EP1028979A1 - Peptides de fusion de region variable qui forment des complexes effecteurs en presence d'antigenes - Google Patents

Peptides de fusion de region variable qui forment des complexes effecteurs en presence d'antigenes

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Publication number
EP1028979A1
EP1028979A1 EP98948515A EP98948515A EP1028979A1 EP 1028979 A1 EP1028979 A1 EP 1028979A1 EP 98948515 A EP98948515 A EP 98948515A EP 98948515 A EP98948515 A EP 98948515A EP 1028979 A1 EP1028979 A1 EP 1028979A1
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European Patent Office
Prior art keywords
antigen
effector
fusion polypeptides
pair
sequences
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EP98948515A
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German (de)
English (en)
Inventor
Walt Mahoney
Greg Winter
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Roche Diagnostics Corp
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Roche Diagnostics Corp
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Publication of EP1028979A1 publication Critical patent/EP1028979A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates generally to the fields of immunochemistry and peptide association. More specifically, it provides a system for obtaining fusion polypeptides with variable regions that drive dimerization in the presence of antigen.
  • Antibody molecules have been designed by evolution to direct a relatively non-specific effector function on to a specific target.
  • the antibody repertory of an individual can be primed against a limitless variety of foreign antigens.
  • the induced antibody Upon revisitation of a previously encountered antigen, the induced antibody will bind and bring into play elements of the complement cascade, or Fc receptor bearing cells with all their capabilities.
  • the contemporary biomolecular chemist has capitalized on the targeting specificity of the antibody for diagnostic and therapeutic purposes. Attaching the antibody with a label permits the detection or quantitation of antigen in a test solution. Attaching the antibody to a drug permits targeting to certain cells or tissues. New ways of delivering an effector function by way of an antibody are clearly of benefit.
  • Immunoassays used in routine clinical measurement involve an antibody specific for an analyte of interest in a biological sample.
  • the detecting of the complex involves a process wherein the complex formed is physically separated from either unreacted analyte, unreacted antibody, or both (U.S. Patent No. 3,646,346).
  • the complex can be first formed in the fluid phase, and then subsequently captured by a solid phase reagent or separated on the basis of an altered physical or chemical property, such as by gel filtration or precipitation.
  • one of the reagents can be attached to a solid phase before contacting with other reagents, and then the complex may be recovered by washing the solid phase free of unreacted reagents.
  • homogeneous assays the presence of the complex is detected by a property which at least one of the reactants acquires or loses as a result of being incorporated into the complex.
  • Homogeneous assays known in the art include systems involving fluorochrome and fluorochrome quenching pairs on different reagents (U.S. Patent Nos. 3,996,345, 4,161 ,515, 4,256,834, and 4,261 ,968); enzyme and enzyme inhibitor pairs on different reagents (U.S. Patent Nos. 4,208,479 and 4,233,401 ); and chromophore and chromophore modifier pairs on different reagents (U.S. Patent No. 4,208,479).
  • a particularly powerful homogeneous assay system is the cloned enzyme donor immunoassay (U.S. Patent No. 4,708,929). Two subunits of the enzyme ⁇ -galactosidase associate to provide the detectable signal, which is quantitatively affected by analyte-specific antibody except in the presence of a sample containing free analyte.
  • the isolated heavy and light chain variable domains (V H and V L ) of an antibody constitute a heterodimer known as the Fv fragment, which contains a single antigen binding pocket. Fv fragments may dissociate at low protein concentrations.
  • Klein et al. measured the equilibrium and kinetic aspects of the interaction of isolated variable and constant domains of IgG, using ultraviolet difference spectroscopy. The equilibriuim binding curve between the light chain variable domain and Fd' (a heavy chain fragment containing the heavy chain variable domain) was 1.2 x 10 6 M "1 at pH 5.4. Subsequently, Hamel et al. found that the association between V H and V L did not depend on antigen specificity, and some variable domains associated better with a counterpart from another antibody molecule.
  • Isolated Fv fragments are expected to have better properties for penetration of solid tumor tissue, lower antigenicity, and improved pharmacokinetics.
  • a single chain variable region scFv
  • scFv single chain variable region
  • Monoclonal antibodies of a non-human species can be humanized by placing the three antigen-binding CDR regions of each V H and V L of the specific antibody into the framework of human V H and V L . See, for example, EP 0329400.
  • U S Patent No 4,859,609 (Dull et al ) constructed hybrid receptors that comprise the ligand binding domain of a predetermined receptor, and a heterologous reporter polypeptide
  • the hybrid receptors are said to be useful for performing assays
  • the ligand binding domain (something other than an immunoglobulin) undergoes a conformational change upon binding of the ligand, which in turn affects the reporter peptide attached on the C-terminal end
  • the model reporter molecule is a phosphorylkinase
  • An assay method is claimed, in which the hybrid receptor is incubated with a test sample, and then a conformational change is correlated with the presence of ligand in the sample
  • the fusion polypeptides of this invention contain a variable region sequence linked to an effector sequence
  • the polypeptides do not form stable complexes in solution, except in the presence of an antigen for which the combined variable region is specific
  • the antigen brings the variable region sequences on two polypeptides together, which in turn drives the effector sequences into juxtaposition Complementation of one effector sequence with the other provides an effector function of therapeutic or diagnostic importance
  • Embodiments of the invention include product embodiments
  • the invention includes a pair of fusion polypeptides that complex with each other in the presence of an antigen, consisting of a first fusion polypeptide comprising a first variable domain sequence linked to a first effector sequence, and a second fusion polypeptide comprising a second variable domain sequence linked to a second effector sequence, wherein complexing between the first and second variable domain sequences in a solution is stabilized if the solution contains the antigen, wherein the first and second effector sequences do not complex with each other in a solution containing antigen when not attached to the first and second variable domain sequences, respectively, and wherein complexing between the variable domains in the first and second fusion polypeptides in the presence of the antigen results in complexing between the effector sequences
  • the polypeptides of the invention are freely soluble in solution, and are not membrane proteins
  • the polypeptides are covalently tethered
  • the invention also includes one or other of the fusion polypeptides
  • Exemplary effector sequences are enzyme fragments and toxin fragments
  • the polypeptides preferably have one or more of the following features 1) conversion of the substrate to the product occurs more rapidly in a solution containing the two fusion polypeptides and the antigen, than in a solution containing the two fusion polypeptides but no antigen, 2) the first and second fusion peptides do not have the catalytic activity except when complexed with each other in the presence of the antigen; 3) the substrate does not promote complexing between the two enzyme fragments.
