WO2000042185A1 - Methodes pour reguler la stabilite de proteines recombinantes, et produits utilises dans ces methodes - Google Patents

Methodes pour reguler la stabilite de proteines recombinantes, et produits utilises dans ces methodes Download PDF

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WO2000042185A1
WO2000042185A1 PCT/US2000/000558 US0000558W WO0042185A1 WO 2000042185 A1 WO2000042185 A1 WO 2000042185A1 US 0000558 W US0000558 W US 0000558W WO 0042185 A1 WO0042185 A1 WO 0042185A1
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protein
antibody
peptide
sequence
antibodies
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Daniel G. Chain
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Mindset Biopharmaceuticals (Usa), Inc.
Mcinnis, Patricia, A.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention is directed to a method for producing recombinant proteins which are stable in vivo and whose stability may be regulated.
  • the present invention is directed not only to the method discussed above, but also to the novel fusion proteins produced by means of the present invention, the DNA encoding such fusion proteins, vectors containing such DNA and suitable for gene therapy, as well as cells in which such vectors have been introduced.
  • Intracellular proteolysis functions to eliminate abnormal proteins, maintain amino acid pools in cells affected by stresses, such as starvation, and generate protein fragments that act as hormones, antigens or other effectors.
  • concentrations of some proteins must vary with time and alterations in the state of a cell, so proteolytic pathways selectively destroy these proteins at the proper time.
  • Conditionally unstable proteins whether long-lived or short-lived, depending on the state of a cell, are often deployed as components of control circuits.
  • many proteins are long-lived as components of larger complexes, such as ribosomes and oligomeric proteins, but are metabolically unstable as free subunits.
  • the ATP-ubiquitin proteasome dependent pathway is the major non-lysosomal proteolytic pathway that functions constitutively to degrade abnormal or damaged proteins.
  • Ubiquitin-mediated proteolysis can also be regulated and is of widespread importance. Regulated proteolysis by the ubiquitin pathway has been implicated in control of the cell cycle, transcription activation, antigen presentation, cell fate and growth, and in the formation and storage of memory. In this multienzyme pathway, protein substrates marked for degradation by conjugation to ubiquitin (a 76-amino acid residue protein) are hydrolyzed by the 26S proteasome, a 2000 kDa proteolytic complex.
  • the post-translational coupling of ubiquitin to other proteins is catalyzed by a family of ubiquitin-conjugating enzymes and involves formation of an isopeptide bond between the C-terminal Gly residue of ubiquitin and the epsilon-amino group of a Lys residue in an acceptor protein.
  • the features of proteins that confer metabolic instability are called degradation signals, or degrons, cf .
  • Varshavsky (1996) Several signals can target specific proteins for degradation.
  • the N-end rule describes a hierarchy of amino acid residues that confer varying degrees of instability and susceptibility of degradation when positioned at the amino terminus of any given protein.
  • the N-end rule proposes that the in vivo half-life of a protein is a function of the N-terminal residue.
  • Varshavsky has described destabilizing residues on proteins, and has classified them as primary, secondary and tertiary. In the case of primary destabilizing residues, the destabilizing activity requires the physical binding of the N- terminal residue by a protein called N-recognin or E3.
  • N-d p the primary destabilizing residues
  • N-d s Secondary destabilizing residues, denoted N-d s , are Asp, Glu and Cys in mammalian cells.
  • the destabilizing activity of the d s residues requires their accessibility to Arg-tRNA-protein transferase.
  • N-terminal Asn and Gin residues are tertiary destabilizing residues, denoted N-d'.
  • the destabilizing activity of N-d fc residues requires accessibility to N- terminal amidohydrolase .
  • a stabilizing N-terminal residue is a default residue, i.e., it is stabilizing because targeting components of an N-end rule pathway do not bind to it or modify it efficiently enough even in the presence of other determinants of an N-degron.
  • Gly, Val and Met are stabilizing residues.
  • Destabilizing N-terminal residues are present in several natural proteins, but their physiological role in regulating protein turnover is limited due to the fact that most naturally synthesized proteins are co-translationally covalently modified to inhibit proteolysis. Thus, while engineered proteins containing destabilizing N-terminal residues are rapidly degraded, the importance of the N-end rule as a regulatory process is questionable at least in mammalian cells, where there is evidence that proteins degraded by the ubiquitin system are recognized by different signals .
  • N-end rule While the N-end rule has been shown to operate in many different systems using engineered protein substances, the physiological role of an N-end rule pathway remains obscure. Recently, mouse and human genes encoding the recognition component of the N-end rule have been identified and cloned, indicating that the pathway plays a role in normal activity of cells. In muscle tissue, moreover, the N- end rule has recently also been shown to be responsible for up to 60% of the ATP-dependent degradation of soluble proteins. In muscle, this process appears to be tightly regulated to maintain the balance between the rates of protein synthesis and degradation that determines its size and functional capacity. Increased proteolysis is the major cause of rapid muscle wasting seen in many pathological states including, for example, sepsis, cancer cachexia, metabolic acidosis, fasting and diabetes.
  • Intracellular expression and targeting of recombinant antibodies can be used to block biological functions or confer new phenotypic traits, such as viral resistance.
  • the treatment of cancer, denervation atrophy and infectious disease are among several possible therapeutic applications of ectopic antibody expression.
  • Intracellular targets for inactivation by antibodies include retrovirally- infected cells, such as HIV- infected cells, where the targets are the virally-encoded protein; for example, one can use antibodies against structural proteins, such as the envelope glycoproteins and gag proteins.
  • Other preferred targets include oncogenes, such as growth factor receptors, receptors, growth factors, and the like.
  • Ectopic antibody expression can be used to test causes of disease by creating transgenic animals which generate such antibodies.
  • the constant domains of antibody chains perform functions that are neither needed nor necessarily exploited for intracellular immunization.
  • simpler antibody forms such as Fab or single-chain Fv (ScFv) fragments, can be used, as well as whole antibodies for intracellular targeting.
  • Antibodies are generally targeted for secretion by the N-terminal hydrophobic leader sequences that are cleaved off as the molecule traverses the endoplasmic reticulum (ER) . Removal of the hydrophobic leader sequence of the antibody chain, or its substitution with a hydrophilic sequence, prevents translocation of the antibody into the ER and restricts it to the cytosol. If a nuclear localization signal or mitochondrial targeting signal is incorporated, then antibodies can also be localized to the nucleus or the mitochondria .
  • a single-chain antibody or single-chain Fv incorporates the complete antibody binding portion of an antibody in a single polypeptide chain of minimal size, e.g., with an approximate molecular weight of 26,000.
  • the antigen- combining site is part of the Fv region, which is composed of the V H and V L variable domains on separate heavy and light chains. Efforts over two decades have indicated that Fv fragments can only rarely be prepared from IgG and IgA antibodies by proteolytic dissection.
  • the first step in this generation involves obtaining the genes encoding V H and V L domains with the desired binding properties. These V genes may be isolated from a specific hybridoma cell line, selected from a combinatorial V-gene library, made by V gene synthesis or generated by phage display.
