WO1994006934A1 - Procede de derivation des epitopes - Google Patents

Procede de derivation des epitopes Download PDF

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Publication number
WO1994006934A1
WO1994006934A1 PCT/US1993/008210 US9308210W WO9406934A1 WO 1994006934 A1 WO1994006934 A1 WO 1994006934A1 US 9308210 W US9308210 W US 9308210W WO 9406934 A1 WO9406934 A1 WO 9406934A1
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Prior art keywords
nucleic acid
antigen binding
immunogen
binding protein
acid species
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Jack D. Keene
Daniel J. Kenan
Donald E. Tsai
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Duke University
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Duke University
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Priority to AU48426/93A priority Critical patent/AU4842693A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • This invention relates to methods of making epitopes and nucleic acids embodying the epitopes so made.
  • a first aspect of the present invention is a method of generating a nucleic acid molecule which is immunologically cross-reactive with an immunogen, which immunogen is not a nucleic acid (e.g. , a peptide) .
  • the method comprises combining an antigen binding protein which binds the immunogen (e.g. , an antibody, a T cell receptor) with a degenerate pool of nucleic acid species, and then recovering a nucleic acid species bound by said binding protein from the degenerate pool.
  • a second aspect of the present invention is an isolated nucleic acid which inhibits complex formation between an antigen binding protein and an immunogen, which immunogen is not a nucleic acid.
  • the nucleic acid inhibits complex formation between a self peptide autoantigen and an antigen binding protein.
  • Figure 1 is a schematic diagram of a process of the instant invention.
  • Figure 2 shows D10 DNA construct (upper) (SEQ ID NO:l) and the degenerate RNA transcript (lower) (SEQ ID NO:2) used to select the D10 RNA epitope (SEQ ID NO:3).
  • Degenerate nucleotide positions in the predicted RNA secondary structure are represented by "N.”
  • BamHI restriction sites, the T7 promoter, complementary regions representing Ul stem II and the D10 loop sequence are indicated.
  • Figure 3 shows the glO fusion peptide used as an epitope tag.
  • Panel A Western blot of E. coli extracts containing various Ul-A protein constructs. All extracts were produced from induced E. coli cells containing recombinant pET-8c T7 expression vectors in accordance with known techniques. See F. Studier et al., Meth. Enzym. 185, 60-89 (1991) . Ul-A cDNA was cloned into pET-8c either attached or not attached to the glO peptide.
  • Panel B Immunoprecipitation and competition analysis of [ 35 S]-methionine-labeled Ul-A and glO-Ul-A proteins expressed by in vitro transcription, and subsequent translation in rabbit reticulocyte lysates.
  • Lanes: 1 and 2 show the amount of Ul-A and glO-Ul-A translation product, respectively, added to each immunoprecipitation reaction; 3: blank; 4 and 5: Ul-A and glO-Ul-A precipitated with serum EW (reactive with Ul-A) ; 6 and 7: Ul-A and glO-Ul-A, respectively, precipitated with anti-glO serum.
  • lanes 8-12 glO-Ul-A was precipitated with anti-glO serum in the presence of varying amounts of competitors as follows: lanes 8-11 contained the glO peptide at concentrations of 27.8, 83.3, 250 and 750 nM, respectively; lane 12 contained a control peptide (sequence GKSRGFAFVEFK-amide) (SEQ ID NO:4) at a concentration of 25 n ⁇ . The faint lower band in all lanes represents either a degradation product or a premature translation termination product and is seen consistently in Ul-A translations.
  • Figure 4 shows the D10 RNA epitope binds specifically to gio antibodies.
  • Various antisera were used to precipitate either [ 32 P] labeled in vitro transcribed D10 RNA or control transcripts. Bound RNA was analyzed on a denaturing 6% polyacrylamide gel by autoradiography. Lanes 1-4 are precipitations of D10 RNA rfith the following antisera: 1, anti-glO serum; 2, preimmune serum; 3, no antibody; 4, anti-glO serum.
  • transcripts were precipitated with the anti-glO serum as follows: 5, a control RNA identical to the DIO epitope but with loop sequence 5 ' -CACCAUAUAA-3 • (SEQ ID NO: 5) ; 6, an unrelated RNA; 7, an RNA containing the loop sequence 5'-CUGACCCCGU-3 • (SEQ ID NO:6) ; 8-11, supernatants from the immunoprecipitations shown in lanes 4-7, diluted to approximate radioactive equivalents.
  • Figure 5 shows competition analysis of gio peptide and DIO RNA epitopes for binding by the glO antibodies.
  • [ 32 P] labeled in vitro transcribed DIO RNA or [ 35 S] labeled in vitro translated glO fusion protein (glO-UlA) was immunoprecipitated by the anti-glO serum in the presence of various competitors.
  • DIO RNA and glO fusion protein bound in the immunoprecipitations were analyzed using denaturing polyacrylamide gels and autoradiography.
  • Panel A DIO RNA immunoprecipitations with: l, ( no competitor; 2, no gio antiserum added; 3, 125 g of glO peptide; 4, 125 mg of bovine serum albumin; 5, 125 mg of control peptide (sequence ETPEEREERRR) (SEQ ID NO:7) .
  • Panel B DIO immunoprecipitations after incubation with increasing amounts of glO peptide. Lanes: 1, no competitor; 2, 37 nM; 3, 74 nM; 4, 148 nM; 5, 222 nM; 6, 444 nM.
  • Panel C immunoprecipitations of a glO containing fusion protein (glO-Ul-A) using various competitors.