  • Also embodied is a method of preparing a pair of fusion polypeptides comprising the steps of: a) selecting a first variable domain sequence and a second variable domain sequence that form a complex that is stabilized in a solution if the solution contains the antigen; b) selecting a first effector sequence and a second effector sequence that do not complex with each other in a solution containing the antigen; c) preparing a first fusion polypeptide in which the first variable domain sequence is linked to the first effector sequence, and a second fusion polypeptide in which the second variable domain sequence is linked to a second effector sequence; and d) confirming that the first fusion polypeptide forms a complex with the second fusion polypeptide that is stabilized in a solution if the solution contains the antigen, and that upon binding between the polypeptides, enzyme activity is reconstituted.
  • Diagnostic embodiments include a method of measuring the amount of an antigen in a sample, comprising the steps of preparing a reaction mixture containing the sample, a pair of fusion polypeptides, and a substrate for the effector enzyme of the fusion polypeptides, and then measuring any product formed in the reaction mixture.
  • FIG. 1 is a block diagram illustrating the procedure for measuring an antigen concentration in a sample according to the present invention.
  • FIG. 2 is a bar graph indicating the amount of V H phage bound to the biotinylated V L or biotinylated hen egg lysozyme (HEL), in the presence of variable analysis.
  • FIG. 3 is a calibration curve for the concentration of HEL prepared on the basis of the amount of V H phage bound to the biotinylated V L which is immobilized on solid-phase in a plate.
  • FIG. 4 is a graph plotting the HEL concentration vs. the absorption of samples which shows the amount of alkaline phosphatase-labeled V H bound to the biotinylated V L which is immobilized on solid-phase in a plate.
  • FIG. 5 is a graph plotting the HEL concentration vs. the increase in fluorescence intensity ratio between fluorescein-labeled V H and Rhodamine X-labeled V L .
  • FIG. 6 is a line drawing representing two fusion polypeptides of this invention interacting in the presence of an antigen.
  • the variable region sequences (indicated by the solid lines) drive interaction of the effector sequences (indicated by the dotted lines), which I reconstitutes enzymatic activity.
  • the combined variable region is specific for the model antigen hen egg lysozyme, and the effector sequences are monomer subunits of mitochondrial malate dehydrogenase.
  • FIG. 7 is a half-tone reproduction of a gel showing the size of the cloned encoding region for mitochondrial malate dehydrogenase.
  • This invention provides fusion peptide pairs with the property that they associate with each other when a particular substance (termed the "antigen") is present.
  • a particular substance termed the "antigen”
  • effector sequences come together in a way that can create a useful chemical or biological effect, or constitute a completed labeling complex that reflects the presence of the antigen
  • each fusion peptide of the invention can be part of a larger protein or molecular complex, each fusion peptide minimally comprises the following elements:
  • a driver of the complexation reaction which is a variable domain sequence, with the property that it forms a stable complex with the opposing variable domain on the opposing peptide in the presence of the antigen;
  • variable domain sequence a reporter of the complexation reaction, termed the effector sequence, which does not substantially associate with the effector on the opposing peptide unless the variable sequences are complexed, and which has an enzymatic, chemical or biological property of interest.
  • a covalent linkage between the variable domain sequence and the effector sequence which can be a peptide bond, a polypeptide linker sequence, or any other type of chemical structure covalently connecting the variable domain and the effector in a manner that permits the fusion peptide to have the required functional activity.
  • FIG. 6 shows the predicted three-dimensional structure of the polypeptide backbone of a fusion polypeptide pair which is in the complexed configuration.
  • the two solid lines show V H and V L domains (left and right) of a monoclonal antibody specific for the antigen hen egg lysozyme.
  • the domains associate along an interface of opposing ⁇ -pleated sheets, which form an antigen binding pocket oriented towards the bottom of the drawing.
  • Each variable domain is coupled at the C-terminal end to a monomer subunit of malate dehydrogenase, shown by dashed lines.
  • the monomer subunits of this enzyme normally self-associate along an interface to form an active enzyme complex.
  • the subunit interface is modified to prevent self-association, but permit association when driven by the variable region domains.
  • the presence of antigen in the mixture can be detected by providing a substrate for the active enzyme, and measuring product formation.
  • polypeptides of the invention are obtained by a method that includes the following steps, which will each be discussed in turn.
  • variable domain sequences are usually V H and V L domains, although other combinations are possible (for example, homologous or heterologous V L -V L pairs in the Bence Jones configuration, and T cell variable region pairs).
  • the variable domain sequences may correspond to a complete intact variable region domain, or may be longer and shorter in length, or incorporate amino acid changes, inserts, or deletions. Typically but not invariably, each variable domain will have the three CDR regions found in intact variable region.
  • Sensitive to alterations are segments that make up the antigen binding site and the interface between the variable region pair, and changes should be made so as not to impair the required binding properties.
  • variable region domains can be of human origin, mouse origin, or of any other species, or they can be artificial sequences designed as a chimera or consensus of multiple species. Variable regions of human origin (or having human framework residues) are of interest for therapeutic applications, in order to minimize unwanted immunogenicity. Also of interest are variable regions of camel origin, or variable regions modified to incorporate camelid mutations which decrease the affinity between variable regions.
  • the "antigen" to which the variable region pairs bind can be a small molecule drug or hapten, protein, nucleic acid, carbohydrate, proteoglycan, glycolipid, or any structure which can be used to select the variable region pairs or binds the variable region pair with sufficient affinity and specificity.
