22, 47, 49, 51, 53, 55, 57, 59, 61 or any specific regions thereof, including, for example, LRRNT (e.g., nucleic acids that encode SEQ ID NOs: 9-15), LRR, LRCCT (e.g., nucleic acids that encode SEQ ID NOs: 16-19), or the connecting peptide. More specifically, provided herein is a nucleic acid comprising SEQ ID NOs:21, 23, 45, 46, 48, 50, 52, 54, 56, 58, 60 and 32 or degenerate variants or complements thereof.
  • isolated nucleic acids comprising a sequence that hybridizes under highly stringent conditions to all or any portion of SEQ ID NOs:21, 23, 45, 46, 48, 50, 52, 54, 56, 58, 60 or 32 or the complement of SEQ ID NOs:21, 23, 45, 46, 48, 50, 52, 54, 56, 58, 60 or 32.
  • the hybridizing portion of the hybridizing nucleic acids is typically at least 15 (e.g., 20, 20, 40, or more) nucleotides in length.
  • the hybridizing portion is at least 80% (e.g., 90% or 95%) identical to the a portion of the sequence to which it hybridizes.
  • Hybridizing nucleic acids are useful, for example, as cloning probes, primers (e.g., PCR primer), or a diagnostic probe.
  • Nucleic acid duplex or hybrid stability is expressed as the melting temperature or Tm, which is the temperature at which a probe dissociates from a target DNA. This melting temperature is used to define the required stringency conditions. If sequences are to be identified that are related and substantially identical to the probe, rather than identical, then it is useful to first establish the lowest temperature at which only homologous hybridization occurs with a particular concentration of salt (e.g., SSC or SSPE). Assuming that a 1% mismatching results in a 1° C.
  • salt e.g., SSC or SSPE
  • the temperature of the final wash in the hybridization reaction is reduced accordingly (for example, if sequences having more than 95% identity are sought, the final wash temperature is decreased by 5° C.).
  • the change in Tm can be between 0.5 and 1.5° C. per 1% mismatch.
  • Highly stringent conditions involve hybridizing at 68° C. in 5 ⁇ SSC/5 ⁇ Denhardt's solution/1.0% SDS, and washing in 0.2 ⁇ SSC/0.1% SDS at room temperature.
  • Moderately stringent conditions include washing in 3 ⁇ SSC at 42° C. Salt concentrations and temperature can be varied to achieve the optimal level of identity between the probe and the target nucleic acid. Additional guidance regarding such conditions is readily available in the art, for example, in “Molecular Cloning: A Laboratory Manual,” Third Edition by Sambrook et al., Cold Spring Harbor Press, 2001.
  • nucleic acids having 80-99% identity i.e., 80, 81, 82 . . . 99%
  • Methods of determining percent identity are known in the art and are as described below in the context of amino acids.
  • compositions including primers and probes, which are capable of interacting with the VLR gene, or comparable genes.
  • the primers are used to support DNA amplification reactions.
  • the primers will be capable of being extended in a sequence specific manner.
  • Extension of a primer in a sequence specific manner includes any methods wherein the sequence and/or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer.
  • Extension of the primer in a sequence specific manner therefore includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions that amplify the primer in a sequence specific manner are preferred.
  • the primers are used for the DNA amplification reactions, such as PCR or direct sequencing. It is understood that in certain embodiments the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner. Examples of primers taught herein include, but are not limited to, 1) 5′-CCACCATGTGGATCAAGTGGATCGCC-3′ (SEQ ID NO:30) and 2) 5′-GAGAGCTAGCTCAACGTTTCCTGCAGAGGGC-3′ (SEQ ID NO:31). Such primers can also be used as hybridization probes as discussed above.
  • the first primer contains a consensus Kozak sequence ahead of the start codon for optimum translation. It is also preferably 5′ phosphorylated such that the PCR product can be cloned into blunt-end restriction enzyme sites.
  • the second primer possesses a restriction enzyme site. The resulting PCR product can then be digested with restriction enzyme and cloned into the expression vector.
  • the restriction enzyme site in the second primer is an NheI restriction site, since these sites have not been found in any of the characterized VLRs to date.
  • expression vectors comprising the nucleic acids that encode VLR or fragments or variants thereof.
  • these expression vectors further comprise an expression control sequence operably linked to the nucleic acid encoding the VLR or fragment or variant thereof.
  • a vector that comprises a nucleic acid that encodes an antigen specific polypeptide e.g., nucleic acids that encode SEQ ID NOs:5 or 22.
  • cultured cells comprising the expression vectors.
  • Suitable expression vectors include, but are not limited to, pLPCX and pIRES-PURO2 (both from Clontech Laboratories, Inc., Mountain View, Calif.).
  • the expression vector can include both a VLR encoding nucleic acid and an antibiotic resistance gene from the same transcript by utilizing an internal ribosome entry site (IRES) sequence. This allows for efficient selection of stable cell lines.
  • IRS internal ribosome entry site
  • VLR5 SEQ ID NO:6
  • VLR5 SEQ ID NO:6
  • VLR5 W127Y VLR5 W127Y
  • VLR5 Y55R/W127Y Other VLRs can be similarly modified using the methods provided herein. Methods of making and screening multiple variants include, for example, in vitro affinity maturation using phage, yeast, bacterial or ribosome display techniques.