  • the single-chain Fv is formed by connecting the component V genes with an oligonucleotide that encodes an appropriately-designed linker peptide, such as Gly4-Ser3 (SEQ ID NO:l) .
  • the linker bridges the C- terminus of the first V region and the N-terminus of the second, ordered as either V H -linker-V L or V L -linker-V H .
  • the scFv binding site can faithfully replicate both the affinity and specificity of this parent antibody combining site.
  • the half-life of an antibody chain depends on the intracellular compartment in which it is located. Secreted antibodies and antibody domains are generally stable. ScFvs which have been retained in the endoplasmic reticulum are, however, rapidly degraded. Similarly, the half-life of antibodies in the cytosol appears to be rather short, particularly in the absence of the antigen. This instability presents significant limitations in the use of intracellular immunization to block antigen function, particularly when high levels of expression are required over a long period. Varshavsky and coworkers have investigated means for varying the half-lives of a variety of proteins. For example, Baker et al, 5,766,927, disclose inhibitors of protein degradation in living cells using dipeptides .
  • the half-life of intracellular proteins is increased in living cells by contacting the cells with a dipeptide regulator having an amino-terminal amino acid residue which is the same or similar to the amino terminal residue of the intracellular protein.
  • a DNA construct which includes a nucleotide sequence encoding the desired amino acid sequence of the regulator can be introduced into the cell in which inhibition of the degradation of a specific type or class of proteins is desired. It thus acts as a competitive inhibitor for the degron protein which carries degradation of the proteins to be protected.
  • Varshavsky et al, 5,763,212; Wu et al, 5,538,862; and Wu et al, 5,705,387 disclose a method for regulating degradation of a recombinant protein using a heat activated degron, i.e., a destabilizing N-terminal amino acid residue which becomes a substrate of the N-end rule pathway only at temperatures high enough to result in at least partial unfolding of the protein.
  • the DNA encoding the heat- inducible N-degron module can be linked covalently at its 3' end to the 5' end of a DNA sequence encoding a protein or peptide of interest.
  • the heat- inducible N-degron module and any protein or peptide linked to the C-terminus of the heat-inducible N-degron module are rapidly degraded by enzymatic components of the N-end rule proteolytic pathway only after the degron has been activated by heating.
  • degradation of a protein bearing an N- degron can be inhibited by prebinding the protein with a low molecular mass ligand which binds to the protein with high affinity.
  • Another way to regulate degradation of a recombinant protein in a cell is to transform the cell with an expression construct encoding a fusion protein comprising the protein of interest linked at its N-terminus to an N- degron. The cell is then contacted with an inhibitor at a concentration sufficient to achieve a predetermined intracellular concentration. Without the inhibitor, degradation of the expressed fusion protein within the cell would result. When degradation is desired, the administration of inhibitor is stopped.
  • Baker et al patents 5,683,904; 5,494,818; and 5,212,058 disclose ubiquitin-specific proteases which specifically cleave at the C-terminus of the ubiquitin moiety in a ubiquitin fusion protein.
  • a ubiquitin fusion protein can be a naturally-occurring fusion protein or a fusion protein produced by recombinant DNA technology. Bachmair et al, 5,646,017; 5,496,721; 5,196,321;
  • 5,132,213; and 5,093,242 disclose methods of designing or modifying protein structure at the protein or genetic level to produce the specified amino-termini in vivo or in vi tro .
  • Genes encoding the proteins can be made to encode an amino acid of the desired class at the amino- terminus so that the expressed protein exhibits a predetermined amino-terminal structure which renders it either metabolically stable or unstable with respect to the N-end rule pathway of proteolytic degradation.
  • Conventional techniques of site-directed mutagenesis for addition or substitution of appropriate codons to the 5' end of an isolated or synthesized gene can be used to provide a desired amino-terminal structure for the encoded protein. So that the protein expressed has the desired amino acid at its amino- terminus, the appropriate codon for a stabilizing amino acid can be inserted or built into the amino-terminus of the protein-encoding sequence.
  • the protein having a predetermined amino- terminal amino acid residue is produced by expressing the protein or polypeptide in a host cell as a fusion protein wherein the amino terminus of the protein or polypeptide is fused to ubiquitin, and the fusion protein is specifically cleavable by a protease at the junction of ubiquitin with the amino-terminal amino acid residue of the protein or polypeptide.
  • the fusion protein is contacted with an extract containing a protease which specifically cleaves the ubiquitin fusion protein at the junction of ubiquitin and the amino-terminal amino acid residue of the protein or polypeptide.
  • a fusion protein including the desired amino acid terminal sequence fused to ubiquitin is produced, and then a protease which cleaves at the ubiquitin site is used to cleave ubiquitin from the protein of interest .
  • lactacystin is an irreversible inhibitor that reacts selectively with active site threonine residues in proteasomes to block the bulk of protein degradation in mammalian cells.
  • Carbobenzoxyl-leucinyl-leucinyl-leucinyl is a membrane permeant peptide aldehyde that binds reversibly to multiple active sites in proteasomes.
  • a major impediment to the development of effective gene therapy using intracellular antibody expression is the inability to achieve a high level of expression in recipient cells.
  • artificially-engineered proteins such as single-chain antibodies which are unstable in their free form, are produced intracellularly by expressing the single-chain antibodies with stabilizing residues.
  • the proteins are stabilized against proteolysis .
  • the present invention is intended to solve the problem of the instability of ectopic antibodies in cells.
  • scFv's are intrinsically unstable
  • a search of protein sequence databases with immunoglobulin heavy and light variable chain sequences was conducted. This search surprisingly showed that the vast majority of such sequences possess destabilizing residues at the start of the variable region. The residues become destabilizing when they are exposed as the free N-terminal residues.
  • the initiator methionine residues of the genetically engineered antibodies are somehow removed intracellularly, perhaps by the action of a methionine aminopeptidase enzyme or similar cleaving enzymes.
  • the in vivo half-life of an intracellular protein is a function of its N-terminal amino acid residue.
  • the present invention provides a technique for generating proteins or peptides with a specified N-terminus in vivo or in vitro .
  • amino acid exposed at the N- terminus of an intracellular protein has been shown to be one crucial determinant that specifies whether a protein will be long- or short-lived in vivo, and particularly intracellularly.
  • individual amino acids can be categorized as either stabilizing or destabilizing amino acids with respect to the half-life they confer upon a protein when exposed at the protein's N-terminus.
  • Destabilizing amino acid residues confer short half-lives, down to a few minutes for some of the destabilizing amino acids, while stabilizing amino acid residues confer long half-lives of many hours.
  • the N-terminus of a single-chain antibody can be designed to increase the intracellular half-life of the single-chain antibody when it is expressed in the cell.
  • Genes encoding single-chain antibodies are made to encode an amino acid of the desired class at the N-terminus, after the initiation methionine, so that the expressed single-chain antibody exhibits a predetermined amino-terminal structure which renders it metabolically stable with respect to the N-end rule of proteolytic degradation once the methionine is metabolically removed.
  • the single chain antibody can also be stabilized by fusing it to a peptide sequence containing a stabilizing residue at the N-terminus, after the initiation methionine.
  • the stabilizing fusion protein or peptide sequence is attached upstream of the native destabilizing residue at the N-terminus of the antibody region.