  • An antigen which induces the dimerization of a variable region pair is also referred to as a "driver antigen”.
  • Raising and selecting variable regions with the specificity for a particular antigen is standard practice in the art.
  • General techniques used in raising, purifying and modifying antibodies, and the design and execution of immunoassays, are found in Handbook of Experimental Immunology (D.M. Weir & C.C. Blackwell, eds.); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); David Wild, ed., The Immunoassay Handbook (Stockton Press NY, 1994); and R. Masseyeff, W.H. Albert, and N.A. Staines, eds., Methods of Immunological Analysis (Weinheim: VCH Verlags gesellschaft mbH, 1993).
  • the immunogen is optionally modified to enhance immunogenicity, for example, by aggregating with glutaraldehyde or coupling to a carrier like KLH, and then mixed with an adjuvant, preferably Freund's complete adjuvant for the first administration, and Freund's incomplete adjuvant for booster doses.
  • an adjuvant preferably Freund's complete adjuvant for the first administration, and Freund's incomplete adjuvant for booster doses.
  • the most common way to produce monoclonal antibodies is to immortalize and clone a splenocyte or other antibody-producing cell recovered from an animal that has been immunized.
  • the cione is immortalized by a procedure such as fusion with a non- producing myeloma, by transfecting with Epstein Barr Virus, or transforming with oncogenic DNA.
  • the treated cells are cloned and cultured, and clones are selected that produce antibody of the desired specificity. Specificity testing is performed on clone supernatants usually by immunoassay.
  • Other methods for obtaining specific variable regions from antibodies or T cells involve contacting a library of immunocompetent cells or viral particles with the target antigen, and growing out positively selected clones. Immunocompetent phage can be constructed to express immunoglobulin variable region segments on their surface. See Marks et al., New Engl. J. Med.
  • Phage of the desired specificity are selected by adherence to antigen attached to a solid phase, and then amplified in E. coli.
  • variable regions with the property of antigen-dependent association involves assaying the association of one of the variable regions with the other in the presence and absence of antigen.
  • Solid phase enzyme or fluorescein labeled association tests are quite appropriate, and fully described in USSN 08/663,922 by Ueda et al., which is hereby incorporated herein in its entirely.
  • the association between heavy and light chains is due in large part to association between C H1 and C L . It is estimated that about 1 in 10 variable region pairs have sufficiently low association constant when detached from the constant regions for use in this invention without further association.
  • the association constant is predicted to be a function of interacting residues along the interface. Accordingly, variable domains that have an antigen-dependent association can be obtained for any antigen, using this selection strategy.
  • the selected variable region pair should have an association constant of one variable region for the other should be at least 10-fold higher in the presence of antigen, and is progressively more preferred if it is at least about 10 2 , 10 3 , 10", or 10 5 fold higher.
  • Association in the absence of antigen is generally less than 10 8 M "1 and preferably less than 10 6 M "1 .
  • Association of the variable regions for each other in the presence of antigen, and association of antigen for the variable region complex is generally over 10 8 M " ⁇ preferably above 10 10 M ⁇ 1 , and more preferably above 10 12 M "1 .
  • Association constants can be modified, if desired, by altering amino acids along the interface. It is not necessary to measure the affinities to practice the invention, as long as a sufficient difference is observed in the presence or absence of antigen in the intended context.
  • the effector sequences have the property that they do not associate with each other when in their fusion polypeptides, except when driven together by the variable region domains. In most instances, the effector sequences will also not associate with each other when not connected with the variable region domains, regardless of whether antigen is present. When driven together in the fusion polypeptides, the effector sequences will interact with each other at an interface typically separated by less than 10 Angstroms. Association of the effector sequences for each other when not driven by antigen is generally less than 10 8 M "1 and preferably less than 10 6 M "1 in the environment of its intended use. Where the effector sequences are complementing enzyme fragments, the enzyme substrate (or other component of the reaction mixture) should not be able to induce association of the effector sequences.
  • Low levels of undriven association are more tolerable and may even be of assistance when the association of the variable regions for each other in the absence of antigen is negligible.
  • the degree of association can be measured by techniques known in the art, such as gel filtration, blotting techniques, and quantitative solid-phase separation assays.
  • a particularly convenient method for measuring the association constant is using a BIAcoreTM SPR biosensors made by Pharmacia (Uppsala, Sweden) according to manufacturers directions (see in particular the BIAtechnology handbook). While effector sequences with these properties may be prepared by any known technique, such as de novo computer modeling, it is more convenient to start with self-associating fragments or subunits of a protein with the desired effector function.
  • the protein is then modified to prevent the association but maintain a surface that can act as an interface when the units are driven together.
  • modification There are several types of modification that can be used. If subunit association is stabilized by disulfide bonds, then the cysteines can be blocked or preferably replaced with another amino acid. If there is strong subunit association due to noncovalent forces, then it may be possible to reduce the polypeptide length of each subunit until the association constant is sufficiently low. In a third approach, the interface residues are mutated in various test combinations.
  • the effector substance will have a degree of identity with the native enzyme subunit or fragment, generally of the order of at least about 70%, and possibly at least about 80% or 90%. Identity is calculated as the percent of amino acids in the consecutive sequence of the native molecule that are preserved in the same order (with no penalty for gaps or inserts) in the adapted form, and is independent of enzyme specificity. Where the native enzyme is made up of non-identical fragments or subunits, the polypeptide pair will usually also have non-identical effector sequences.
  • the polypeptide pair can have identical or non-identical effector sequences, independently of the variable region sequences, which are usually non-identical. For instances where the effector sequence are non-identical, the choice of which effector sequence to attach to which variable domain sequence is determined empirically.
  • the interacting effector sequences complement each other to provide an effector function of some chemical or biological interest, such as formation of an epitope, a ligand for a receptor, an enzyme, or a toxin.
  • Contexts where each of these embodiments are relevant are described in a later section.
  • DHFR dihydrofolate reductase
  • NADPH NADPH
  • Roles of several amino acids in DHFR function have been elucidated, and the crystal structure has been obtained.