  • VLR variants that have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity to a stated sequence.
  • identity can be calculated after aligning the two sequences so that the identity is at its highest level.
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection.
  • a sequence recited as having a particular percent identity to another sequence refers to sequences that have the recited identity as calculated by any one or more of the calculation methods described above.
  • a first sequence has 80 percent identity, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent identity to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent identity to the second sequence as calculated by any of the other calculation methods.
  • VLR variants and derivatives can involve amino acid sequence modifications.
  • amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants.
  • Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues.
  • Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule.
  • variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the polypeptide, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.
  • Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis.
  • Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues.
  • Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues.
  • substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct.
  • the mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure.
  • substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Table 1 which shows conservative substitutions.
  • Substitutional or deletional mutagenesis can be employed to insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).
  • Deletions of cysteine or other labile residues also may be desirable.
  • Deletions or substitutions of potential proteolysis sites, e.g. Arg is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.
  • Certain post-translational derivatives are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton (1983) Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86), acetylation of the N-terminal amine and, in some instances, amidation of the C-terminal carboxyl.
  • antibodies that selectively bind antigen specific polypeptides or VLRs or that selectively bind fragments or variants of antigen specific proteins or VLRs can be used to, for example, to localize VLRs or VLR producing cells. Such antibodies can be indirectly or directly detectably labeled as discussed in more detail below.
  • Such antibodies include, by way of example, antibodies that selectively bind the stalk region or a portion thereof, The antibodies can be monoclonal or polyclonal. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975) or Harlow and Lane (1988) Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York.
  • the immunizing antigen can be an antigen specific polypeptide or any fragment (including for example, the stalk region) or variant thereof.
  • the monoclonal antibodies secreted by the clones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, protein G, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • immunoglobulin purification procedures such as, for example, protein A-Sepharose, protein G, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • a variety of immunoassay formats may be used to select antibodies that selectively bind antigen specific polypeptides or fragments or variants thereof.
  • solid-phase ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with target. See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988), for a description of immunoassay formats and conditions that could be used to determine selective binding.
  • chimeric antibodies single chain antibodies, and hybrid antibodies (e.g., with dual or multiple antigen or epitope specificities), antibody conjugates and antibody fragments (such as F(ab′)2, Fab′, Fab and the like, including hybrid fragments) that selectively bind antigen specific polypeptides.
  • the VLRs and antibodies to VLRs may be directly or indirectly linked to a detectable tag or label.
  • a detectable tag or a label is any tag that can be visualized with imaging or detection methods, in vivo or in vitro.
  • the detectable tag can be a radio-opaque substance, radiolabel, a chemoluminescent label, a fluorescent label, or a magnetic label.
  • the detectable tag can be selected from the group consisting of gamma-emitters, beta-emitters, and alpha-emitters, gamma-emitters, positron-emitters, X-ray-emitters and fluorescence-emitters.
  • Suitable fluorescent compounds include fluorescein sodium, fluorescein isothiocyanate, phycoerythrin, and Texas Red sulfonyl chloride, Allophycocyanin (APC), Cy5-PE, CY7-APC, and Cascade yellow.
  • the detectable tag can be visualized using histochemical techniques, ELISA-like assays, confocal microscopy, fluorescent detection, cell sorting methods, nuclear magnetic resonance, radioimmunoscintigraphy, X-radiography, positron emission tomography, computerized axial tomography, magnetic resonance imaging, and ultrasonography.
  • the label or tag may be directly bound to the VLR or antibody or, alternatively, the label or tag may be indirectly linked using a molecule or other agent that is directly linked to the label.
  • the VLR or antibody may be biotinylated and a subsequent detectable label like a fluorescently labeled strepavidin could be added to bind the biotin.
  • Biotin is detected by any one of several techniques known in the art. For example, the biotin is detectable by binding with a fluorescence-labeled avidin and the avidin is labeled with a phycoerythrin or a catenated fluorescent label to increase the signal associate with each binding event.
  • the antigen specific polypeptides or VLRs, or fragments or variants thereof, or antibodies to the antigen specific polypeptides or VLRs are bound to a solid support or a mobile solid support such as a slide, a culture dish, a multiwell plate, column, chip, array or beads.
  • An array includes one or more multiwell arraying means such as microplates or slides.
  • a mobile solid support refers to a set of distinguishably labeled microspheres or beads.
  • the microspheres are polystyrene-divinylbenzene beads. Sets of microspheres marked with specific fluorescent dyes and having specific fluorescent profiles can be obtained commercially, for example, from Luminex Corporation (Austin, Tex.).
  • the plurality can be a homogeneous or heterogeneous for a selected polypeptide, nucleic acid, or antibody.
  • the LRRs of the polypeptides are highly variable across polypeptides.
  • the plurality can include polypeptides with different binding specificities, based on the variability of the internal LRRs.
  • kits that include a container with polypeptides (soluble or membrane bound form), nucleic acids, or antibodies or a stable or mobile solid support with polypeptides, nucleic acids, or antibodies attached.
  • polypeptides and nucleic acids can be used in a variety of techniques.
  • the polypeptides can be used to detect a selected agent, to block the activity of a selected agent, to purify an agent, as an imaging tool, and as a therapeutic agent.