  • the stabilon is preferably linked to the antibody through a protease-sensitive linker region that can be cleaved by specific restriction proteases.
  • Restriction proteases have well defined recognition signals that are cleaved within the target substrates. Restriction proteases have been used in a number of ways to cleave fusion proteins, for example, in vector targeting. In this case a retroviral vector is fused with a ligand such as Epidermal Growth Factor that binds to receptors on human cells.
  • restriction proteases are Factor Xa (Ile-Glu-Gly-Arg) (SEQ ID NO:35); Enterokinase (Aps4-lys) (SEQ ID NO: 36) ; and matrix metalloproteases MMP (Pro-Leu-Gly-Leu-Trp-Ala) (SEQ ID NO: 37) .
  • the restriction protease can be an endogenous protein in the cell in which the single-chain antibody is expressed or the product of an inducible gene that is co-transfected with the stabilon fusion antibody protein under the control of an inducible promoter.
  • the removal of the stabilon moiety by the restriction protease renders the single chain antibody susceptible to proteolysis by the N-end rule pathway.
  • the amino acids which produce stable N-terminals are glycine, methionine, valine and serine .
  • methionine may be metabolically removed intracellularly, it is preferred to use a stable N-terminal residue other than methionine.
  • the stabilon may be a single stable N-terminal amino acid residue, such as glycine, valine or serine, it is preferably a peptide sequence consisting of two or more stable residues, preferably no more than 10.
  • the peptide may include glycine, valine, serine and methionine in any order, it is preferred that the peptide comprise multiple repeats of the same stable amino acid residue such as, for example, Gly 10 (SEQ ID NO:32), Val 4 (SEQ ID NO:33), Ser 7 (SEQ ID NO:34), etc.
  • Another way to modulate the half-life of an intracellular single chain antibody besides use of a proteolysis-sensitive linker is to use a stabilon comprising multiple repeats of methionine residue.
  • a single chain antibody whose N-terminal comprises ten methionine residues, rather than one would be expected to have a half-life ten times as long as the half-life of a single chain antibody having only the initiator methionine. This is because the mechanism by which the initiator methionine is metabolically removed must be repeated ten- times before the destabilizing residue becomes freely available at the N-terminal.
  • the present invention is preferably directed toward improving the stability of intracellularly expressed single chain antibodies
  • the concept of using a stabilon connected by a proteolysis-sensitive linker to a destabilizing sequence, so that degradation can be initiated upon induction of expression of a restriction protease by means of an inducible promoter has more general applicability. It may be applicable to any protein, whether or not it is initially unstable. Thus, for example, if it is desired to intracellularly express a desired recombinant protein and subsequently have it removed from the cell at will, one can engineer the protein so as to fuse it to a destabilizing residue or sequence connected to a stabilon by means of a proteolysis-sensitive linker.
  • the protein will be stable. Naturally, one would have to verify that the biological activity of the protein of interest is not affected by adding such a fusion sequence to the N-terminus thereof.
  • the inducible promoter of the restriction protease may be activated or an endogenous inducible restriction protease may be activated such that the stabilon will be removed, leaving a destabilizing sequence fused to the N-terminus of the protein; such a destabilizing sequence will cause the protein to be degraded by the N-end rule pathway.
  • Inducible promoters are well-known and used predominantly in transgenic animal technology and in regulating intracellular expression for in vi tro type of experiments.
  • a tetracycline inducible promoter such as the tetracycline responsive promoters taught in U.S. Patent 5,650,298.
  • the tetracycline inducible promoter will be activated, releasing the restriction protease and commencing a chain of reactions which ultimately causes elimination of the genetically engineered protein.
  • Gene therapy can be used to treat illnesses and conditions which have a genetic or metabolic cause or which result from infection. In many of these conditions, cells are either deficient in a protein or produce a dysfunctional protein. Gene therapy treats these conditions by introducing into the appropriate cell DNA coding for the normal gene product or DNA coding for a factor that can neutralize or block the activity of an abnormal functioning molecule. Gene therapy can be affected by receptor-mediated gene delivery transkaryotic implantation, viral shuttle vectors, such as retroviral gene transfer, etc.
  • Viruses have been used to deliver DNA in gene therapy.
  • types of gene therapy in which viruses have been used for transfer are HSV-1 vector mediated transfer of BDNF into cerebellar granule cells, Alonso et al
  • herpes simplex virus type 1 carries a large number of viral functions which can be replaced by foreign genes to create a vector for gene therapy applications .
  • a DNA sequence encoding the single chain antibody, or other protein of interest is ligated to a DNA sequence encoding the amino acid sequence of the stabilon which provides N-terminal stability, and also optionally to DNA encoding the proteolysis-sensitive linker between the DNA encoding the stabilon and the DNA encoding the protein of interest.
  • This DNA sequence is then operably linked to a promoter which will permit expression of the amino acid sequence encoded by the
  • DNA intracellularly into the cell or cells of interest is delivered to the cells of interest either by direct injection or, more preferably, by the use of a conventional vector which is selected so as to carry the DNA selectively into the cells of interest.
  • the protein with the stabilizing N-terminal sequence is expressed intracellularly in the targeted cells. If the protein of interest is a single chain antibody, or an antibody binding region or other ligand binding polypeptide, the expressed protein will then bind to the target antigen or ligand within the cell.
  • the protein bearing the N-terminal stabilon is deemed a "protected" protein for purposes of the present invention.
  • the N-termini of the antibodies are stabilized by adding one or more of glycine, methionine, serine and valine.
  • the vector carrying the engineered DNA is further engineered to carry a DNA segment encoding a second protein along with an associated promoter.
  • the second protein will be the restriction protease for which the proteolysis-sensitive linker is designed.
  • the promoter will be an inducible promoter which will cause the restriction protease to be expressed only upon being subjected to the predetermined induction signal. While this second DNA sequence is preferably located on the same vector, it may also be introduced on a separate vector, either simultaneously with the introduction of the initial vector or separately therefrom.
  • the promoter for the restriction protease may be a constitutive promoter and the DNA encoding the restriction protease enzyme and the promoter DNA operably linked thereto may be introduced at the time that it is desired to remove the initially introduced protein.
  • An advantage of causing the single chain antibody to be removed at will by means of the N-end rule degradation pathway is that it is expected that not only will the antibody be degraded but also the antigen to which it has bound. Thus, it is expected that not only will free antibody be degraded by this pathway, but entire immunoconjugates, thus causing removal of predetermined antigens or proteins bearing predetermined antigens which one wishes to remove from the cellular milieu.
  • the "antibody gene" of the antibody cassettes utilizes a cDNA encoding heavy chain variable (V H ) and light chain variable (V L ) domains of an antibody which can be connected at the DNA level by an appropriate oligonucleotide to bridge the two variable domains, which on translation produces a single polypeptide (referred to as a single-chain variable fragment (sFv) ) capable of binding to a target, such as a protein.
  • the antibody gene does not encode an operable secretory sequence, and, thus, the expressed antibody remains within the cell.
  • a nucleotide sequence encoding an intracellular localization amino acid sequence may be used.
  • antibodies which are useful in treating Alzheimer's Disease may be administered in this manner.