  • the X-ray crystal structure of the beta 2 homodimer of luciferase from V. harveyi has been determined and the active site partly described. Fisher et al., 1996, J. Biol. Chem.
  • Bovine Rnase-A consists of a single chain of 124 amino acids (13,683 mol wt). When subjected to limited digestion by subtilisin, the peptide bond between residues Ala 20 and Ser 21 is cleaved.
  • S-peptide fragments 1-20
  • S-protein fragments 21-124
  • S-peptides 1-20 and S-protein fragments 21-124
  • Simonson et al. 1992, Biochemistry 31:8661
  • Varadarajan et al. 1992, Biochemistry 31:12315
  • Kim et al. 1992, Biochemistry 31 :12304.
  • Effector peptides with a cytotoxic function include bacterial toxins, such as cholera toxin and cholera toxin B subunit, E. coli heat-labile enterotoxin and its B subunit, Bordetella pertussis toxin and the subunits S2, S3, S4, and S5 (in any combination), diphtheria toxin and the ⁇ toxin fragment, shiga and shiga-like toxins, staphylococcal ⁇ -hemolysin, vibrio thermostable direct hemolysin, alpha- sarcin, ricin, and abrin.
  • bacterial toxins such as cholera toxin and cholera toxin B subunit, E. coli heat-labile enterotoxin and its B subunit, Bordetella pertussis toxin and the subunits S2, S3, S4, and S5 (in any combination)
  • diphtheria toxin and the ⁇ toxin fragment such as cholera tox
  • cytotoxic effector peptides are RNases, such as colicins; T1 ribonucleases (including fungal ribonucleases); T2 ribonucleases (both plant style and seed RNases and ribosome- inactivating proteins).
  • RNases such as colicins
  • T1 ribonucleases including fungal ribonucleases
  • T2 ribonucleases both plant style and seed RNases and ribosome- inactivating proteins.
  • RNase A superfamily including RNase A, seminal RNase, RNase dimers, eosinophil-derived neurotoxin, eosinophil cationic protein, onconase, frog ribonucleases, and angiogenin.
  • RNases that are engineered to be cell-type selective by coupling to ligands for cell-surface receptors (cytotoxic ribonuclease chimeras).
  • cytotoxic ribonuclease chimeras For a critical review of RNase chimeras, the reader is referred to Youle et al., 1993, Crit. Rev. Ther. Drug Carrier Syst. 10:1-18.
  • Prior et al. (1996, Bioconjugate Chem. 7:23-29) have described a chimeric molecule of pseudomonas exotoxin, conjoined to barnase. This protein is toxic to cells due to its RNase activity, which is delivered to the cell by way of the endotoxin delivery pathway.
  • a critical consideration in choosing the effector sequences is the position of the terminating residues. Since it is necessary that the variable region sequences be able to drive the two enzyme effector sequences together, the three-dimensional distance between terminating residues of the interacting effectors optimally matches that of the interacting variable regions.
  • the most usual configuration of the fusion peptides is for the C-terminus of each variable region to be linked to the N-terminus of each effector, although other configurations are possible. It is also possible to trim a few residues from the variable regions or the effectors, or both, to enhance the match of the spread.
  • the opposite approach that is, adding a linker sequence between the variable sequence and the effector sequence on one or both chains — becomes increasingly more difficult with increasing length of the linker.
  • fusion polypeptide is a polypeptide made up of two or more amino acid sequences that do not normally occur together (or at least in the same configuration) in a naturally occurring protein.
  • Fusion polypeptides are typically prepared by expressing a recombinant polynucleotide encoding it, either by PCR-type amplification or using a suitable expression vector, but polypeptide synthesis or conjugation of separate polypeptides using a cross-linking agent can also be used.
  • the fusion proteins of this invention are designed to be freely soluble in solution, and are not membrane proteins.
  • the testing of candidate fusion polypeptides involves verifying that the polypeptide chains assemble under the proper conditions, and that the effector sequences interact upon assembly.
  • the polypeptides should preferentially complex with each other in the presence of the analyte, but complexation should not be inducible by other possible compounds in the reacting environment, such as substrate or receptor for the effector sequences.
  • the ability of the effector sequences to "complex" or interface with each other can be measured by techniques such as diference spectroscopy or circular dichroism.
  • complexing of the subunits is inferred from the ability of the assembled polypeptide pair to provide the activity of the combined effector sequences: catalytic activity of the expected specificity, if the effector is an enzyme, or cytotoxic activity, if the effector is a toxin.
  • This invention also includes "tethered" compositions.
  • the polypeptide pair is interconnected through an interchain covalent bond or bridge.
  • the bridge can be a disulfide bond, a peptide bond between amino acid side chains, or a chemical moiety created by treatment of the polypeptides with a crosslinking agent.
  • the bridge is selected and positioned so as not to stabilize dimerization of the variable domain or of the effector domain, permitting the polypeptides to dissociate and pivot around the connection point. As a result, the effector will still not be in the fully active form until driven into the correct position by association of the variable domain sequences with antigen. When antigen is present, however, the reaction is nearly bimolecular.
  • Candidate polypeptide pairs that show low levels of activity can be adapted in this fashion by incorporating an additional cysteine into each of the opposing chains along the interface.
  • suitable position to create the tethering effect without causing the effector to assume a permanently activated form is near the base of the variable domain sequences.
  • variable region domains can be substituted for another atop the effector sequence.
  • CDRs of a variable region of a new specificity can be casetted into the framework of a proven fusion polypeptide, taking care to avoid disturbing the association properties of the variable domain interface.
  • modification the features of the effector sequences by substitution or mutation. For example, where the effector is an enzyme, mutations near the catalytic site can be used to change the substrate specificity. Hogan et al.
  • Enzymes can also be displayed on filamentous phage (Soumillion et al., Appl. Biochem. Biotechnol.), which may allow for efficent screening of mutants against a new substrate.
  • the fusion polypeptide pairs of this invention have a number of applications in both clinical medicine and research. Two applications of particular interest are as biopharmaceuticals and as assay reagents.