  • compositions comprising the polypeptides or nucleic acids and a pharmaceutically acceptable carrier.
  • the compositions can also be administered in vivo.
  • the compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like.
  • parenterally e.g., intravenously
  • intramuscular injection by intraperitoneal injection
  • transdermally extracorporeally, topically or the like.
  • the exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
  • Parenteral administration of the composition is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Pat. No. 3,610,795, which is incorporated by reference herein.
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the polypeptide, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (21st ed.) ed. David B. Troy, publ. Lippicott Williams & Wilkins 2005.
  • an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
  • compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • variable lymphocyte receptors and variable lymphocyte receptor fragments and variants can also be administered to patients or subjects as a nucleic acid preparation (e.g., DNA or RNA) that encodes the variable lymphocyte receptor or variable lymphocyte receptor fragment or variant, such that the patient's or subject's own cells take up the nucleic acid and produce and secrete the encoded variable lymphocyte receptor or variable lymphocyte receptor fragment.
  • a nucleic acid preparation e.g., DNA or RNA
  • compositions and methods which can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems.
  • the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes.
  • Transfer vectors can be any nucleotide construction used to deliver nucleic acids into cells (e.g., a plasmid), or as part of a general strategy to deliver genes, e.g., as part of recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)).
  • plasmid or viral vectors are agents that transport the disclosed nucleic acids, such as VLR into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered.
  • Viral vectors are, for example, Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors.
  • Retroviruses include Murine Maloney Leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector.
  • the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
  • This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions.
  • steps in methods of making and using the disclosed compositions are discussed throughout the application, it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
  • subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian.
  • a mammal e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent
  • fish e.g., a fish, a bird or a reptile or an amphibian.
  • a bird or a reptile or an amphibian e.g., a fish, a bird or a reptile or an amphibian.
  • patient or subject may be used interchangeably and can refer to a subject with a disease or disorder.
  • patient or subject includes human and veterinary subjects.
  • VLR includes mixtures of two or more VLRs, and the like.
  • compositions e.g., a polypeptide, cell or nucleic acid
  • compositions e.g., a polypeptide, cell or nucleic acid
  • the polypeptides, or fragments thereof can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding the polypeptide (e.g., in a cell or in a cell-free translation system), or by chemically synthesizing the polypeptide.
  • Ranges may be expressed herein as from about one particular value, and/or to about another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent about, it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
  • Optional or optionally means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
  • polypeptide As used herein, polypeptide, protein, and peptide are used interchangeably to refer to amino acid sequences.
  • VLRs that Specifically Bind Bacillus anthracis
  • VLR-positive lymphocytes from Bacillus anthracis exosporium immunized lamprey were harvested and their RNA isolated. Primers were used to amplify VLR cDNAs which were cloned into an expression vector and transformed into bacteria. Selected colonies were screened by PCR with VLR specific primers. The heterogeneous size of the PCR products indicated the diversity of the VLR cDNA library. Plasmids were purified from individual colonies and transfected into HEK-293 cells, which were tested for VLR expression by Western blotting of detergent-soluble cell lysates with anti-VLR mAb under reducing conditions. The first six VLRs expressed were composed of monomeric VLR units of different sizes ( FIGS.
  • the secretion of recombinant VLRs into the culture medium of transfected HEK-293 allowed screening of VLR clones for antigen binding by ELISA.
  • the C-terminal domain of BclA is the dominant epitope recognized by monoclonal antibodies derived from B. anthracis exosporium immunized mice. BclA is also recognized by polyclonal VLRs in the plasma of immunized lamprey.
  • ELISA plate wells were coated with the following antigens: purified recombinant BclA-CTD, wild-type Bacillus anthracis spores, BclA-deficient Bacillus anthracis spores, and spores from Bacillus cereus whose BclA-CTD differs by 15 (out of 134) amino acids from the BclA-CTD of Bacillus anthracis .
  • the supernatant of VLR4 transfected HEK-293 cells reacted specifically with recombinant BclA-CTD and wild-type B. anthracis spores ( FIG. 4 ).
  • VLR4 does not recognize BclA-deficient B. anthracis spores or the B. cereus BclA protein that has extensive homology to B. anthracis BclA ( FIG. 4 ).
  • VLR4 and VLR5 differ by only twenty amino acids, even though the former recognizes BclA-CTD and the latter does not ( FIG. 5A ). Amino acid differences are noted at positions predicted to be located on the inner surface of the VLR solenoid structure and to have been selected for during evolution (Alder, et al., Science 310:1970, 2005).
  • the VLR-4 transfected HEK-293 cells express both membrane-bound VLR and secreted VLR multimers ( FIG. 1A ).
  • Lampreys were immunized with 1 ⁇ 10 7 type O human erythrocytes once a week for four weeks. One week following the last immunization, lamprey plasma was collected. Two CHO cell lines were also employed, one transfected with ⁇ 1,2-fucosyltransferase to produce the H antigen on the surface of CHO cells and the other transfected with the vector alone (Prieto et al., J Biol. Chem. 1997 Jan. 24; 272(4):2089-97.) Cells were first incubated in 1:10 dilution of lamprey plasma or 1:50 of the monoclonal antibody 92 FR A2, which is specific for the H antigen.
  • VLR-B clones Isolation of antigen specific VLR-B clones.