  • Panel D immunoprecipitation of glO-UlA fusion protein after incubation with increasing amounts of D10 RNA.
  • Figure 6 shows immunoprecipitation of RNAs tagged with the DIO RNA epitope.
  • RNA In vitro transcribed [ 32 P] radiolabeled RNA was immunoprecipitated with anti-glO serum or with an anti-Ul RNA serum (EW) . Bound RNA was analyzed using a denaturing 6% acrylamide gel and autoradiography: Panel A: The DIO DNA construct (Fig. 2) was cloned into the BamHI site of PGEM-3zf (+) to produce tagged vector RNA, and transcripts with different 3' termini were generated with SP6 RNA polymerase.
  • the anti-glO serum was used to precipitate the following transcripts (lanes) : 1, Ul RNA (negative control); 2, DIO RNA; 3-5, increasing lengths of DIO tagged vector RNA; 6-10, supernatants from lanes 1-5, respectively. Approximate nucleotide sizes are indicated by arrows.
  • Panel B Ul RNA was tagged with the DIO epitope by replacing loop III, sequence CAAAUGU (SEQ ID NO:9), with the sequence UGGUGGAGCA (SEQ ID NO:10) (construct Ul-3Dx) .
  • EW anti-Ul RNA serum
  • Amino acid sequences disclosed herein are presented in the amino to carboxy direction, from left to right. The amino and carboxy groups are not presented in the sequence.
  • Nucleotide sequences are presented herein by single strand only, in the 5' to 3 ' direction, from left to right.
  • epitope refers to a portion of a molecule which has a three-dimensional structure on an exposed surface to which an antibody can specifically bind, whether in the context of said molecule or as a portion thereof.
  • immunogen refers to a compound capable of eliciting an immune response, whether or not that compound is intentionally used to induce an immune response.
  • antigen binding protein refers the members of the immunoglobulin superfamily.
  • Members of the immunoglobulin superfamily include, but are not limited to, major histocompatibility complex molecules, cell adhesion molecules (including both neuronal cell adhesion molecules and cellular cell adhesion molecules) virus receptors such as picornavirus receptors (e.g., poliovirus receptors, rhinovirus receptors) , growth factor receptors (e.g. , interleukin receptors, lymphokine receptors) , T cell receptors (e.g., alpha-beta class and gamma-delta class T cell receptors) , and antibodies. Antibodies and T cell receptors are currently preferred.
  • antibodies refers to all types of immunoglobulin ⁇ , including IgG, IgM, IgA, IgD, and IgE.
  • the antibodies may be monoclonal or polyclonal and may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e . g. , M. Walker et al., Molec. Immunol 26, 403-11 (1989) .
  • Antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in Reading U.S. Patent No. 4,474,893, or Cabilly et al., U.S. Patent No. 4,816,567.
  • Antibodies may also be chemically constructed according to the method disclosed in Segel et al. , U.S. Patent No. 4,676,980.
  • the term antibodies further includes fragments which retain the specific binding characteristics of the antibody from which they are derived, with such fragments including, for example, Fab, F(ab')., and Fv fragments, and the corresponding fragments obtained from antibodies other than IgG.
  • fragments are produced by known techniques.
  • monoclonal Fab fragments may be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, e . g. , W. Huse, Science 246, 1275- 81 (1989) .
  • the present invention provides a method of generating a nucleic acid molecule which is immunologically cross-reactive with a non-nucleic acid immunogen by combining an antigen binding protein which binds said immunogen with a degenerate pool of nucleic acid species (i.e., under conditions which permit the binding of a nucleic acid species to the antigen binding protein) and then recovering a nucleic acid species bound by the antigen binding protein from the degenerate pool.
  • An embodiment of this method is schematically illustrated in Figure l, the steps of which are explained in detail below.
  • suitable anti-peptide antibodies e.g., anti-glO antibodies
  • polyclonal antibodies used to carry out the present invention may be produced by immunizing a suitable animal (e.g., rabbit, goat, etc.) with a non-nucleic acid immunogen antigen for which a nucleic acid epitope is desired, collecting immune serum from the animal, and removing the polyclonal antibodies from the immune serum, in accordance with known procedures.
  • Monoclonal antibodies used to carry out the present invention may be produced in a hybridoma cell line according to the technique of Kohler and Milstein, Nature 265, 495-97 (1975).
  • a solution containing the appropriate antigen may be injected into a mouse and, after a sufficient time, the mouse sacrificed and spleen cells obtained.
  • the spleen cells are then immortalized by fusing them with myeloma cells or with lymphoma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells.
  • the hybridoma cells are then grown in a suitable media and the supernatant screened for monoclonal antibodies having the desired specificity. Variations and refinements of these techniques can be employed to produce other types of antibodies, as noted above.
  • T cell receptors are structurally and functionally analogous to antibodies, and can be manipulated in much the same way as antibodies. See generally A. Williams and A. Barclay, Ann. Rev. Immunol . 6, 381-405 (1988); S. Brostoff and M. Howell, Clin . Immunol . & Immunopathol . 62, 1-7 (1992).
  • Non-nucleic acid immunogens other than peptides which may be employed include glycoproteins, fats, lipids, viruses (e.g., rhinovirus) , polysaccharides, carbohydrates, and allergens. Allergens include pollen, mold, spores, insects, epidermal particles, dust, etc. See, e . g. , Greer Laboratories, Inc., Allergenic Extracts Allergy Supplies & Services , 2-4 (April 1, 1992) (Greer Laboratories, Inc., P.O. Box 800, Lenoir, NC, USA 28645-0800; tel. 704-754- 5327) . Peptides are preferred, with the term "peptide" as used herein referring to a peptide as a discrete molecule or residing in a protein.