  • Other antibodies can be introduced into a cell for intracellular expression also.
  • Such antibodies may be those against structural proteins, such as envelope glycoprotein and gag protein, against tat, rev, nef, vpu and/or vpx regulatory proteins.
  • Other targets include oncogenes, such as trans-membrane growth factor receptors, receptors, growth factors, membrane associated guanine nucleotide binding proteins, etc.
  • a DNA sequence containing nucleotides coding for a protein or peptide of interest, as well as nucleotides which code for an amino acid sequence at the N-terminus of the protein or peptide selected to increase the half-life of the protein or peptide are operably linked to a promoter that will permit expression of the protein or peptide in the cell(s) of interest (protein or peptide cassette).
  • the protein or peptide with the protected N-terminus is expressed intracellularly.
  • a DNA sequence containing a sufficient number of nucleotides to code for the portion of an antibody capable of binding to a target, as well as nucleotides which code for an amino acid sequence at the N- terminus of the antibody portion selected to increase the half-life of the antibody portion, are operably linked to a promoter that will permit expression of the antibody in the cell(s) of interest (antibody cassette), and the cassette is delivered to a cell. Thereafter, the antibody with the protected N-terminus is expressed intracellularly and binds to the target, thereby disrupting the target from its normal actions.
  • the present invention provides a method of targeting a particular molecule (target molecule) , preferably a receptor site or an undesired protein.
  • This method comprises the intracellular expression of a single- chain antibody which has been stabilized and which is capable of binding to the specific target (e.g., a target protein), wherein the vector encoding the antibody preferably does not contain sequences coding for its secretion.
  • target molecule preferably a receptor site or an undesired protein.
  • the single-chain antibody can be stabilized against proteolysis by inhibitors of the proteolytic pathway. While any substance that blocks the recognition of the substrate of other components of the ubiquitin pathway can be used, which substances are well known to those skilled in the art, the most common substances are those which block the proteasome, thus preventing the degradative final step of the ubiquitin pathway.
  • the preferred embodiment of the present invention is directed to a method and a vector for modulating the degradability of intracellularly targeted antibodies and fragments thereof, particularly single-chain antibodies, as described below in detail.
  • the present invention is also generally applicable to a method and a vector for modulating the degradability of any intracellularly expressed protein or peptide as would be well appreciated by those in the art.
  • a protein or peptide which includes an antibody or a fragment thereof, that is unstable intracellularly with respect to the N-end rule of proteolytic degradation as reported by Varshavsky (1996) can be stabilized by inserting into the gene encoding the protein or peptide a nucleotide sequence encoding a stabilon sequence immediately after the N-terminal ATC encoding the initiation methionine residue. In this way, even if the N-terminal initiation methionine residue is removed, the presence of a stabilon sequence at the new N-terminus of the protein or peptide stabilizes the protein or peptide with respect to the N-end rule of proteolytic degradation.
  • stabilon or “stabilon sequence” is intended to mean an amino acid sequence of one or more amino acid residues, preferably two to ten amino acid residues, which are stabilizing N-terminal residues, such as Gly, Val, Ser and Met, with respect to the N-end rule of proteolytic degradation according to Varshavsky (1996) .
  • the stabilon is a single amino acid residue, it is preferably Gly, Val or Ser.
  • the stabilizing residues Gly, Val, Ser and Met can be combined in any order, although it is preferred that the stabilon sequence include multiple repeats of the same stable amino acid residue, i.e., Gly 10 (SEQ ID NO:32), Val 4 (SEQ ID NO:33), Ser 7 (SEQ ID NO:34), etc. Multiple repeats of Met residues can also be used as the stabilon sequence. While it may be susceptible to successive removal of the N-terminal Met residue by the action of a methionine amino acid peptidase or a similar enzyme, it will still prolong the stability of the protein.
  • N-terminal repeats of Met residues are stabilizing to the extent that Met residues are still available to protect the N-terminus, and this is dependent on the rate at which single Met residues are successively and enzymatically removed from the N-terminus.
  • a stabilon is directed to modulating the degradability of an intracellularly expressed protein or peptide to improve its intracellular stability.
  • it is desirable at some point to remove the protein or peptide when a protein or peptide is an antibody or fragment thereof and it has had sufficient time to bind a target antigen intracellularly, then it may be desirable to degrade the antibody and thereby degrade the antigen as part of an antibody/antigen complex once the antibody has served its purpose. This is important when the antibody cannot be made inhibitory to by binding to the antigen as is often the case since the active sites of a molecule are often not accessible to the surface. That is, the antibody does not necessarily achieve its function simply by binding to the antigen. Because the active site of the antigen is usually buried and, therefore, is not immunogenic, most antibodies are not inhibitory. Degradation of the antibody/antigen complex is another way to block function of the antigen when the antibody itself is not inhibitory.
  • modulating the degradability of a protein or peptide includes the ability to later destabilize and degrade a previously stabilized or naturally stable protein or peptide.
  • the protein or peptide can be engineered so as to fuse it to a destabilizing sequence.
  • a destabilizing sequence can be joined to an N-terminal stabilon sequence by means of a protease-sensitive linker region.
  • the protein or peptide naturally contains the destabilizing N-terminal sequence
  • the protein's or peptide ' s own destabilizing sequence can be joined at the N-terminus to a stabilon sequence by means of protease-sensitive linker region.
  • protease-sensitive linker region can be cleaved by an appropriate restriction protease whose expression can be placed under the control of an inducible promoter.
  • the inducible promoter of the restriction protease or an endogenous inducible restriction protease may be activated so that the stabilon is removed by cleavage at the protease-sensitive linker region, thereby exposing the destabilizing sequence at the N-terminus where it will cause the protein to be degraded by the N-end rule pathway.
  • the protease-sensitive linker region is intended to be a sequence which is recognized and cleaved by a restriction protease whereby the protease-sensitive linker region is removed to leave a destabilizing sequence at the N-terminal end of the cleavage site.
  • restriction proteases and their recognition and cleavage sites/sequences that can be used according to the present invention are well known in the art.
  • Non-limiting examples of restriction proteases are Factor Xa (Ile-Glu-Gly-Arg) (SEQ ID NO: 35) ; Enterokinase (Aps4-lys) (SEQ ID NO: 36) ; and matrix metalloproteases MMP (Pro-Leu-Gly- Leu-Trp-Ala) (SEQ ID NO: 37) .
  • a protein or peptide that is otherwise naturally stable intracellularly can be altered by effectively inserting a stabilon sequence, joined to a destabilizing sequence by a protease-sensitive linker region, between the N-terminal initiation Met residue and the rest of the protein or peptide as a unit by recombinant DNA methodology.
  • Such an alteration would provide the ability to remove the stabilon by cleavage at the protease-sensitive linker region with an appropriate restriction protease and expose an artificial destabilizing sequence, which is recognized by the N-end rule proteolytic pathway and positioned N-terminal to an otherwise stabilizing sequence of the naturally stable protein or peptide .
  • destabilizing sequence is intended to mean one or more amino acid residues selected from Arg, Lys, His, Asn, Gin, Asp, Glu and Cys, preferably Arg, Lys and His, according to the destabilizing residues reported by Varshavsky (1996) .