  • a heterologous expression system was developed utilizing HEK-293T cells transfected with full-length VLR-B cDNAs, which spontaneously secrete recombinant oligomeric VLR-B antibodies into the tissue culture supernatant.
  • the secretion of VLR-B clones by HEK-293T cells provided the means to screen a large number of clones for antigen binding using a methodology similar to hybridoma screening.
  • the procedure enables antigen specific VLR-B clones to be isolated utilizing techniques accessible to biological laboratories and requires a time investment comparable to monoclonal antibody production ( FIGS.
  • VLR-B + lymphocytes from blood samples by FACS.
  • RNA was isolated from sorted VLR-B + cells and VLR-B cDNA clones were amplified by PCR with primers specific for constant portions of the VLR-B transcript.
  • the VLR-B cDNAs were cloned into a mammalian expression vector for transient transfection of HEK-293T cells. Tissue culture supernatants were then screened to identify clones that produced antigen-specific VLR-B antibodies by ELISA and flow cytometry.
  • B. anthracis exosporium was chosen as the immunogen because as described herein the C-terminal domain (CTD) of the BclA spore coat protein is the immunodominant epitope recognized by VLR-B antibodies made in the in vivo response.
  • CCD C-terminal domain
  • HEK-293T cells in 24-well plates were transiently transfected with purified plasmid derived from a single bacterial colony so that every well represented a single VLR-B cDNA clone. When purified plasmids containing VLR-B cDNAs from B.
  • anthracis exosporium-immunized lamprey were transfected in this manner and supernatants screened for BclA-CTD binding, 14 of the 212 clones (6.6%) secreted VLR-B antibodies that recognize BclA-CTD and not the GST control protein. Eight of the 14 antigen reactive clones recognized BclA-CTD at levels 10-fold above background ( FIG. 9A ). The specificity of these recombinant VLR-B antibodies was evaluated by testing for binding to B. anthracis and two closely related Bacillus species, B. cereus and B. thuringiensis . The VLR-B antibodies were found to react with B. anthracis spores, and not with B. cereus, B.
  • FIG. 9B shows BclA-deficient B. anthracis spores.
  • VLR-B antibodies that recognized the BclA-CTD recombinant protein (vBa49) did not recognize the B. anthracis spores.
  • VLR-B antibodies None of the seven recombinant VLR-B antibodies that recognized B. anthracis spores reacted with spores of closely related Bacillus species, even though, B. anthracis BclA-CTD differs from B. cereus BclA-CTD at only 14 of 134 amino acids positions, only nine of which are solvent exposed ( FIG. 10D ). Moreover, most of the BclA-CTD sequence disparities involve chemically similar amino acids. When the solvent-exposed amino acid differences were plotted onto the crystal structure coordinates of BclA-CTD, it was noted that the amino acid differences were dispersed over the face of the molecule, rather than being clustered. Since it is unlikely that the VLR-B antibody makes contact with all of the disparate amino acids, we conclude that the VLR-B antibodies can discriminate between related proteins on the basis of a few subtle amino acid variations.
  • VLR-B antibody purification by antigen affinity chromatography The ease with which the VLR-B antibodies detected the BclA-CTD antigen by ELISA and immunofluorescence assays suggested the VLR-B antibody interaction with antigen would be of sufficient strength and stability to facilitate purification by affinity chromatography. Therefore, supernatant from the VLR-4 antibody-producing HEK-293T cell clone was incubated with sepharose beads covalently conjugated to BclA-CTD.
  • VLR-4-secreting cells Having determined the optimal VLR4 binding and elution conditions, stable clones of VLR-4-secreting cells were selected and expanded to obtain larger quantities of the VLR-4 antibody, which was purified by BclA-CTD affinity chromatography and eluted with 0.1M triethylamine pH 11.5.
  • the purified VLR4 antibody retained its ability to bind antigen despite the harsh elution conditions and was stored for >6 months at 4° C. in pH 7.2 MOPS-buffered saline without loss of antigen reactivity.
  • VLR4 band was shown to have a molecular weight of ⁇ 400 kDa.
  • the molecular weight of the monomer was estimated to be 40 kDa, hence suggesting that the oligomer is composed of 10 subunits.
  • the lower molecular weight VLR4 oligomer was estimated to contain eight VLR subunits.
  • a partially reduced band of ⁇ 80 kDa was observed on western blots of supernatants exposed to relatively low concentrations of reducing agents, which suggests the oligomeric VLR-B antibodies may be composed of dimeric subunits ( FIG. 10D ). From these findings, a model was generated in which the quaternary structure of lamprey VLR-B antibody is composed of a disulfide-linked pentamer or tetramer of dimers, much like IgM ( FIG. 1B ).
  • VLR-B cell surface molecules are tethered to the lymphocyte surface by GPI-linkage.
  • the plasmacytoid cells that secrete VLR-B antibodies also express cell surface VLR-B. If the GPI-linked VLR-B on the surface of the cell were liberated by a phospholipase, the cysteines used for oligomer formation should have to be located N-terminal to the GPI cleavage site, because amino acids C-terminal to the GPI cleavage site would be removed by GPI addition in the ER.
  • VLR4 GPI-stop a construct encoding the VLR4 antibody from the start codon to the GPI cleavage site (VLR4 GPI-stop ) was expressed in HEK-293T cells.