  • antibodies may be collected from a human or animal subject without prior specific immunization to produce a nucleic acid epitope to an antigen binding protein where the native epitope bound by that antigen binding protein is unknown.
  • antibodies may be collected from human or animal subjects afflicted with autoimmune disease to produce a nucleic acid epitope which immunologically cross-reacts with the self peptide targeted by autoantibodies in the disorder.
  • autoimmune diseases in human subjects include, but are not limited to, systemic lupus erythematosus, myasthenia gravis, and rheumatoid arthritis.
  • degenerate pools are known, and may be produced in accordance with known techniques. See, e. g. , Blackwell et al., Science 250, 1104-1110 (1990) ; S. Manualr and J. Keene, Proc. Natl . Acad. Sci . USA 85, 3299 (1988) ; Joyce et al. , Nucleic Acids Res . 17, 711-722 (1989) ; Oliphant et al., Methods Enzy ol . 155, 568 (1987).
  • the pool may be formed of DNA molecules or RNA molecules, with pools of RNA molecules currently preferred.
  • nucleotide bases which form the pool may optionally be modified by methylation, O-methylation, provision of base analogues with atypical hydrogen bonding patterns, etc.
  • degenerate pools of nucleic acids comprise a plurality of distinct nucleic acid species in an aqueous solution. Typically, from 16 to 10 10 distinct nucleic acid species are included in the pool, depending on the number of nucleotides being randomized. The precise number is not critical, though it is preferred that the number be sufficiently high to approach complete representation of all the possible members of the randomly represented set. Individual nucleic acid species within the pool will be 2,
  • nucleic acid species in length or more.
  • the nucleic acid species may be linear or may possess some form of secondary structure, such as a stem and loop structure.
  • Each nucleic acid species in the pool includes a degenerate segment of nucleotides, typically of 2, 3, or 4 up to about 25 or 100 nucleotides, in which each degenerate nucleotide position is randomly assigned both with respect to the other nucleotides in that segment of that species and with respect to nucleotides occupying the same position in other species in the degenerate pool.
  • each species in the degenerate pool may include non-random segments, such as primer segments or replication origins for amplification of the pool, though these segments may ultimately be removed from the final selected species as discussed below.
  • Combining the anti-peptide antigen binding protein with the degenerate pool may be facilitated by immobilizing the antigen binding protein on a solid support and contacting the degenerate pool (i.e., the aqueous solution carrying the degenerate pool) to the solid support, all in accordance with known techniques.
  • the step of combining the degenerate pool with the antigen binding protein is followed by the step of separating nucleic acid species bound to said solid support (e.g. , by washing away any unbound nucleic acid species, then eluting nucleic acid species bound to the solid support) ; then producing a pool of complementary nucleic acids from said nucleic acid species separated from said solid support (e.g.
  • a separating step as described above preferably includes a wash step and an elution step.
  • the wash step removes undesired nucleic acid species from the solid support
  • the elution step removes the desired nucleic acid species from the solid support to provide the subset degenerate pool.
  • the elution step may be carried out by any suitable means, such as phenol extraction.
  • the separating step may be carried out at the same wash stringency at each cycle (i.e., as either a high stringency or low stringency wash) , or the wash stringency may be changed between cycles (with stringency typically being adjusted from low stringency to high stringency as the cycles progress) .
  • wash stringency may be increased by increasing the concentration of NaCl or urea in the wash buffer or by increasing the temperature of the wash buffer.
  • buffers containing 150 mM NaCl at 4 * C are considered to provide lower wash stringency
  • buffers as above containing 0.3 M NaCl or greater, or 0.3 M urea or greater, or at temperatures greater than 20" C are considered to provide intermediate to higher wash stringency
  • buffers containing 0.5 M NaCl or greater, or 0.5 M urea or greater, or at temperatures greater than 37" C are considered to provide higher wash stringency.
  • Standard washing buffers also contain 0.05% nonidet P-40 and 50 mM Tris-HCl at pH 7.4, although the detergent, buffer, buffer salts, buffer concentration, and the pH are not critical and can be varied over a wide range. See, e . g. , E. Harlow and D. Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Laboratory 1988) : R. Bentley and J. Keene, Mol . Cell . Biol . 11, 1829 (1991).
  • the amplifying step may be carried out in vivo or in vitro by any suitable means. See generally D. Kwoh and T. Kwoh, Am. Biotechnol . Lab. 8, 14-25 (1990).
  • In vivo amplification may be carried out by standard recombinant DNA techniques, such as by ligating cDNA produced as described above into a plasmid, and then taking that plasmid or pool thereof with inserts and transforming a bacterial culture therewith. See, e . g. , J. Sambrook et al. , Molecular Cloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring Harbor Laboratory) ; J. Ma and M. Ptashne, Cell 51, 113-119 (1987); S. Manual and J. Keene, Proc. Natl . Acad. Sci . USA 85, 3299 (1988).
  • Suitable in vitro amplification techniques include, but are not limited to, polymerase chain reaction (see U.S. Patents Nos. 4,683,202 and 4,683,195 to K. Mullis et al.), ligase chain reaction (see R. Weiss, Science 254, 1292 (1991)), strand displacement amplification (see G. Walker et al., Proc. Natl . Acad. Sci . USA 89, 392-396 (1992); G. Walker et al., Nucleic Acids Res . 20, 1691-1696 (1992)), transcription-based amplification (see D. Kwoh et al., Proc. Natl . Acad Sci .