  • the use of the fusion polypeptides will generally involve converting a substrate to a product in a manner that depends on the presence of an antigen, by creating an environment that contains the antigen, the substrate, and a pair of fusion polypeptides.
  • the environment can be, for example, the microenvironment inside an individual being treated with a therapeutic or pharmaceutical composition according of the invention, or an in vitro environment present in a reaction mixture for an assay.
  • variable region sequences When adapted for use as biopharmaceuticals for human therapy, the variable region sequences, the effector sequences, and the linker sequences (if used) will typically be chosen to resemble human sequences as much as possible, to avoid immunogenicity.
  • the specificity of the variable region and the function of the effector will depend on the nature of the embodiment. Veterinary and ex vivo therapeutic use is also contemplated.
  • the variable region of the combined peptide pair is specific for a small artificial molecule, termed the activator.
  • the effector sequences assemble to form an enzyme which is capable of converting a prodrug into the active form.
  • Both the prodrug and the activator are in the general circulation, with the prodrug in excess, acting as a drug reservoir.
  • the activator is typically inert in its biological effect, except for its ability to assemble the polypeptide pair, which then activates the prodrug. Since the activator is small, it can potentially be administered orally, nasally, or by inhalation. As a result, the prodrug may be administered only on an occasional basis, and then titrated to the effective dose on an ongoing basis using the activator
  • the active form of the prodrug itself is a large molecule that is administered by injection or by some other invasive procedure
  • examples of this include any polypeptide drug, including growth factors such as GM-CSF, EPO, and insulin Polypeptide drugs can be converted into a prodrug according to the strategy outlined in USSN 60/[pend ⁇ ng, attorney docket 33746-30011 00]
  • the strategy involves using a cross-linking agent to form the prodrug into an inactive loop configuration
  • the loop contains either a protease recognition sequence in the ammo acid sequence, or else an enzyme cleavable group within the cross-linker
  • enzyme cleavable cross-linkers are outlined in USSN 08/883,632, and include those that are cleavable by glycosidase, phosphatase, amidase or esterase
  • the combined effector sequences of the polypeptide pair mediating the prodrug activation would have the corresponding catabo c activity for either the peptide recognition sequence
  • the variable region of the combined peptide pair is specific for a target substance in a particular tissue or on a particular cell
  • Suitable target substances include tissue specific antigens, such as one of the CD markers for certain cells in the hematopoietic line, the asialoglycoprotein receptor on hepatocytes, integ ⁇ ns on blood vessel walls, or IgE on mast cells and IgE-secreting lymphocytes
  • Other target substances include malignancy markers like prostate-specific antigen, carcinoemb ⁇ onic antigen, and gangliosides (enriched on melanoma cells), or mfectivity markers, such as viral core proteins presented by some infected cells
  • the corresponding effector can take several forms In one form, it is a toxin, which assembles only on cells having the marker, inducing cell-specific lysis In another form, it is a prodrug activator The prodrug will be in the general circulation, but will only be activated near the surface of the target cell, by
  • compositions suitable for human use are sterile and substantially free of mycobacte ⁇ a
  • administration is typically intravenous or intramuscular, although other routes are possible, or the composition can be administered locally near the site of the intended effect
  • Formulation of the composition typically includes both fusion polypeptides of the associating pair, although they can also be administered at separate sites or at separate times
  • one or both polypeptides can be substituted with naked DNA or an expression vector having the corresponding encoding sequence, permitting expression of the polypeptides in situ
  • the polypeptide pair will have a variable region specificity for an antigen to be detected or quantified in a sample.
  • the effector sequences may simply assemble to form an epitope, which can then be labeled with a secondary reagent, but more typically will directly form an active enzyme.
  • One assay embodiment is a reagent for immunohistochemistry of tissue sections. The variable region will be specific for an antigen in the target tissue, and the enzyme will catalyze the precipitation of a stain or an electron-dense particle. In this fashion, the tissue can be developed with a single reagent containing the polypeptide pair, either mixed with or followed by substrate, avoiding the multiple incubation and washing steps of indirect immunostaining.
  • Another assay embodiment is a separation type assays.
  • any of the classic immunoassays using solid phase capture or other types of separation can be adapted and simplified using the polypeptide pairs of this invention.
  • a plastic surface is coated with an antigen- specific capture antibody, the surface is contacted with the sample, and then the surface is contacted with the polypeptide pair. Presence of antigen in the sample is revealed by the immediate conversion of substrate to product, mediated by antigen-induced assembly of the polypeptides. There is no washing step required between contacting the bound antigen with the polypeptides, and supplying the substrate.
  • the fusion polypeptides of this invention are particularly well adapted for homogeneous immunoassays.
  • a quantitative assay simply involves preparing a reaction mixture containing the sample, the polypeptide pair, and the substrate.
  • the rate of conversion of substrate to product will be directly related to the number of assembled polypeptide pairs, which in turn will be directly related to the amount of antigen in the sample.
  • the dimerization constant of the polypeptides in the absence of antigen is low, the components of the reaction mixture can in principle be mixed in any order.
  • the two polypeptides and the substrate can be precombined, and then added to the sample, whereupon the development reaction will begin immediately.
  • the product formed (or substrate consumed) can be measured at a certain time after mixing, or the rate can be measured following initiation of the reaction, either manually or in an automated procedure.
  • the reaction mixture with the sample and the reagents is first incubated at 0 to 4°C, and then warmed to 37°C.
  • Antigen binding by variable regions is often fairly independent of temperature, whereas conversion of product is often temperature-sensitive, depending on the activation energy of the conversion reaction. By manipulating the temperature in the manner described, the conversion of substrate will begin only upon warming, by which time the binding reaction may be close to equilibrium.
  • Suitable biological or clinical samples include but are not limited to urine, plasma, serum, and histological sections.
  • Suitable antigens for detecting or quantifying include those that correlate with certain clinical conditions, such as ferritin, prostate specific antigen, alpha fetoprotein, carcinoembrionic antigen, hCG, prolactin, thyroid stimulating hormone, progesterone, T3 and T4, free T3 and T4, aldosterone, insulin, and so on.