  • the resultant wild-type VLR4 (VLR4 WT ) and VLR4 GPI-stop molecules were separated by non-reducing SDS-PAGE and their molecular weights were determined by western blotting with anti-VLR-B mAb (4C4).
  • This analysis confirmed the VLR4 WT molecular weight of >225 kDa, while the VLR4 GPI-stop molecule migrated as a ⁇ 40 kDa monomer ( FIG. 11A ). This observation suggested that the cysteines that are used for VLR-B oligomerization are located C-terminal of the GPI cleavage site.
  • the purified VLR4 antibody was separated by reducing SDS-PAGE and visualized by Gelcode Blue staining. This allowed the VLR4 antibody to be excised from the acrylamide gel before acetylated by iodoacetamide to prevent disulfide bond re-formation and digestion with trypsin. The trypsinized peptides were then separated by reverse phase chromatography for sequencing by MS/MS.
  • VLR4 GPI-stop as a monomer allowed investigation of the contribution to antigen binding by the individual VLR4 antibody units.
  • BclA-CTD coated wells were incubated with supernatants containing the oligomeric VLR4 WT or monomeric VLR4 GPI-stop antibodies.
  • the oligomeric VLR4 antibody induced a strong binding signal, indicative of a tight interaction with BclA-CTD, while the monomeric VLR4 antibody form interacted with BclA-CTD to yield a barely detectable signal above the background ( FIG. 11D ).
  • VLR-B antigen binding site The concave surface of VLR-B is composed of parallel ⁇ -strands, one each from LRR-NT, LRR1, LRR-V(s), LRRVe, and LRR-CP.
  • the parallel ⁇ -strands of the concave surface have been proposed to be the antigen binding site because the highest sequence variability is observed there. Therefore, it was determined whether the amino acid residues responsible for antigen binding are located on the ⁇ -strands of the concave surface of VLR-B.
  • the availability of multiple BclA-CTD specific VLR-B clones provided the means for this test using site directed mutagenesis. Four of the recombinant VLR-B antibodies against BclA-CTD exhibited high sequence identity.
  • VLR4 Three of these bind to BclA-CTD with high avidity (VLR4, vBA41, vBA191), while the other (VLR5) binds this antigen weakly.
  • the weakly binding VLR5 antibody differs from the consensus sequence of the high avidity anti-BclA-CTD clones at six of the twenty possible hyper-variable amino acid positions on the concave surface (H34, Y55, T58, Q101, S103, W127) ( FIGS. 12A and 12B ).
  • VLR5 H34N VLR5 Y55R
  • VLR5 T58I VLR5 Q101H VLR5 S103A
  • VLR5 W127Y VLR5 W127Y
  • This assay was conducted by flowing transfectant supernatants containing the various VLR-B antibodies over BclA-CTD covalently coupled to a Biacore chip.
  • the other mutant VLR5 antibodies displayed an equivalent or slightly weaker binding avidity than VLR5 wt antibody.
  • VLR-B anti-H antigen monoclonal VLR-B
  • vRBC-36 antigen binding site
  • the vRBC-36 model was constructed by homology-based modeling to hagfish VLR-B (PDB ID: 206R) crystal structure data using SWISS-MODEL (http://swissmodel.expasy.org/). Hypervariable amino acid positions are highlighted purple. The red arrow denotes a depression on the concave surface that is the likely contact surface of the fucose sugar that distinguishes the H antigen from other carbohydrate moieties.
  • Table 2 lists the amino acids encoded by the hypervaiable residues of each LRR molecule.
  • LRR Residues LRRNT SRDT (SEQ ID NO: 33) LRR1 DHYI (SEQ ID NO: 34) LRRV SGYE (SEQ ID NO: 35) LRRV TGDV (SEQ ID NO: 36) LRRV CCFE (SEQ ID NO: 37) LRRVe QDAH (SEQ ID NO: 38) LRR-CP GFYH (SEQ ID NO: 39)
  • Monoclonal anti-VLR antibodies and recombinant VLR antibody Two mouse monoclonal antibodies were produced by hyper-immunization of mice with a recombinant VLR-B invariant stalk region protein produced in E. coli and subsequent fusion of regional lymph node cells with the non-productive Ag8.653 myeloma variant.
  • the 4C4 antibody was also reactive with VLR-B protein by Western blotting.
  • a recombinant monoclonal VLR-B antibody (mVLR-RBC36) with human H antigen specificity was obtained by isolating RNA from the leukocytes of lamprey immunized with blood group O erythrocytes for production of cDNA with Superscript III (Invitrogen, Carlsbad, Calif.,). Primary and nested PCR was then carried out with primers specific for the VLR-B locus followed by cloning of PCR amplicons into the vector pIRESpuro2 (Clonetech, Mountain View, Calif.) and bacterial transformation.
  • BSA immunizations were injections of 10 ⁇ g of BSA in 50 ⁇ l of one of the following vehicles: sterile 0.66% PBS, 200 ⁇ g Al(OH) 3 absorbed with protein for four hours before injection, or emulsions with Ribi and Titermax Gold (Sigma, St. Louis, Mo.) adjuvants prepared according to manufacturer's protocol.