  • a desired nucleic acid species may be amplified and/or sequenced and synthesized in accordance with known techniques.
  • a complementary nucleic acid (e.g., a cDNA) to the nucleic acid species may be produced by reverse transcription and the desired nucleic acid species produced in greater quantities by recombinant techniques.
  • the immunological cross-reactivity of the recovered nucleic acid species with the non-nucleic acid immunogen it mimics may be confirmed by suitable immunoassay, such as blocking assays or competition experiments, carried out in accordance with known techniques.
  • suitable immunoassay such as blocking assays or competition experiments, carried out in accordance with known techniques.
  • the foregoing method provides an isolated nucleic acid which inhibits complex formation between an antigen binding protein and a non-nucleic acid immunogen.
  • Binding of the nucleic acid to such an antigen binding protein can be routinely determined in a standard competition assay in vitro , with nucleic acids of the invention having dissociation constants (K d s) of 10 '5 , 10 "7 or 10 "8 up to 10 "12 or 10 "u moles per liter.
  • K d s dissociation constants
  • the format of competition assay is not critical, though enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA) are particularly convenient.
  • Nucleic acids of the invention have association constants (K a s) which make them useful as inhibitors of the binding of non-nucleic acid immunogens to antigen binding proteins (such as antibodies) , with the K fl s for such nucleic acids ranging from 10 5 , 10 7 , 10 8 , or 10 9 up to 10 10 or 10 12 liters per mole, with these values being determinable in the same manner as given above with respect to K d s.
  • K a s association constants
  • the nucleic acid itself may mimic any of a broad variety of non-nucleic acid immunogens: for example, one embodiment of the foregoing is an isolated nucleic acid which inhibits complex formation between a self peptide autoantigen and an antigen binding protein, wherein said antigen binding protein is from a human or animal subject which expresses said self peptide.
  • antigen binding proteins may be obtained from human subjects afflicted with an autoimmune disease, as noted above.
  • isolated nucleic acids of the present invention may be of any length, typically of from 2, 3, 4, 5, or 6 nucleotides in length or more. Again there is no particular upper limit on the length of the isolated nucleic acid, with nucleic acids of 50, 100, or 200 or more nucleotides being suitable. As above, the isolated nucleic acid may be linear or may possess some form of higher order structure, such as a stem and loop structure.
  • the isolated nucleic acid may be modified from that initially retrieved from the degenerate pool, such as by removing primer segments or other portions thereof which are not critical for binding, or by making minor modifications to the structure of one or more of the individual nucleotides in the nucleic acid itself such as methylation, O- methylation, provision of nucleotide analogues with atypical patterns of hydrogen bonding, other modifications to prevent nucleophilic attack on the phosphodiester bond, and the like.
  • Isolated nucleic acids of the invention can be used in a variety of ways.
  • the isolated nucleic acid may be conjugated, either directly or indirectly and either covalently or non-covalently, to a molecule to be tagged thereby (i.e., a "tagged molecule").
  • the tagged molecule itself may be, for example, a protein or a heterologous nucleic acid.
  • the tagged molecule can then be detected with antigen binding proteins, particularly antibodies, known to bind that isolated nucleic acid.
  • Nucleic acids of the invention may be used in methods of detecting an antigen binding protein which binds a predetermined non-nucleic acid immunogen. Such methods comprise contacting a biological sample suspected of containing the antigen binding protein to a nucleic acid, which nucleic acid is capable of inhibiting complex formation between the antigen binding protein and said non- nucleic acid immunogen, under conditions which permit the formation of a reaction product; and then detecting the presence or absence of the reaction product.
  • Biological samples taken from human or animal subjects for use in this method are generally biological fluids such as serum, blood plasma, or ascites fluid.
  • the sample taken from the subject can be a tissue sample (e.g., biopsy tissue; scrapings; etc.) .
  • any suitable assay format can be used to carry out the detection of the reaction product, examples being radioimmunoassay, immunofluorescence methods, enzyme-linked immunoassays, and the like.
  • Those skilled in the art will be familiar with numerous specific immunoassay formats and variations thereof which may be useful for carrying out the method disclosed herein. See generally E. Maggio, Enzyme- Immunoassay , (1980) (CRC Press, Inc. , Boca Raton, FL) .
  • Nucleic acids of the present invention may be used to produce an immune response to a non-nucleic acid immunogen in a human or animal (e.g. , dog, cat, horse, goat, rabbit) subject.
  • the nucleic acid serves as a surrogate immunogen for the non-nucleic acid immunogen.
  • the method comprises administering a nucleic acid to the subject, which nucleic acid is capable of inhibiting complex formation between an antigen binding protein and the immunogen, with the nucleic acid being administered in an amount effective to induce an immune response in said animal to said immunogen.
  • Techniques for enhancing the immunogenicity of the nucleic acid which are known in the art may, if desired, be employed.
  • Subjects may be administered nucleic acids for this purpose to simply raise stocks of antibodies, or for therapeutic purposes to subjects in need of such treatment.
  • Administration to a subject may be carried out by any suitable means, such as by subcutaneous injection, intravenous injection, intraperitoneal injection, and nasal spray.
  • the amount of nucleic acid administered will depend upon factors such as route of administration, species, use of booster administrations, etc., but is generally between 50 micrograms to 5 milligrams per kilogram subject body weight, and more typically is between 50 micrograms and 200 micrograms per kilogram subject body weight.
  • Nucleic acids of the invention may be employed in methods of blocking complex formation between a non- nucleic acid immunogen and an antigen binding protein (again, typically an antibody) which binds the non-nucleic acid immunogen.