  • drugs administered in therapy or drugs of abuse Drugs of abuse include LSD and other halucinogens, amphetamines, barbiturates, cannabinols, and the like.
  • the enzyme formed upon assembly of the polypeptides will preferably a reaction which involves a deep color change in the solution.
  • a substrate of particular interest for homogeneous assays of this invention is X-gal.
  • Reagents used in the assays of this invention can be packaged separately or in any combination into kit form to facilitate distribution.
  • the reagents are provided in suitable containers, and typically provided in a package along with written instructions relating to assay procedures.
  • This example describes binding experiments conducted using variable region sequences from anti-hen egg lysozyme (anti-HEL) monoclonal antibody with the designation HyHEL-10.
  • the Fv fragment was previously known to form a trimolecular complex of 39 kDa in size, as measured by exclusion chromatography. Its association constant (K a ) measured by titration microcalorimetry was previously reported as high as 4.2 x 10 8 M "1 at 30°C.
  • Fv fragments were expressed by BL21(DE3) transformed with pKTN2, essentially as described in Tsumoto et al. (J. Biol. Chem 269:28777, 1994).
  • V L and V H were separated in an FPLC MonoQ HR5/5 anion exchange column (Pharmacia) using a buffer gradient (20 mM Tris buffer pH
  • V L 8.8 containing 0 to 2 M NaCI), the V L being essentially non-adsorbing under these conditions.
  • variable region sequences were expressed separately, precipitated with 65% ammonium sulfate, dissolved in water, dialized into 0.1 M phosphate buffer, and optionally further purified using a DEAE resin.
  • the reader is referred to Ueda et al., Nature Biotechnology 14:1714, 1996.
  • the binding kinetics were analyzed using the SPR biosensors BIAcoreTM or BiacoreTM 2000 (Pharmacia) at room temperature. Measurements were generally performed with a continuous flow of 5 ⁇ l/min of HBS buffer (10 mM HEPES, pH 7.44, 150 mM NaCI, 3.4 mM EDTA, 0.05% TweenTM 20).
  • HBS buffer 10 mM HEPES, pH 7.44, 150 mM NaCI, 3.4 mM EDTA, 0.05% TweenTM 20.
  • a minimum amount of the purified protein was immobilized by amine coupling to a CM5 sensor chip using 5mM formate pH 3.2 as buffer. Samples with various combinations of V H and HEL concentrations were diluted in HBS and injected. To avoid denaturation as much as possible, stock V H solution was thawed, diluted, and centrifuged at 15 krpm for 5 min at 4 degrees immediately before use.
  • V L K49T The monoclonal antibody with this mutation (V L K49T), which is analogous to HyHEL-8 V L retains antigen binding affinity (Lavoie et al.).
  • the mutant V L was expressed, purified, and immobilized on the sensor chip.
  • the association rate constant obtained by the curve fitting based on simple bimolecular model varied from 6.36 x 10 3 /M/sec (at 1.4 ⁇ M) to 9.28 x 10 4 /M/sec (at 88 nM) depending on HEL concentration.
  • the apparent equilibrium association constant K. 2 was calculated to be 2.3 x 10 8 to 3.4 x 10 9 /M, which is close to the value obtained by calorimetry.
  • V L -Doma ⁇ n Polypeptide of HyHEL-10 From the vector plasmid pKTN2 (Tsumoto, K et al , J Biol Chem 69, 28777-28782, 1994) which encodes pel B signal peptide sequence upstream o the structural genes of V H and V L of the antibody HyHEL-10 which is specific to HEL, the 670 bp portion thereof encoding the pe1B, V L and ssi transcription termination sequence were cleaved by the restriction enzymes, Nhel and EcoRI, and purified in agarose electrophoresis This DNA fragment was ligated with the DNA fragment obtained by digesting the vector pET20b having a T7 promoter (Novagen Inc ) by using the restriction enzymes Xbal and EcoRI, to prepare V L -express ⁇ on vector pETVLhel Then, E coll BL21 (DE3) having T7 polymerase gene on its genome was transformed with this vector, and the transformant
  • a vector plasmid pluck 2001 was prepared by altering the vector portion pTZ18U of pluck 2000 (Japanese Patent Publication No 129516/1995) to pTZ19U
  • This pluck 2001 possesses pe1 B signal peptide sequence, the part of V L and V H structural genes of HyHEL-10, c-myc tag, C- terminal of M13 phage gene 3 protein (g3p), M13 origin ampicillin resistance gene, and an origin derived from plasmid pUC series
  • the following operations were performed to remove the V L structural gene from the vector plasmid pluck 2001 That is, in order to combine the pel B signal sequence and V H structural gene with the downstream c-myc tag and g3c C-terminus in matching reading frame of their codon, PCR reaction was conducted by using 1 ng of pluck 2001 as template and 50 pmol each of primers identified in SEQ ID NOS 1 and 2, respectively, in a 100 ⁇ l of reaction liquid containing 2 5-un ⁇ t Pfu DNA
  • V L and HEL Immobilizing of V L and HEL to a Microtiter Plate, and ELISA using the V H -Phage: In order to immobilize V L s and HEL to microtite plate, they were biotinylated with biotin NHS
  • the streptavidin plate thus obtained was washed twice with PBS containing 0.1% TweenTM 20 (PBS-T), and in succession, the biotinylated lysozyme which was diluted with PBS so that the concentration thereof was 10 mg/l. was poured by 100 ⁇ 1 into the plate and preserved at room temperature for one hour. After removing the solution, the plate was rinsed two times using PBS-T. Into this plate were added 10 ⁇ 1 of sample in which 0, 0.1 mg/ml or 10 mg/l of V L s were contained in PBS, and 90 ⁇ I of V H -phage solution which had been mixed with the equivalent amount of binding buffer 30 minutes before, and the resulting mixture was incubated at 37°C for one hour.