  • the recombinant DNA molecules, where a promoter is operably linked to a gene encoding a desired protein or peptide for intracellular expression is altered so as to insert a sequence encoding (1) a stabilon, (2) a stabilon joined to a protease-sensitive linker region, or (3) a stabilon joined to a destabilizing sequence through a protease-sensitive linker region, can be readily constructed in view of the high level of skill of those in the art with guidance from a wealth of standard reference texts on recombinant DNA technology, such as Ausubel et al (1987- 1998) , Maniatis et al (1989) , Watson et al (1987) , Darnell et al (1985) , Lewin (1985) , Old et al (1981) , Berger et al (1987) , etc.
  • a DNA molecule is said to be "capable of expressing" a protein or peptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information, and such sequences are “operably linked” to nucleotide sequences which encode the protein or peptide.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression.
  • the regulatory regions needed for gene expression in general include a promoter region, as well as the DNA sequences which, when transcribed into RNA, signal the initiation of protein synthesis. These regions normally include those 5'-non-coding sequences involved with initiation of transcription and translation.
  • a promoter region is, thus, operably linked to a DNA sequence if the promoter is capable of effecting transcription of that DNA sequences.
  • a "promoter sequence” is the sequence of the promoter which is found on the DNA and is transcribed by the RNA polymerase .
  • Suitable promoters which are intended to include inducible promoters, include, but are not limited to, the metallothionine promoter (Brinster et al, 1982) , the heat shock protein promoter, elastase I gene control region, which is active in pancreatic acinar cells (Swift et al, 1984; Ornitz et al, 1986; MacDonald, 1987), the insulin gene control region, which is active in pancreatic beta cells (Hanahan, 1985) , the immunoglobulin gene control region, which is active in lymphoid cells (Grosschedl et al, 1984; Adames et al, 1985; Alexander et al, 1987) , the mouse mammary tumor virus control region, which is active in testicular, breast, lymphoid and mast cells (Leder et al, 1986), the albumin gene control region, which is active in the liver (Pinkert et al, 1987) , the alpha-feto
  • Vectors for delivery of a gene encoding a desired intracellularly expressed protein or peptide operably linked to a promoter and a second gene which encodes a restriction protease under the control of an operably linked inducible promoter, either together on the same vector or separately, are well known in the art.
  • These vectors include mammalian expression vectors, such as a herpes vector, an adenovirus vector, a pox vector, a retroviral vector and other vectors taught above as vectors for gene therapy.
  • the vector carrying a gene encoding such a desired protein or peptide is preferably also engineered to carry a gene encoding a restriction protease for which the protease-sensitive linker region is designed.
  • the gene encoding the restriction protease may be located on a separate vector which may be introduced into the host cell simultaneously with the vector carrying the gene encoding the desired intracellularly expressed protein or peptide or separately therefrom.
  • an inducible promoter operably linked to the protease gene is used.
  • an inducible promoter include mouse mammary tumor virus promoter (induced by dexamethasone) , metallothionine promoter (induced by zinc) , yeast gal4 promoter (induced by galactose) , heat shock gene promoter, steroid hormone-responsive promoter, and tetracycline-responsive promoters.
  • the lac repressor/operator inducer system of E. coli has been reported to function in eukaryotic cells.
  • a suitable inducible promoter can be selected by those of skill in the art, taking into account that the inducer or inducing event should not be significantly toxic or harmful to the host in which it is administered.
  • the "antibody gene" of the antibody cassette uses a cDNA encoding heavy chain variable and light chain variable domains of an antibody which are connected at the DNA level by an appropriate oligonucleotide as a bridge of the two variable domains, which on translation form a single polypeptide (e.g., a single-chain variable fragment (sFv) ) which is capable of binding to a target, such as a protein.
  • the single-chain variable fragment is substituted at the N-terminus in order to increase the half-life of the single-chain variable fragment once the fragment has been formed in the cell .
  • the term "antibody” refers to at least that portion of an immunoglobulin capable of selectively binding to a target, such as an antigen or other protein.
  • the term includes single-chain antibodies.
  • the antibody is expressed from a DNA sequence which contains a sufficient number of nucleotides coding for the portion of antibody capable of binding to the target, as well as an amino acid sequence which extends the half-life of the antibody, i.e., which protects the antibody.
  • This DNA sequence is also referred to as the antibody gene.
  • the antibody gene is operably linked to a promoter to permit expression of the protected antibody in the cells of interest. Promoters are well known in the art and can readily be selected depending upon the cell type to be targeted. For example, when the function of a target protein is a result of its overexpression, by "turning on the promoter" one can selectively cause expression of the antibody.
  • the entire sequence of antibody gene and promoter is also referred to as an antibody cassette.
  • the antibody cassette is delivered to the cell using an appropriate vector to carry the cassette into the cell(s) of interest. Once the cassette is introduced into the cell, the protected antibody is expressed in the cell. The expressed protected antibody can then bind to the target antigen. Because the antibody is protected, there is sufficient time to bind to the target antigen before proteolysis destroys the antibody.
  • proteins and peptides that otherwise would be immediately subjected to proteolysis in the cytosol can be expressed in protected form so that they are available for a sufficient length of time to bind their target antigen.
  • Antibodies used in the present invention can be antibodies to almost any type of biological molecule.
  • the antibodies used can be antibodies to intermediate metabolites, sugars, lipids, autocoids and hormones, as well as macromolecules, such as complex carbohydrates, phospholipids, nucleic acids, such as RNA and DNA, and proteins.
  • macromolecules such as complex carbohydrates, phospholipids, nucleic acids, such as RNA and DNA, and proteins.
  • the preferred target molecules for antibodies used in the present invention include proteins, RNA, DNA and haptens . More preferably, the targets are proteins, RNA and DNA. Still more preferably, the target is a protein. It should be understood, however, that the present invention is not directed to such antibodies per se, but to means for protecting them against prompt intracellular degradation, and the stabilized antibodies so produced.
  • compositions of the present invention include a recombinant DNA molecule containing a gene for the antibody or molecule of interest along with a nucleotide sequence for the amino acid at the N-terminus which prolongs the half-life of the antibody or molecule of interest in vivo, in association with a means of gene delivery.
  • an antibody may preferably be an antibody such as that disclosed in WO 98-44955.
  • Such antibodies are useful, for example, as medicaments for treating diseases or conditions of the central nervous system, such as Alzheimer's Disease.
  • the present invention is particularly useful for producing protected single-chain antibodies.
  • These single- chain antibodies can be single-chain composite polypeptides having end-specific peptide binding capability and comprising a pair of amino acid sequences homologous or analogous to the variable regions of an immunoglobulin light and heavy chain (linked V H -V L or single-chain Fv) , and including the amino acid(s) at the N-terminus which impart stability to the antibodies in vivo.
  • V H and V L may copy natural monoclonal antibody sequences, or none or both of the chains may comprise a CDR-FR construct of the type described in U.S. Patent 5,091,513.
  • the separate polypeptides analogous to the variable regions of the light and heavy chains are held together by a peptide linker.