  • Such methods comprise contacting the antigen binding protein to a nucleic acid, which nucleic acid inhibits complex formation between the antigen binding protein and the non-nucleic acid immunogen.
  • the contacting step may be carried out in vitro (again typically by combining constituents in an aqueous solution) , or may be carried out in vivo in a human or animal subject. Where carried out in vivo, the subject, dosage, route of administration, and other parameters may be as given above in connection with a method of inducing an immune response. Where carried out in vitro, again, numerous different formats for carrying out such blocking experiments will be known to those skilled in the art, as also discussed above.
  • Nucleic acids may be prepared for administration to a subject as a pharmaceutical composition comprising the nucleic acid in a pharmaceutically acceptable carrier. Preparation is typically carried out by intimately admixing the nucleic acid with the carrier. The nucleic acid is included in an amount sufficient to achieve the intended effect: i.e., induce an immune response or block complex formation between an antigen binding protein and an immunogen, as explained above.
  • Pharmaceutically acceptable carriers may be solid or liquid carriers, such as sterile pyrogen-free phosphate-buffered saline solution.
  • the carrier may optionally contain one or more adjuvants, such as aluminum hydroxide, aluminum phosphate, plant and animal oils, etc.
  • the vaccine formulation may contain one or more stabilizer, exemplary being carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran, and glucose, proteins such as albumin or casein, and buffers such as alkaline metal phosphates and the like.
  • stabilizer exemplary being carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran, and glucose, proteins such as albumin or casein, and buffers such as alkaline metal phosphates and the like.
  • nucleic Acid Sets Useful as Polyvalent Vaccines The techniques described above may be used to provide a method of generating a set of nucleic acid species useful as immunogens, where at least two members of the set are not immunologically cross-reactive with one another.
  • immunologically cross-reactive is meant that the cross-reactive compounds or species bind to the same antigen binding protein at the same site (including overlapping sites) with the binding affinities give herein (e.g., they compete for binding with one another).
  • the method comprises collecting a plurality of antigen binding proteins from a human or animal subject, which antigen binding proteins are either antibodies or T cell receptors; combining the antigen binding proteins with a degenerate pool of nucleic acid species; and then recovering a plurality of nucleic acid species bound by the antigen binding proteins from the degenerate pool to produce the set.
  • the nucleic acid species are immunologically cross-reactive with compounds which are not nucleic acids, and at least two members of the set are immunologically cross-reactive with different immunogens.
  • Such methods are particularly useful for developing polyvalent vaccines, i.e., vaccines capable of inducing an immune response to different epitopes on a common immunogen and/or vaccines capable of inducing an immune response to different immunogens.
  • the collecting step may be carried out by any suitable means, such as by harvesting T cell lymphocytes from the subject, by collecting immune serum from the subject, or both.
  • the type of immune response elicited by the vaccine can be biased towards cellular or humoral by means of the type of antigen binding protein collected.
  • the antigen binding proteins may be collected from a subject afflicted with the disease or by immunizing a suitable subject with an immunogen for that disease prior to the collecting step.
  • this method provides the advantage that an infectious agent or toxin need not be isolated or identified in order to generate a vaccine against it.
  • the technique may be employed with any disease, including diseases of viral, bacterial, protozoan, or other icrobial origin.
  • antigen binding proteins collected from a plurality of subjects, all previously afflicted with the same disease or infected with the same disease-causing organism are pooled to ultimately provide a polyvalent vaccine directed against numerous variants or serotypes of that disease.
  • a polyvalent vaccine directed against numerous variants or serotypes of that disease.
  • one or more elderly subjects who lived through the influenza epidepmic of 1918 will have the combined immunological experience to resist any potential challenge by the swine flu virus, H1N1.
  • the combined immunological repertoire of this population of individuals which includes antibodies to multiple determinants of the virus such as the surface antigens, hemagglutinin (HI) and neuraminidase (Nl) , can be captured from their pooled sera using this invention and that repertoire passed by immunization of an immunologically naive subject(s) using the polyvalent vaccine.
  • the entire immunological repertoire of any population which has developed immune resistance to any pathogen, known or unknown could be similarly passaged using such polyvalent vaccines derived from their combined sera. It is one advantage of this invention that the vaccine recipients need not be subject to exposure to the pathogen or to any materials extracted from a pathogen.
  • the nucleic acid vaccine is rendered pure by simple extraction using any of several standard procedures such as emulsification in phenol.
  • Another example of the practice of this invention is using rhinoviruses which cause the common cold. It has not been possible to develop satisfactory vaccines to rhinoviruses because approximately 30 different, but related serotypes exist. The viral antigenic determinants can change abruptly and mutate in order to escape immune survaillance. However, in the combined experience of a population of individuals resides a record of immunological response to all of the various serotypes of the rhinovirus. Thus, the present invention will allow the derivation of a polyvalent vaccine to rhinovirus using the combined sera of a population of individuals experienced with infection by the many serotypes of rhinovirus. Such a vaccine will provide broad protection to the recipient for all rhinovirus challenges.
  • This invention embodies an additional advantage for the production of a polyvalent vaccine in that the selected nucleic acid mimetics will contain in the sub pool structural variants of the original immunogen which can be used to immunize the subject against unanticipated variants of a pathogen.
  • the protozoan agent of African sleeping sickness, Trypanosoma brucei contains on its surface VSG antigens that can undergo spontaneous antigen switching allowing the pathogen to escape immune survellance.
  • the present invention provides for the derivation of polyvalent nucleic acid vaccines which mimic subtle variations of the original antigen.