  • BSA coated beads BSA was conjugated to 1 micron carboxylate polystyrene beads with the carbodiimide kit according to manufactures protocol (Polysciences, Warrington, Pa.) with lipopolysaccharide, lipoteichoic acid, and peptidoglycan (Invivogen, San Diego, Calif.) being added before injection.
  • Erythrocytes were from B6 mice or human blood group 0 donors and were washed three times prior to injection.
  • washed erythrocytes (5 ⁇ 10 6 ) mixed with lamprey plasma at varying dilutions were allowed to settle in conical bottom microwell plates for 1 hour before visual assessment of agglutination after tilting the plate at 80° C.
  • VLR reactivity with H antigen was determined by incubating CHO cells that were stably transfected with constructs for ⁇ 1,2-fucosyltransferase or vector alone with test plasma samples. The CHO cells were then stained by incubation with 4C4 VLR mAb and goat anti mouse Ig (H+L)-RPE (Southern Biotech, Birmingham, Ala.) for 10 minutes each before analysis of immunofluorescence using a CyanTM flow cytometer (Cytomation, Fort Collins, Colo.).
  • test samples were mixed with the 4C4 anti-VLR mAb conjugated to sepharose or CHO cells (3 ⁇ 10 6 ) fixed by paraformaldehyde for one hour at 4° C. Beads or cells were spun down and the supernatant transferred to a new test tube before repeating the adsorption process prior to the analysis of antigen reactivity by agglutination or western blot assays.
  • ELISPOT analysis of VLR secreting cells Microwells in 96 well plates (Millipore, Billerica, Mass.) were coated overnight at 4° C. with 100 ⁇ l of 50 ⁇ g/ml of recombinant BclA C-terminal domain protein (ref) then blocked with 1% BSA in PBS for 2 hours at 37° C. before adding test cell suspensions in IDMEM (Mediatech, Herndon, Va.) supplemented with 10% FBS, L-glutamine, penicillin, streptomycin, insulin, and transferrin for 18 hours at 25° C. in 5% CO2. The cells were then washed away with PBS before adding 1 ⁇ g/ml VLR antibody in 1% BSA for one hour at 37° C.
  • IDMEM Mediatech, Herndon, Va.
  • Plasma samples (1 ⁇ l) were electrophoresed on a 10% SDS page gel with or without 2-mercaptoethanol before transfer onto a nitrocellulose membrane which was blocked with 3% milk followed by incubation with the 4C4 anti-VLR mAb for one hour. The membranes were then washed 5 times with PBS-0.5% tween before adding goat anti-mouse HRP (Southern Biotech, Birmingham, Ala.) and a final wash one hour later. A SuperSignal chemiluminescent kit (Pierce, Rockford, Ill.) was used to detect VLR-antibody conjugates.
  • VLR-B stalk region specific monoclonal antibodies 6C3 IgM isotype
  • 4C4 IgG2b isotype
  • BSA bovine serum albumin
  • BSA was conjugated to the surface of polystyrene beads, 1 ⁇ 10 8 of which were injected either alone or together with 1 ⁇ g each of lipopolysaccharide, lipoteichoic acid, or peptidoglycan.
  • An immunization protocol was used that elicited a strong VLR humoral response to anthrax exosporium proteins: primary immunization followed by booster immunization two weeks later and collection of plasma samples for testing at four weeks. None of these methods of BSA immunization resulted in the production of VLR-B antibodies that could be detected by ELISA (n+30, 4-5 per immunization group).
  • the immunized lamprey did not respond with the lymphoblastoid transformation of circulating lymphocytes that was observed after hyperimmunizating lamprey with anthrax exosporium.
  • BSA as a model protein immunogen thus failed to induce a VLR-B antibody response, even when given with adjuvants, in aggregated form or coated onto the surface of a solid matrix.
  • Lamprey produce agglutinating VLR-B antibodies in response to mammalian erythrocytes.
  • lamprey were immunized intraperitoneally with either mouse or human erythrocytes.
  • erythrocyte hemagglutinin responses were elicited that were antigen dose dependent and specific for the donor erythrocyte immunogen ( FIG. 14A , Table 3).
  • VLR-B antibodies Carbohydrate H antigen specificity of VLR-B antibodies to blood group O erythrocytes.
  • Previously studies suggested the hemagglutinins made by lamprey that were immunized with human blood group O erythrocytes were specific for the H trisaccharide cell surface antigen that defines this blood type.
  • To test for H antigen specificity of the VLR-B antibodies CHO cells were employed that were stably transfected with the ⁇ 1,2-fucosyltransferase enzyme that generates the H trisaccharide. Animals immunized with blood group O erythrocytes were shown to produce VLR-B antibodies that recognized CHO cells expressing the H trisaccharide antigen ( FIG.
  • the H antigen specific VLR-B antibodies are disulfide-linked multimers.
  • a recombinant VLR-B antibody with H antigen specificity was generated.
  • a VLR-B cDNA library was prepared from blood leukocytes of immunized animals and individual VLR-B clones were transfected into HEK 293T cells. When the transfected cells were screened for clones producing antigen specific VLR-Bs, one clone was identified that produced a VLR antibody that reacted with H antigen + CHO cells and not with H antigen ⁇ CHO cells ( FIG.
  • VLR antibody is a large multimeric protein of >250 kDa that is composed by multiple individual VLR-B subunits of 35 kDa linked together by disulfide bonds ( FIG. 15B ).