  • Single-chain FV single-chain FV
  • Methods of production of such single-chain antibodies may be accomplished in accordance with the methods described, for example, in U.S. Patents 4,946,778; 5,091,513; and 5,096,815.
  • nucleotide sequences are included in the gene which ensure that the antibody produced will have the entire N-terminus.
  • the antigen binding or variable region is formed by the interaction of the variable heavy and variable light domains at the amino termini of the chains.
  • the smallest fragment containing a complete binding site is referred to as Fv and is a heterodimer of the variable light and variable heavy domains.
  • Fv fragment containing a complete binding site
  • a gene coding for such an antibody can be used as long as the fragment retains sufficient binding ability as compared to the parent antibody.
  • the host ' s immune system is used to prepare the antibody which will bind to a specific molecule, such as a target protein, by standard immunological techniques.
  • the antibody is prepared using the protein, or an immunogenic fragment thereof, or a peptide chemically synthesized based upon such protein. Any of these sequences can optionally be conjugated to keyhole limpet hemocyanin and used to raise an antibody in another mammal, such as mice, rabbits, rats and hamsters. Thereafter, the animals are sacrificed, and their spleens are obtained.
  • Monoclonal antibodies are produced using conventional fusion techniques for making hybridoma cells, typically by fusing an antibody- producing cell with an immortal cell line, such as a myeloma cell, to produce the hybrid cell.
  • Antibodies are also prepared by in vi tro immunization, such as using a culture of spleen cells, injecting an antigen, and then screening for an antibody produced to the antigen.
  • spleen cells are harvested and incubated in medium together with the desired antigen at a concentration typically of about 1 ⁇ g/mL. Thereafter, an adjuvant, depending upon the results of the filter immunoplaque assay, is added to the cell culture. The spleen cells are incubated with the antigen for about three to five days and then harvested.
  • a label for the antibodies such as a labelled second antibody, such as rabbit anti-mouse IgA, IgG or IgM.
  • a reagent such as horseradish peroxidase-avidin.
  • the DNA encoding any antibody or active fragment that is specific for the antigen of interest can be used in generating the recombinant antibody-encoding DNA molecules for use according to the present invention.
  • the C-terminal end-specific monoclonal antibodies disclosed in WO 96/25435 may be used to obtain the recombinant antibody-encoding DNA molecules according to the present invention.
  • mRNA may be isolated from hybridomas producing monoclonal antibodies determined to be selective for the antigen of interest.
  • cDNA is synthesized, and the nucleotide sequence encoding the variable domains of the monoclonal antibody may then be cloned using the polymerase chain reaction (PCR) with primers based on the conserved sequences at each end of the nucleotide sequences encoding the V domains of immunoglobulin heavy chain and light chain.
  • PCR polymerase chain reaction
  • a recombinant gene encoding a recombinant single- chain Fv antibody molecule is constructed, for example, by joining nucleotide sequences encoding the V H and V L domains with a nucleotide sequence encoding a peptide interchain linker (Biocca et al, 1993; Duan et al, 1994; Mhashilkar et al, 1995; Marasco et al, 1993; Richardson et al, 1995; U.S.
  • a known antibody to the target protein can be used, the most preferred example being antisenilin (see WO 98-44955) .
  • a gene to at least the antigen binding portion of the antibody is synthesized.
  • the gene preferably does not contain the normal signal peptide sequences.
  • the gene also encodes an intracellular localization sequence, such as one for the endoplasmic «t:eticulum, nucleus, nucleolar, etc.
  • a localization sequence such as the KDEL sequence, is used.
  • the antibody gene preferably also does not encode functional secretory sequences.
  • Variable light and variable heavy domain genes can be constructed using any of the antibodies obtained as above. Alternatively, commercially available variable heavy and variable light chain domain libraries can be used. In referring to the antibody gene, this term can encompass genes for both the heavy chain and light chain regions of the antibody gene.
  • the gene is operably linked to at least one promoter which results in its expression. Any well-known promoter that permits expression in mammalian cells can be used. These promoters are well known in the art and include promoters, such as CMV, a viral LTR, such as the rous sarcoma virus LTR, HIV-LTR, LTLV-1LTR, the SV40 early promoter, E.
  • coli lac UV5 promoter and the herpes simplex thymidine kinase (tk) promoter.
  • the combination of the antibody DNA sequence and the promoter is referred to as the antibody cassette.
  • Phage Display technology The production of recombinant antibodies is much faster compared to conventional antibody production and they can be generated against an enormous number of antigens. In contrast, in the conventional method, many antigens prove to be non- immunogenic or extremely toxic, and therefore cannot be used to generate antibodies in animals. Moreover, affinity maturation (i.e., increasing the affinity and specificity) of recombinant antibodies is very simple and relatively fast. Finally, large numbers of different antibodies against a specific antigen can be generated in one selection procedure.
  • Phage Display libraries To generate recombinant monoclonal antibodies one can use various methods all based on Phage Display libraries to generate a large pool of antibodies with different antigen recognition sites.
  • a library can be made in several ways: One can generate a synthetic repertoire by cloning synthetic CDR3 regions in a pool of heavy chain germline genes and thus generating a large antibody repertoire, from which recombinant antibody fragments with various specificities can be selected.
  • One can use the lymphocyte pool of humans as starting material for the construction of an antibody library. It is possible to construct naive repertoires of human IgM antibodies and thus create a human library of large diversity. This method has been widely used successfully to select a large number of antibodies against different antigens. Another possibility is to prepare so- called patient libraries.
  • IgG libraries can be generated, which will contain IgG antibodies of high specificity and with high affinity. Construction of specialized libraries can be used to generate antibodies from patients with, for example, certain tumors for selection of anti-tumor activity.
  • PDGF denotes platelet-derived growth factor
  • FHJF denotes fibroblast growth factor
  • EFG denotes epidermal growth factor
  • M-CSF denotes mononuclear-phagocyte growth factor.
  • the family includes src, fgr, yes, lck, hck, fyn, lyn and tkl .
  • oncogenes (sometimes referred to as ones)
  • some oncogenes undergo a mutation from a proto-onc (normal gene for normal protein) to an one (gene whose protein can cause malignant transformation) which appears to result in malignant transformation of cells.
  • point mutations at the ras gene at the codons for the ras p21 at residue positions 12, 13 and 61 have resulted in mutant ras p21 proteins which are associated with various cancers.
  • Antibodies specific to many of these ras mutants are known.
  • expression of viral proteins can lead to disease resulting in illness and even death.
  • the virus can be either an RNA or a DNA virus.
  • RNA virus retroviruses
  • retroviruses are typically classified as being part of one of three subfamilies, namely, oncoviruses, spumaviruses, and lentiviruses .
  • Infection by an oncovirus is typically associated with malignant disorders.
  • the viral proteins encoded include the gag, pol and envelope proteins.
  • the virus contains oncogenes which encode a protein capable of malignant transformation of a cell in culture. Lentiviruses result in infection which is generally slow and cause chronic debilitating diseases after a long latency period.
  • genes encoding the gag, pol and envelope structure proteins they also encode a variety of regulatory proteins .
  • the RNA and/or DNA of the virus can take over the cell machinery to produce the virally-encoded protein.