  • some selected ligands in the vaccine resemble the exact original non-nucleic immunogen while others resemble subtle variations of the original immunogen.
  • nucleic acids each of which nucleic acids inhibits complex formation between an antigen binding protein and an immunogen, wherein the antigen binding protein is either an antibody or a T cell receptor, and wherein at least two members of the set do not bind to the same antigen binding protein.
  • Members of the set will have the characteristics as given above: e.g., bind to the antigen binding protein at a K d of from 10 '5 or 10 "7 to 10 "u moles per liter.
  • the set may be provided in an aqueous carrier solution, may be provided in the form of a cDNA library encoding the set as described above, or may be provided in a pharmaceutically acceptable carrier as described above.
  • the set is essentially free of other nucleic acids which do not possess such binding characteristics, though other ingredients can of course be added to the set which do not detract from the function thereof.
  • the techniques described above can also be adapted for generating tools for the rational design of drug compounds. Such techniques are particularly useful where other structural information on the drug compound is unavailable.
  • the method generates a plurality of nucleic acid species which are immunologically cross- reactive with a drug compound (which compound is not a nucleic acid, and which compound possesses at least two epitopes) .
  • the method comprises immunizing an animal with the drug compound according to methods described above, then collecting antigen binding proteins that bind the compound, then combining the antigen binding proteins with a degenerate pool of nucleic acid species, and then recovering a plurality of nucleic acid molecules bound by the antigen binding protein from the degenerate pool, wherein at least two of the nucleic acid species do not bind to the same antigen binding protein.
  • the method may be employed with any drug compound which presents a plurality of epitopes thereon, including (but not limited to) peptides, glycoproteins, fats, lipids, polysaccharides, and carbohydrates, including chemical analogues thereof.
  • the foregoing techniques provide a set of isolated nucleic acid species which inhibits complex formation between an antigen binding protein and a drug compound as described above, wherein at least two of the nucleic acid species do not bind to the same antigen binding protein.
  • the characteristics of the members of the set are as given above: i.e., they bind to the antigen binding protein at a K d of from 10 "5 or 10 "7 to 10 "u moles per liter.
  • the set may be provided in an aqueous carrier solution, as a cDNA library encoding the same, or in a pharmaceutical carrier.
  • the set may be screened itself for drug analogs, or may be used to vaccinate a suitable host subject as discussed above to generate additional complementary mimetic surface ligands. Again, the set is essentially free of other nucleic acids which do not possess such binding characteristics, though other ingredients can of course be added to the set which do not detract from the function thereof.
  • Mimetic conformational selection can be used to improve the biological efficacy of a compound such as a receptor binding molecule, by providing a pool of structural variants which themselves possess biological activity as agonists or antagonists.
  • insulin which binds to an insulin receptor can serve as the immunogen and insulin-binding antibodies can be collected for use in conformational selection.
  • Mimetic nucleic acid ligands selected using the antibodies are structural analogus of insulin and can also be utilized as functional analogs of insulin in biological assays and therapeutic regimens.
  • structural analysis of selected mimetic ligands by any of several known methods (i.e., co- crystallographic analysis) will provide a means to correlate variations in biological function of the mimetic molecule with its structural features.
  • mimetic nucleic acid ligands that always display receptor agonist activity should conserve certain structural features. These surfaces can be modeled against the original drug compound in order to rationally engineer optimal drug design. The foregoing is explained in greater detail in the following non-limiting examples.
  • the peptide was coupled to keyhole limpet hemocyanin (Sigma) using the crosslinker MBS (3-maleimidobenzoyl-N- hydroxysuccinimide ester, Boehringer Mannheim Biochemicals) .
  • MBS crosslinker
  • a high-titer antiserum was obtained from rabbits immunized with the peptide-carrier conjugate in accordance with standard techniques.
  • the specificity of the antiserum was characterized using Western blot and immunoprecipitation methods previously reported (J. Chambers and J. Keene, Proc. Natl . Acad. Sci . USA 82, 2115-2119 (1985); R. Bentley and J. Keene, Mol . Cell . Biol .
  • Figure 3 demonstrates that the serum was specific for proteins containing the glO fusion peptide as assayed by immunoblot and immunoprecipitation.
  • Figure 3A shows recognition by the anti-glO serum of glO-tagged Ul-snRNP A protein (glO-Ul-A) in Western blot analysis. The anti-glO serum reacted with only the glO-Ul-A fusion protein (Fig.
  • Anti-glO antibody binding could be inhibited by excess free glO peptide (Fig. 3B, lanes 8-11) , but not by a control peptide (lane 12) , further demonstrating that the interaction was specific for the glO peptide sequence.
  • these results demonstrate that the glO fusion peptide can function as an epitope tag recognized by the anti-glO serum.
  • a more demanding test of an epitope tag is recognition of the tagged molecule within the context of a acromolecular complex.
  • the potential complications of using an epitope tag are its interference in assembly of a complex or its inaccessibility within an assembled complex.
  • RNP complexes formed in vitro between glO-Ul-A and Ul RNA Previous studies have used the glO tag to im unoprecipitate RNP complexes formed in vitro between glO-Ul-A and Ul RNA.
  • Other RNP complexes analyzed using the glO epitope include those formed by U1-70K, U2-B" , U2-A' , and Ro-RNP 60kD.
  • RNP particles formed by these tagged proteins in vivo also are accessible to recognition of the epitope by the anti-glO serum as assessed by immunoprecipitation and immunofluorescence (data not shown) .
  • RNA epitope tag would be equally useful for studying complexes containing RNA.