  • PBS-T PBS containing 0.1% TweenTM 20
  • V L 1-0 means that the batch 1 of biotinylated V L s was immobilized onto solid-phase in a plate, and V H -phages and O ⁇ g/ml of HEL were added thereto for incubation.
  • the batch 1 of biotinylated V L s was prepared by mixing 500 ⁇ g of purified V L in 350 ⁇ 1 of 0.1 mo1/1 NaHC0 3 : pH8.
  • V L 1 150 mmo1/1 NaCI and 100 ⁇ g of biotin-NHS in 3.5 ⁇ l of DMSO were mixed, reacting at room temperature for 30 minutes, and thereafter, dialyzing into PBS containing 0.02% sodium azide.
  • V L 1 measurements were made with samples having HEL concentrations of final 0.1 and 10 ⁇ g/ml, respectively.
  • V L 2 prepared using I ⁇ l of biotin-NHS solution instead of 3.5 ⁇ 1 of biotin-NHS solutions of V L 1
  • V L 2 prepared using I ⁇ l of biotin-NHS solution instead of 3.5 ⁇ 1 of biotin-NHS solutions of V L 1
  • V L s with a concentration of 0 ⁇ g/ml, together with V H -phages, was added thereto for incubation.
  • Tested concentrations of V Ls were 0 ⁇ g/ml, 1 ⁇ g/ml and 10 ⁇ g/ml.
  • the V H -phages were prepared from three kinds of phage samples of independent colonies (phage 1 , 2 and 3). As shown in FIG.
  • Example 3 Separation assays using enzyme reporter molecules.
  • the chromosome DNA of E. coli XL1-blue was extracted by the known method (Sambrook, Fritsh, Maniatis, "Molecular Cloning, Ver. 2", 1989), and the 1450 bp portion encoding alkaline phosphatase (the gene: PhoA, EC3.1.3.1) was amplified by PCR.
  • the oligonucleotides of SEQ ID NOS. 3 and 4 were used as PCR primers. More specifically, 35 cycles of PCR processes were conducted with 1 ng chromosome DNA of E.
  • coli XL1-Blue as template DNA in a 100 ⁇ 1 of reaction liquid containing 50 pmol each of the primers, and 2.5 unit of Tag DNA polymerase (Perkin Elmers).
  • the alkaline phophatase gene fragments obtained by the PCR were digested with the restriction enzyme NotI, and purified in agarose electrophoresis.
  • the expression vector pET20 b (Novagen Inc.) was digested with NotI, treated with the phosphatase from bovine small intestine (Takara) and ligated with the alkaline phosphotase DNA fragment. From the restriction enzyme analyses of the resultant plasmid, it was confirmed as the target plasmid and was named pAP.
  • V H -Alkaline Phosphatase-Expression Plasmid From the vector plasmid p'CTN2, the 480 bp portion encoding peIBb signal sequence and V H was amplified by PCR. To add 3'-terminal Hindlll site to the PCR products, the oligonucleotides of SEQ ID NOS. 5 and 6 were used as PCR primers. More specifically, PCR reactions were conducted by using pKTN2 plasmid as template DNA in a I00 ⁇ 1 of reaction liquid containing the primers of each 50 pmol, and 2.5 unit Pfu DNA polymerase.
  • V H -AP V H -alkaline phosphatase
  • the E. coli BL21(DE3)LysS having T7 polymerase on its genome was transformed with pV H AP by the calcium chloride method, and was cultivated at 37°C overnight in an LB medium containing 1.5% agar and antibiotics (50 mg/l ampicillin and 34 mg/l chloramphenicol), to form colonies. These colonies were transferred to a 5 ml of LB medium containing antibiotics (50 mg/l ampicillin and 34 mg/l chloramphenicol), and were cultured at 28°C overnight again. The cells reached to saturation density were collected by centnfugation, and were suspended in a 50 ml of fresh LB medium containing antibiotics (50 mg/l carbenicillin and 34 mg/l chloramphenicol).
  • V H -AP chimeric protein was purified in two stages, one by TALONTM Metal Chelating Column, using 6 x His sequence encoded at the C-terminus of the protein, and the second by the negative ion exchange column.
  • the TALONTM metal chelating resin by Clontech Laboratories Inc.; 2 ml for the column head
  • 20 ml of protein solution were stirred gently in a 50 ml tube at 4°C for one hour to adsorb the protein having a polyhistidine sequence onto the column.
  • the resin was transferred to a 5ml column, where it was washed and eluted with 20 mM MES-Na buffer.
  • V H -Alkaline Phosphatase Using a V L plate prepared similarly as in Example 1 , 10 ⁇ l of sample containing various concentrations of HEL in PBS and 90 ⁇ 1 of V H -AP chimeric protein solution (4 ⁇ g/ml in O.IM Tris HCI: pH ⁇ .O) were added to their respective wells, and incubated at room temperature for two hours.
  • FIG. 4 This figure plots the absorbance against the final concentrations of HEL at one hour after the reaction, with the plotted figures being the means of the two measurements. As is evident from this figure, it was confirmed that the method of this invention assure highly sensitive and reproducible measurement of antigen substance with a final concentration of I ng/ml.
  • the measurement sensitivity achieved by using this alkaline phosphatase as a reporter molecule is approximately tenfold of that recorded in Example 1 , wherein filamentous phages were employed.
  • Example 4 Homogeneous assays using fluorochromes.
  • E. coli BL21 (DE3) was transformed with pKTN2, and cultured at 30°C in a 5 ml of LB medium containing 50 m/1 ampicillin followed by successive culture in 50 ml and one liter of medium, until the cell density reached saturation.
  • V H and V L polypeptides 50 mM Tris-HCI (pH8.8), and separated into V H and V L polypeptides in a negative ion exchange chromatography column (MonoQ HR5/5, Phamarcia Co.). Since few of the V L are adsorbed to the column, the V H and V L could be separated relatively easily, with the non-binding fractions taken as V L and the binding fractions as V H . The purity of both polypeptides after separation was found to be 90% or higher when confirmed by SDS polyacrylamide electrophoresis.