  • HTLV-1 is a retrovirus which is the etiological agent of adult T-cell leukemia-lymphoma (ATLL) , an aggressive neoplasm of CD4 + T-cells.
  • ATLL adult T-cell leukemia-lymphoma
  • the viral proteins expressed by such a virus result in the transformation of the cell .
  • the tax and rex gene and gene products appear to be significant with respect to tumorigenicity . Thus, they are a preferred grouping of target molecules.
  • HIV comprises a family of lentiviruses, including
  • HIV-1 and HIV-2 which are the etiological agents of immunodeficiency diseases, such as the acquired immune deficiency syndrome (AIDS) and related disorders (Barre- Sinoussi et al, 1983; Gallo et al, 1984; Levy et al, 1984).
  • the Epstein-Barr virus has been linked to a number of tumors, such as selected outbreaks of Burkitt ' s lymphoma, nasopharyngeal cancer, and B-lymphomas in immunosuppressed individuals (zur Hausen, 1991) .
  • Hepatitis B virus has been linked to hepatocellular cancer (zur Hausen, 1991) .
  • the X open reading frame of the virus seems to be involved. Accordingly, an antibody that targets this region or expression products from this region would be preferred in the present method.
  • Papillomaviruses have been linked to anogenital cancer.
  • the E6 and E7 genes appear to be involved and are good targets for a stabilized antibody according to the present invention.
  • nucleic acid such as a DNA provirus
  • RNA of the virus By binding to the RNA of the virus, one can interfere with its expression of viral protein.
  • Anti-nucleotide antibodies have been extensively studied ⁇ cf . PCT WO94/02610) , and the antibodies have the same basic features .
  • antibodies can be produced and/or screened by standard techniques, such as by using RNA, to create a library containing antibodies.
  • RNA messenger RNA
  • This nucleic acid sequence is present in the 5'LTR and is responsive to tat, resulting in enhanced expression of viral protein.
  • the proteins of the present invention are available for sufficient time in the desired location to perform their desired function.
  • binding to a protein that must be further processed can significantly reduce the cleavage of the protein into its active components.
  • a viral envelope protein e.g., HIV gpl60
  • the capsid protein e.g., the HIV capsid protein
  • the capsid protein is modified co-translationally by addition of the fatty acid myristic acid. It appears that myristic acid is involved in the attachment of the capsid precursor protein to the inner surface of cells. In HIV proviruses, which have been altered so that they are not capable of adding this myristic acid, the provirus is not infective.
  • proteins such as surface receptors, transmembrane proteins, etc.
  • these proteins include neu and envelope glycoproteins, such as those of the primate lentiviruses, such as HIV-1 or
  • HIV-2 By using antibodies that can be delivered to such a region of the cell and be specific for a particular protein, one can disrupt the function of the protein without disrupting other cellular functions.
  • the PDGF- /2 and FGF-like factors produced by sis and int-2 pass through the endoplasmic reticulum. These factors are involved in many cancers.
  • the present method of intracellularly generating stable antibodies can be used to disrupt a function that is undesirable at a particular time.
  • the MHC class I and class II molecules are important in the immune system's recognition of antigens.
  • immune recognition particularly from MHC class II molecules, can cause problems, such as in organ transplants.
  • the host immune response can be down-regulated.
  • These molecules can preferably be targeted at different points in their processing pathway.
  • this method can be designed by the skilled artisan without undue experimentation.
  • the HIV-1 envelope gene directs the synthesis of a precursor polyglycoprotein termed gpl60.
  • This protein is modified by addition of multiple N- linked sugars as it enters the endoplasmic reticulum.
  • the glycosylated envelope protein precursor is then cleaved within the Golgi apparatus to yield a mature envelope protein comprised of an exterior glycoprotein, gpl20, and a transmembrane protein, JP41.
  • the envelope glycoprotein complex is anchored to the virion envelope and infects the cell membrane by gp41 through non-covalent interactions. Following binding of the gpl20 exterior glycoprotein to the CD4 receptor, the fusion of viral and host cell membrane allows virus entry.
  • the fusogenic domain of the gpl20/gp41 complex is thought to reside at the amino terminus of gp41 because this region exhibits sequence homology with a fusogenic domain of other viral proteins and because mutations in this region inactivate the virus and prevent viral fusion. While the processed gpl20 and gp41 are transported to the cell surface and secreted as part of the virion of the virus, sometimes referred to as viral particles, the uncleaved gpl60 is delivered to lysosomes for degradation. The cleavage process normally is relatively inefficient.
  • the method of using stabilized intracellular antibodies to bind to the newly synthesized gpl60 in the lumen of the endoplasmic reticulum and inhibit its transport to the Golgi apparatus greatly reduces the amount of protein available for cleavage to gpl20 and gp41. Accordingly, the viral particles produced have greatly diminished amounts of gpl20 and gp41 on their surface, and these particles are not considered to be infectious.
  • This discussion of the HIV-1 gpl60/gp41 proteins exemplifies use of the present invention for other envelope proteins and processed proteins. The same techniques used herein can be adapted by known techniques based upon the present disclosure without undue experimentation.
  • HIV infection of cell cultures typically generates an acute and/or chronic infection. In both cases, virus is produced and is released by budding at the cellular membrane.
  • An acute infection is typically characterized by a cytopathic effect manifested by vacuolization of cells and formation of syncytia and consequent cell lysis.
  • tissue cultures the cytopathic effects of HIV-1 consist of multinucleated giant cell (syncytium) formation and the lysis of single cells.
  • syncytium multinucleated giant cell (syncytium) formation and the lysis of single cells.
  • Syncytium formation is mediated solely by the HIV-1 envelope protein expressed on the infected cell surface.
  • the envelope binds to the CD4 receptor present on adjacent cells and then, via a fusion reaction analogous to that involved in virus entry, the opposed cell membrane are fused, forming heterokaryons .
  • Single cell lysis also depends on effective membrane fusion induced by the envelope glycoprotein, as some mutations in the gp41 amino terminus result in replication competition viruses that are attenuated for both syncytium formation and single cell lysis. It has also been reported that amino acid changes in gpl20 which affect processing of the gpl60 precursor can decrease single cell lysis, and that single cell lysis requires adequate levels of CD4 expression, independent of the level of viral protein expression or viral DNA in the infected cell. In addition, the HIV envelope glycoprotein has been implicated by a number of other individuals in explaining the onset of the associated immunodeficiency infected individuals.
  • Siliciano et al (1988) have shown that a subset of CD4 + gpl20-specific clones manifest cytolytic activity and lyse uninfected autologous CD4 + T-cells in the presence of gpl20 in a process that is strictly dependent upon CD4 mediated uptake of gpl20 by T-cells. Since gpl20 can be shed from infected cells, this CD4 -dependent autocytolytic mechanism can contribute to the profound depletion of CD4 + T- cells in AIDS patients. Others have shown that an autoimmune idiotypic network develops in HIV-1 infections which leads to the development of autoimmune antibodies that destroy CD4 + T- cells.
  • gpl20 This autoimmune mechanism develops because of the sequence homologies between gpl20 and class II MHC molecules.