  • the anti-glO serum was presented with a degenerate pool of RNA containing 1,048,576 unique species, assuming incorporation of four different nucleotides at 10 randomized loop positions (Fig. 2) . These RNAs were transcribed from approximately 1 x 10 11 molecules of degenerate oligodeoxynucleotide template (D. Tsai et al., Nucl . Acids Res . 19, 4931-4936 (1991)). Assuming that most templates are transcribed at least once, all possible RNA species should be redundantly represented.
  • RNA was prepared by in vitro transcription of PCR-generated templates in accordance with known techniques (D. Tsai et al. , Nucl . Acids Res . 19, 4931-4936 (1991)).
  • the transcripts were immunoprecipitated with the anti-glO serum as follows: Protein A Sepharose beads (Sigma, 4 mg per 50 ml reaction) were washed in NT2 buffer (50 ⁇ UM Tris [pH 7.4], 150 mM NaCl, 0.05% Nonidet P-40) (C. Query et al., Cell 57, 89-101 (1989)), mixed with 2 ml of anti-glO serum, incubated on ice for 10 min, and washed in NT2.
  • NT2 buffer 50 ⁇ UM Tris [pH 7.4], 150 mM NaCl, 0.05% Nonidet P-40
  • RNA precipitations were performed with either protein A Sepharose beads alone, pre-immune serum, or anti-glO serum.
  • the D10 RNA bound only to the anti-glO serum (Fig. 4, lane 1) , confirming that binding is specific for the post-immune antiserum.
  • Immunoprecipitation experiments with various RNA species suggest that the anti-glO serum is specific for the DIO RNA sequence. For example, an unrelated RNA was not recognized (Fig. 4, lane 6) , nor were RNAs containing the same stem but with different loop sequences (lanes 5 and 7) .
  • the DIO RNA is recognized by the anti-glO serum, the selection procedure theoretically could recover RNA bound to any surface of any antibody molecule in the serum. Since the pre-immune serum showed no reactivity towards the DIO RNA, the most likely RNA-binding surface is the antigen-combining site of the glO-specific antibodies. This possibility was tested by competition experiments using the glO peptide and the DIO RNA.
  • RNAs were analyzed on 6% denaturing acrylamide gels and proteins on 10% SDS-PAGE gels, followed by autoradiography.
  • Antibody-RNA complexes were formed in the presence of competitor glO peptide or control peptide (Fig. 5A) , or with increasing amounts of glO peptide (Fig. 5B) , and uncompeted RNA was recovered by immunoprecipitation.
  • the D10 RNA was examined for its ability to inhibit complex formation between a glO fusion protein and the glO-reactive antibodies.
  • the D10 RNA was able to compete with the glO fusion protein for binding to the antibody (Fig. 5C, lane 4) .
  • An unrelated RNA was not able to compete for the antibody combining site (Fig. 5C, lane 5) .
  • increasing amounts of DIO RNA were able to progressively compete with an [ 35 S] labeled glO fusion protein (Fig. 5D, lanes 1-6) .
  • the DIO DNA sequence (Fig. 2) was cloned into pGEM-3zf(+) and various length transcription templates produced by truncation 3' to the DIO epitope.
  • RNA was synthesized in vitro from these templates, and the anti-glO serum was used to immunoprecipitate the DIO-tagged pGEM-3zf(+) RNAs. All fusion RNAs were precipitated by the anti-glO serum (Fig. 6A, lanes 2-5) , while a control RNA was not precipitated (lane 1) . Therefore, the DIO sequence is functional as an RNA epitope tag in these contexts.
  • RNA sequences within the pool of artificially randomized 10-mer loops approaches that of RNA sequences within a HeLa cell; however, recognition of an RNA epitope within the cellular milieu may encounter different constraints than recognition within a pool of in vitro transcripts.
  • DIO epitope in cellular extracts
  • [ 32 P] labeled HeLa cell extracts and [ 3 P] labeled deproteinized HeLa cell RNA were prepared in accordance with known techniques (R. Bentley and J. Keene, Mol . Cell . Biol . 11, 1829-1839 (1991)) .
  • [ 32 P] labeled in vitro transcripts were mixed with either HeLa cell extract or HeLa cell RNA under the DIO binding conditions described above.
  • the anti-RNA reactivities in the anti-glO serum and in the anti-Ul RNA patient serum (EW) were normalized by using 1 ml of a (1:5) dilution of serum EW and 10 ml of the anti-glO serum.
  • Antibody-RNA complexes were analyzed as described above.
  • Ul-3Dx gift of J. Snedeker
  • transcripts of exogenous NEU1 RNA were not immunoprecipitated by the anti-glO serum (Fig. 6B, lane 8 and 13) ; however, they were recognized by a patient serum, EW, that binds the second stem-loop of Ul RNA (lane 5) .
  • the anti-glO serum was not reactive with HeLa cell RNA (Fig. 6B, lane 7) .
  • RNA Seguences from a Degenerate Pool This example demonstrates that antibodies taken directly from a subject afflicted with an autoimmune disease can be used to generate nucleic acid species which bind thereto.
  • a degenerate pool of RNA sequences is created using synthetic DNA oligomers that are randomized in either of three different contexts representing linear unstructured RNA or in the framework of the natural Ul stem II, from positions 50-89, containing degenerate loops of 10 or 13 nucleotides as described in detail previously (D. Tsai et al., Nucl . Acids Res . 19, 4931-4936 (1991)). All three RNA structural contexts share identical PCR primer regions at the 5' and 3 1 termini.