  • V H and V L Labeling of V H and V L with Fluorochrome: 285 ⁇ g of purified V H (0.5 ml ) was dialyzed with 0.2 M sodium phosphate (pH7.0) and 0.1 M
  • the molar ratio of protein to the dye was 1 :2.
  • the equivalent volume of 1 M Tris-HCL (pH7. 5) was added to terminate the reaction.
  • the mixture was placed to gel filtration in PD-10 column (Pharmacia Co. ) equilibrated with 0.2 M sodium phosphate (pH7.0) and 0 1 M NaCI to remove the dyes which had not been reacted.
  • Example 5 Construction of fusion peptides with a malate dehydrogenase effector
  • a pair of fusion polypeptides is obtained that have enzymatic effector sequences based on mitochondrial malate dehydrogenase.
  • Three-dimensional computer modeling was performed using known amino acid sequences, and X-ray crystallographic data available from the Brookhaven database. The sequences of the heavy and light chain variable regions of monoclonal antibody HyHEL-10 was imposed on the crystal structure of the intact Fv fragment.
  • Various candidate enzymes with homologous or heterologous subunits were reviewed to determine whether the distance spanned by the ends of the polypeptide chains matched the distance spanned by the two C-terminal amino acids of the Fv fragment. Results are shown in Table 1 :
  • Malate dehydrogenase has a number of advantages for this type of construct.
  • the 3D structure is known to 1.87 Angstroms; it is a homodimer with a distance between termini suitable for fusing to V H and V L .
  • the fused proteins can optionally be brought closer together, if necessary, by deletions of a few amino acids at the N termini of the enzyme or the C termini of the V H and V L , without any predicted loss in activity.
  • Malate dehydrogenase can be used for sensitive assays with a detection limit in the picomolar range. It is not present in plasma or other biological fluids likely to be tested in a standard clinical assay. It is a proven label in other clinical chemistry technologies, and is stable. Mitochondrial malate dehydrogenase is allosterically regulated. Moreover, the mechanism of catalysis is understood, which should facilitate adaptation to other substrates where desirable.
  • FIG. 6 shows the predicted three-dimensional structure of the polypeptide backbone of a fusion polypeptide pair which is in the complexed configuration.
  • the two solid lines show V H and V L domains (left and right) of the anti-HEL antibody.
  • the domains are predicted to associate in the manner shown.
  • the malate dehydrogenase homodimer structure is shown by the dashed lines. The structure has been rotated so that the two N-termini of the enzyme correspond with the C-termini of the variable region sequences. Fusion of the separate variable region domains each to a malate dehydrogenase subunit (with the possible removal of a few amino acids) can be done without distorting either structure or disturbing the interaction between subunits.
  • FIG. 7 shows the successful amplification of the mitochondrial malate dehydrogenase (MDH) encoding region from a cDNA library.
  • PCR primers were prepared that hybridize to flanking sequences in the cloning vector.
  • Track 1 (no band): cDNA prepared with cytoplasmic MDH-specific primers, amplified with mitochondrial MDH specific primers.
  • Track 2 ( ⁇ 1 kb band): cDNA prepared with cytoplasmic MDH-specific primers, amplified with cytoplasmic MDH specific primers.
  • Track 3 (no band): cDNA prepared with mitochondrial MDH-specific primers, amplified with cytoplasmic MDH specific primers.
  • Track 4 ( ⁇ 1 kb band): cDNA prepared with mitochondrial MDH-specific primers, amplified with mitochondrial MDH specific primers.
  • Tracks 6-8 (no bands): controls.
  • Track 9 (ladder): molecular weight standards.
  • SEQ. ID NOS:7 and 8 provide the amino acid sequence and partial nucleic acid sequence of the heavy chain of HyHEL-10.
  • SEQ. ID NOS:9 and 10 provide the amino acid sequence and nucleic acid sequence of the light chain of HyHEL-10.
  • SEQ. ID NOS:11 and 12 provide the mouse MDH amino acid sequence and nucleic acid sequence.
  • SEQ. ID NOS:13 and 14 provide the pig MDH amino acid sequence and nucleic acid sequence.
  • MDH variants are designed in which various amino acids at the MDH subunit interface are substituted so as to lessen the dimerization constant.
  • the interface is readily identified from the structure shown in FIG. 6, and about 3 residues are changed in various combinations.
  • Degree of association is determined by either direct molecular weight analysis, or by BIAcoreTM binding, as outlined in Example 1. Candidates that do not dimerize on their own are then rescreened for dimerization in the presence of the substrate malate.
  • Recombinant polynucleotides are then prepared, in which L 108 of the light chain or His 116 of the heavy chain are attached to the N-terminal of candidate modified MDH sequences.
  • the expressed fusion polypeptides are tested for the criteria of antigen-driven but not substrate-driven association, and the antigen-dependent ability of the fusion polypeptides to catalyze conversion of malate. Further iterations of sequence alteration and testing is undertaken as necessary that adjust the amino acids at the effector subunit interface or the linkage between the variable domain sequences and the effector sequences to optimize the properties of the polypeptide pair.
  • a stable phage-display system using a phagemid vector phage display of hen egg-white lysozyme (HEL), Escherichia coli alkaline phosphatase, and anti-HEL monoclonal antibody, HyHEL-10. Biochem. Biophys. Res. Comm. 218:682-687.

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Abstract

Les polypeptides de fusion faisant l'objet de cette invention contiennent une séquence de région variable liée à une séquence effectrice. Ces polypeptides ne forment pas de complexes stables en solution, sauf en présence d'un antigène. Lors du contact avec l'antigène, les séquences de région variable se fixent l'une à l'autre, ce qui a pour conséquence de mettre en juxtaposition les séquences effectrices. L'association d'une séquence effectrice avec l'autre fournit une fonction effectrice, par exemple une activation enzymatique, qui peut servir à un grand nombre d'applications thérapeutiques ou diagnostiques.
EP98948515A 1997-11-14 1998-09-24 Peptides de fusion de region variable qui forment des complexes effecteurs en presence d'antigenes Withdrawn EP1028979A1 (fr)

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