  • VLR-B + lymphocytes in the lamprey Tissue distribution of VLR-B + lymphocytes in the lamprey.
  • tissue distribution of the VLR-B + cells was determined by immunohistochemical staining using the two antibodies that are specific for the invariant stalk region of VLR-B.
  • Discrete localization of VLR-B + cells was observed using monoclonal 6C3 anti-VLR-B antibody in the kidney and typhlosole, two hematopoietic organs, as well as in the gills.
  • VLR-B + lymphocytes were not detected in the epithelium of the intestine, which in the larval filter-feeding stage is a straight tube beginning near the last gill slit and terminating at the cloaca. Over most of its length, the intestine is folded like an elongated horseshoe over the typhlosole, which is comprised primarily by hematopoietic lineage cells lining the blood filled sinuses. The VLR-B + lymphocytes were found to be dispersed throughout the typhlosole, wherein they exhibited greater morphological diversity and variability in staining intensity than the VLR-B + lymphocytes in other tissues ( FIG. 17A ).
  • VLR-B + cells were intermixed with other hematopoietic cells in the kidneys, which extend over most of the body length and flank the lateral and dorsal surfaces of the lamprey intestine.
  • the VLR-B + lymphocytes were most abundant in the most ventral aspects of the kidneys.
  • the gills displayed the greatest accumulation of VLR + lymphocytes in terms of the density of positively staining cells.
  • the VLR-B + cells were especially abundant within the vessels located at the gill bases.
  • the immunofluorescence staining pattern of these intravascular lymphocytes was suggestive of extensive intracellular VLR-B accumulation ( FIG. 17B ).
  • VLR-B staining in the tissue sections was also consistently evident along the inner surface of blood vessels and sinuses, reflecting the abundant pool of circulating VLR-B antibodies, and was not evident in the intercellular spaces outside of the vasculature.
  • VLR-B bearing cells were examined further by the staining of viable cells in fluid suspensions freshly prepared from the blood, kidney, and typhlosole.
  • light scatter characteristics were used to examine the lymphocyte-like cells by immunofluorescence flow cytometry, 15-35% of the blood cells in the ‘lymphocyte gate’ were VLR-B + , ⁇ 50% were VLR-B + in the kidney cell suspensions, and 15-30% were VLR-B + in the typhlosole ( FIG. 17C ).
  • the VLR-B + cells from blood and kidney consistently expressed relatively high VLR-B levels, whereas VLR-B + cells from the typhlosole exhibited greater variability and lower levels of cell surface VLR-B.
  • VLR-B + lymphocyte morphology and gene expression profile The VLR-B + and VLR-B ⁇ cells within the ‘lymphocyte gate’ were isolated by fluorescence activated cell sorting and examined by transmission electron microscopy.
  • the VLR-B + cells in these studies resembled small lymphocytes in jawed vertebrates in that they have a relatively large nucleus, which contains a compacted chromatin concentrated in a peripheral pattern, surrounded by a narrow rim of cytoplasm that contains relatively few distinguishable organelles, such as mitochondria.
  • VLR-B + and VLR-B ⁇ populations of cells were also used to compare their gene expression profiles.
  • the purified VLR-B + cells were found to express VLR-B transcripts, and not VLR-A transcripts.
  • the VLR-B ⁇ cells in the ‘lymphocyte gate’ expressed VLR-A and not VLR-B transcripts ( FIG. 18 ).
  • CD45 and GATA were found to be expressed at higher levels in the VLR-B ⁇ population, whereas the TCR-like, CD4-like, and TNFR14 genes were expressed at higher levels in the VLR-B + lymphocytes.
  • VLR-B + cells and VLR-A + cells represent distinct lymphocyte populations, confirm that lymphoid lineage cells preferentially express the TCR-like and CD-4-like genes and suggest that the TNFR14 gene may also be preferentially expressed by lymphocytes.
  • VLR-B + lymphocyte responses to in vivo antigenic stimulation Immunization of lamprey with a cocktail of antigens and phytomitogens was shown to induce a global lymphoblastoid response. This type of lymphoblastoid response was reproduced by injecting the lamprey larvae with a large dose (>2 ⁇ g) of anthrax exosporium intraperitoneally ( FIG. 19A ). When blood cells from these animals were stained with the anti-VLR-B antibodies, most of large lymphoblastoid cells were found to be VLR-B + , although the level of cell surface VLR-B was noticeably diminished ( FIG. 19B ). This result suggested that, given in sufficient dosage, the anthrax exosporium can serve as a mitogen for lamprey lymphocytes.
  • the frequency of antigen binding cells was determined before and after immunization with anthrax exosporium.
  • fluorescence labeled anthrax spores were used to detect antigen-binding VLR-B + cells.
  • a small subpopulation of the VLR-B bearing lymphocytes ( ⁇ 1%) in naive animals were found to bind B. anthracis or B. cereus spores
  • a four-fold increase of B. anthracis binding VLR-B + cells was observed following immunization with B. anthracis exosporium ( FIGS. 20A and 20B ) and the frequency of B. cereus - binding cells was unchanged.
  • VLR-B + and VLR-B ⁇ subpopulations of cells from immunized lamprey were separated according to their light scatter characteristics and evaluated by ELISPOT assays for their ability to secrete VLR-B antibodies to the BclA-CTD antigen.