  • a selection procedure is performed consisting of three cycles of successive transcription, RNA immunoprecipitation with serum from a patient afflicted with systemic lupus erythematosus (SLE) , reverse transcription, and PCR as described above. Sequencing of multiple clones, each representing a selected RNA species, reveals several RNA species which bind to antibodies in EW patient serum.
  • SLE systemic lupus erythematosus
  • MOLECULE TYPE cDNA

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Abstract

L'invention se rapporte à un procédé de production d'une espèce d'acides nucléiques qui ont, immunologiquement une réaction croisée avec des immunogènes acides non nucléiques. Le procédé consiste à combiner une protéine fixant l'antigène qui lie ledit immunogène à un groupe dégénéré d'espèces d'acides nucléiques, et à récupérer ensuite une espèce d'acides nucléiques liés à la protéine fixant l'antigène à partir du groupe dégénéré. L'invention se rapporte également à l'espèce d'acides nucléiques ainsi obtenue, ainsi qu'à son utilisation pour marquer des molécules destinées à la détection immunologique, à détecter des anticorps contre des immunogènes d'acides non nucléiques prédéterminés, à bloquer la formation du complexe entre une protéine fixant l'antigène et un immunogène d'acide non nucléique, et à induire une réponse immune vis-à-vis de l'immunogène chez l'homme ou l'animal. Les immunogènes préférés sont des peptides et les protéines préférées fixant les antigènes sont des anticorps.
PCT/US1993/008210 1992-09-11 1993-08-31 Procede de derivation des epitopes Ceased WO1994006934A1 (fr)

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5688935A (en) * 1990-06-11 1997-11-18 Nexstar Pharmaceuticals, Inc. Nucleic acid ligands of tissue target
US5712375A (en) * 1990-06-11 1998-01-27 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: tissue selex
US5750342A (en) * 1990-06-11 1998-05-12 Nexstar Pharmaceuticals, Inc. Nucleic acid ligands of tissue target
US5763566A (en) * 1990-06-11 1998-06-09 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: tissue SELEX
US5789157A (en) * 1990-06-11 1998-08-04 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: tissue selex
US5864026A (en) * 1990-06-11 1999-01-26 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: tissue selex
US6114120A (en) * 1995-05-03 2000-09-05 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: tissue selex
US6127119A (en) * 1990-06-11 2000-10-03 Nexstar Pharmaceuticals, Inc. Nucleic acid ligands of tissue target
WO2001048480A1 (fr) * 1999-12-28 2001-07-05 Keene Jack D Procedes servant a isoler et a caracteriser des complexes endogenes de proteines-arnm (rnpm)
US7964356B2 (en) 2007-01-16 2011-06-21 Somalogic, Inc. Method for generating aptamers with improved off-rates
US8703416B2 (en) 2008-07-17 2014-04-22 Somalogic, Inc. Method for purification and identification of sperm cells
US8815517B2 (en) 1999-12-28 2014-08-26 Ribonomics, Inc. Methods for identifying functionally related genes and drug targets
US8975026B2 (en) 2007-01-16 2015-03-10 Somalogic, Inc. Method for generating aptamers with improved off-rates

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991019813A1 (fr) * 1990-06-11 1991-12-26 The University Of Colorado Foundation, Inc. Ligands d'acide nucleique

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991019813A1 (fr) * 1990-06-11 1991-12-26 The University Of Colorado Foundation, Inc. Ligands d'acide nucleique

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5688935A (en) * 1990-06-11 1997-11-18 Nexstar Pharmaceuticals, Inc. Nucleic acid ligands of tissue target
US5712375A (en) * 1990-06-11 1998-01-27 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: tissue selex
US5750342A (en) * 1990-06-11 1998-05-12 Nexstar Pharmaceuticals, Inc. Nucleic acid ligands of tissue target
US5763566A (en) * 1990-06-11 1998-06-09 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: tissue SELEX
US5789157A (en) * 1990-06-11 1998-08-04 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: tissue selex
US5864026A (en) * 1990-06-11 1999-01-26 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: tissue selex
US6127119A (en) * 1990-06-11 2000-10-03 Nexstar Pharmaceuticals, Inc. Nucleic acid ligands of tissue target
US6114120A (en) * 1995-05-03 2000-09-05 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: tissue selex
WO2001048480A1 (fr) * 1999-12-28 2001-07-05 Keene Jack D Procedes servant a isoler et a caracteriser des complexes endogenes de proteines-arnm (rnpm)
US6635422B2 (en) 1999-12-28 2003-10-21 Ribonomics, Inc. Methods for isolating and characterizing endogenous mRNA-protein (mRNP) complexes
US7504210B2 (en) 1999-12-28 2009-03-17 Ribonomics, Inc. Methods for isolating and characterizing endogenous mRNA-protein (mRNP) complexes
US8143002B2 (en) 1999-12-28 2012-03-27 Ribonomics, Inc. Methods for isolating and characterizing endogenous mRNA-protein (mRNP) complexes
US8815517B2 (en) 1999-12-28 2014-08-26 Ribonomics, Inc. Methods for identifying functionally related genes and drug targets
US7964356B2 (en) 2007-01-16 2011-06-21 Somalogic, Inc. Method for generating aptamers with improved off-rates
US8975026B2 (en) 2007-01-16 2015-03-10 Somalogic, Inc. Method for generating aptamers with improved off-rates
US9382533B2 (en) 2007-01-16 2016-07-05 Somalogic, Inc. Method for generating aptamers with improved off-rates
US8703416B2 (en) 2008-07-17 2014-04-22 Somalogic, Inc. Method for purification and identification of sperm cells

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