  • Cells isolated from the blood, kidney and typhlosole were placed in culture for 18 hours before their evaluation for VLR-B antibody secretion.
  • the cells which secreted BclA-CTD specific antibodies were found only among the VLR-B + cells with the highest forward and side light scatter characteristics, a finding that indicated their relatively large cell size ( FIG. 21A ).
  • VLR-B producing cells When large VLR-B producing cells were isolated for evaluation by transmission electron microscopy, they were found to have plasmacytoid morphology featuring extensive cytoplasm with multiple organelles and an expanded network of rough endoplasmic reticulum ( FIG. 21B ). In a typical response to anthrax exosporium, four weeks after the first immunization, the VLR-B antibody secreting cells were most abundant in either the blood or the kidney and were less abundant in the typhlosole. These observations indicate that immunization with an effective immunogen induces antigen-specific lymphocytes to undergo lymphoblastoid transformation, proliferation, and differentiation into plasmacytoid cells that secrete antigen-specific VLR-B antibodies.
  • influenza virus lamprey were immunized intraperitoneally with 25 ⁇ g of formalin fixed influenza virus diluted in 50 ⁇ l of 2/3 PBS. Two weeks later, the animals received a secondary injection with the same amount of immunogen. Plasma samples were collected two weeks after the secondary immunization and tested for reactivity.
  • ELISA assays were carried out by coating plates overnight at 4° C. with 10 ⁇ g/mL formalin killed influenza virus diluted in PBS. Purified adenovirus was used to coat plates as a control. Plates were then washed and blocked with 3% BSA. Detection of VLR-B antibodies was carried out using monoclonal anti-VLR-B (4C4) antibody followed by a secondary antibody with enzyme conjugate.
  • Viral neutralization activity was tested by hemagglutinin inhibition assays and plaque neutralization assays.
  • lamprey immunized with influenza virus produce VLRs specific for immunogen.
  • Table 4 shows influenza virus hemagglutinin inhibition and neutralization titers for plasma from immunized and na ⁇ ve lamprey.
  • VLPs virus-like particles
  • Env HIV envelope protein
  • VLR monoclonal anti-VLR-B (4C4) antibody followed by a secondary antibody with enzyme conjugate.
  • lamprey immunized with HIV VLPs produce VLR-B antibodies specific for HIV envelope protein gp120 subunit.

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Publication number Priority date Publication date Assignee Title
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020025554A1 (en) * 1999-12-08 2002-02-28 Millennium Pharmaceuticals, Inc. A Massachusetts Corporation Novel leucine rich repeat-containing molecules and uses therefor

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR1601438A (ja) 1968-10-17 1970-08-24
US6521448B1 (en) * 1997-08-19 2003-02-18 Diacrin, Inc. Porcine MHC class I genes and uses thereof
US6960651B2 (en) 1999-06-29 2005-11-01 Millennium Pharmaceuticals, Inc. TANGO 332 polypeptides
US6903201B2 (en) * 2001-01-05 2005-06-07 Curagen Corporation Proteins and nucleic acids encoding same
US8039588B2 (en) * 2004-05-21 2011-10-18 The Uab Research Foundation Variable lymphocyte receptors
AU2016203994B2 (en) 2006-08-02 2018-06-07 The Uab Research Foundation Methods and compositions related to soluble monoclonal variable lymphocyte receptors of defined antigen specificity
EP2049564B1 (en) 2006-08-02 2017-05-17 The UAB Research Foundation Methods and compositions related to soluble monoclonal variable lymphocyte receptors of defined antigen specificity

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020025554A1 (en) * 1999-12-08 2002-02-28 Millennium Pharmaceuticals, Inc. A Massachusetts Corporation Novel leucine rich repeat-containing molecules and uses therefor

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Adler et al. Science, vol. 310, pp. 1970-1973, December 2005 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10239936B2 (en) 2006-08-02 2019-03-26 The Uab Research Foundation Methods and compositions related to soluble monoclonal variable lymphocyte receptors of defined antigen specificity
US10870692B2 (en) 2006-08-02 2020-12-22 The Uab Research Foundation Methods and compositions related to soluble monoclonal variable lymphocyte receptors of defined antigen specificity
US11945857B2 (en) 2006-08-02 2024-04-02 The Uab Research Foundation Methods and compositions related to soluble monoclonal variable lymphocyte receptors of defined antigen specificity
WO2015120363A1 (en) * 2014-02-10 2015-08-13 Emory University Expression of chimeric polypeptide with variable lymphocyte receptors on immune cells and uses for treating cancer
US10323077B2 (en) 2014-02-10 2019-06-18 Emory University Expression of chimeric polypeptide with variable lymphocyte receptors on immune cells and uses for treating cancer
US11136369B2 (en) 2014-02-10 2021-10-05 Emory University Expression of chimeric polypeptide with variable lymphocyte receptors on immune cells and uses for treating cancer
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US11384134B2 (en) 2014-05-02 2022-07-12 Emory University Recombinant vector and expression system comprising a nucleic acid encoding a variable lymphocyte receptor (VLR)
WO2023010060A2 (en) 2021-07-27 2023-02-02 Novab, Inc. Engineered vlrb antibodies with immune effector functions

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