  • immunodeficiency diseases such as HIV-1
  • gpl20 has been shown to cause an increase in intracellular calcium and neuronal toxicity, an effect which might be mediated by activation of the nuclear endonuclease.
  • CD4 receptors on uninfected cells leading to an abortive cell activation and, thus, trigger apoptosis.
  • the envelope glycoprotein can act as a superantigen, binding only the variable- ⁇ region of the T- cell antigen receptor, thereby inducing massive stimulation and expansion of such T-cells, followed by deletion of energy.
  • effects associated with AIDS can be alleviated and retarded.
  • Intracellular expression of a stabilized antibody to its target results in an antibody that binds the target, e.g., envelope glycoprotein or tat protein, respectively, in the cell and prevents further processing.
  • the present method is highly specific and does not adversely affect cellular functioning.
  • a mutant envelope protein that contains a single point mutation that abolishes the protein's ability to bind to this antibody will be processed normally in cells that constitutively express the protein.
  • single-chain antibodies to other proteins will not affect the processing of the envelope protein.
  • the present method permits using an antibody specific to a particular portion and results in a process that can be tailored for specific diseases.
  • the antibody can be under the control of a promoter that is specifically activated by the target (e.g., an HIV LTR) , thereby only turning on the antibody when the target is present.
  • a promoter that is specifically activated by the target (e.g., an HIV LTR)
  • Other types of inducible promoters are known in the art and can be selected and used based upon the present disclosure.
  • the use of the present stabilized antibodies does not affect processing of other proteins.
  • the antibody to the HIV envelope glycoprotein does not bind other envelope glycoproteins and does not prevent processing of such a protein.
  • the processing of an unrelated envelope glycoprotein, such as Bunyavirus envelope glycoprotein will not be affected.
  • cytoplasmic side of a membrane receptor there is through the cytoplasmic tail that signal transduction occurs.
  • signal transduction occurs.
  • the neu/erbB-2 receptor or G protein receptor one can target the loop or cytoplasmic tail, thereby preventing such single transduction.
  • Stabilized fragments of antibodies to activated receptors such as to phosphorylated amino acids, can be used, thus reducing the pool of target receptors .
  • the stabilized antibodies bind specifically to the target, e.g., a protein, and, thus, effectively compete with other molecules that also form complexes with the protein.
  • the antibodies of the present invention must retain at least about 75% of the binding effectiveness of the complete antibody to the target, i.e., have constant as well as variable regions. More preferably, the fragments have at least 85% of the binding effectiveness of the complete antibody. Still more preferably, the fragments have at least 90% of the binding effectiveness of the complete antibody. Even more preferably, the fragments have at least 95% of the binding effectiveness of the complete antibody.
  • the method of the present invention is broadly applicable to a wide range of target molecules, including proteins, RNA, DNA, haptens, phospholipids, carbohydrates, etc.
  • the target molecules can be present in a wide range of hosts, such as animals, birds and plants.
  • the target is found in animals, including humans.
  • the species is one that has commercial importance, such as fowl, pigs, cattle, cows, sheep, etc.
  • the target molecule is found in humans.
  • antibodies are able to recognize an almost limitless number of foreign molecules, in nature, antibodies recognize structures exterior to the cell. Once synthesized, antibodies are secreted into the surrounding fluid or remain bound to the outer cell membrane. In the present invention, the stabilized antibodies are expressed and these antibodies retain the ability to specifically bind to a target intracellularly.
  • Specificity for a particular target can be obtained by using the immune system itself .
  • the target or an antigenic portion thereof is used, or a hapten-carrier complex to generate an antibody. This can be accomplished by standard techniques.
  • the antigen binding or variable region is formed by the interaction of the variable heavy (V H ) and variable light (V L ) domains at the amino termini of the chains .
  • V H variable heavy
  • V L variable light
  • the smallest fragment containing a complete binding site is referred to as Fv and is a heterodimer of the V H and V L domains.
  • Fv variable heavy chain binding domain
  • dAbs heavy chain binding domain
  • single domain antibodies a gene coding for such a stabilized antibody fragment as long as the stabilized fragment retains sufficient binding ability compared to the parent antibody.
  • the stabilized fragment contains at least a V H and V L heterodimer.
  • variable domains are each folded into a characteristic structure composed of nine strands of closely packed ⁇ -sheets. The structure is maintained despite sequence variation in the V H and V L domains.
  • Analysis of antibody primary sequence data has established the existence of two classes of variable region sequences, hypervariable sequences and framework sequences .
  • the framework sequences are responsible for the correct ⁇ -sheet folding of the V H and V L domains, and for the interchain interactions that bring the domains together.
  • Each variable domain contains three hypervariable sequences which appear as loops.
  • the six hypervariable sequences of the variable region, three from the V H and three from the V L form the antigen binding site, and are referred to as a complementarity determining region.
  • V H and the V L chain of interest it is possible to express these proteins in bacteria and rapidly test their function.
  • One method is by using hybridoma mRNA or splenic mRNA as a template for PCR amplification of such genes.
  • the binding affinity (K d ) should be at least about 10 "7 L/M, more preferably at least about 10 "8 L/M.
  • the genes encoding the light chain and the heavy chain encode a linker to make a single- chain antibody (sFv) .
  • the sFv will properly fold even under the reducing conditions sometimes encountered intracellularly.
  • the sFv typically comprises a single peptide with the sequence V H -linker-V L or V L -linker-V H .
  • the linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation.
  • the linker should be able to span the 3.5 nm distance between its points of fusion to the variable domains without distortion of the native Fv conformation.
  • the amino acid residues constituting the linker must be such that it can span this distance and should be five amino acids or larger.
  • the amino acids chosen also need to be selected so that the linker is hydrophilic so that it does not get buried in the antibody.
  • the linker should be at least about 10 residues in length. Still more preferably, it should be about 15 residues in length. While the linker should not be too short, it also should not be too long, as that can result in steric interference with the combining site. Thus, it preferably should be 24 residues or less.
  • the linker (Gly- Gly-Gly-Gly-Ser) 3 (SEQ ID NO: 2) is a preferred linker that is widely applicable to many antibodies as it provides sufficient flexibility.
  • Other linkers that can be used are listed in PCT WO94/02610, the entire contents of which are hereby incorporated by reference .
  • the gene does not encode the normal leader sequence for the variable chains as it is preferred that the antibody is not expressed with a leader sequence.
  • the nucleotides coding for the binding portion of the antibody preferably do not encode the antibody's secretory sequences, i.e., the sequences that cause the antibody to be secreted from the cell. These sequences can be contained in the constant region.
  • the immune system can prepare a stabilized antibody which will bind to a specific molecule, such as a target protein, by standard immunological techniques, such as by using the protein, or an immunogenic fragment thereof, or a peptide chemically synthesized based upon such protein. Any of these sequences can be conjugated, if desired, to keyhole limpet hemocyanin (KLH) and used to raise an antibody in animals, such as mice, rabbits, rats and hamsters. Thereafter, the animals are sacrificed and their spleens are obtained. Monoclonal antibodies are produced by using standard fusion techniques for making hybridoma cells. This typically involves fusing an antibody-producing cell (i.e., spleen) with an immortal cell line, such as a myeloma cell, to produce the hybrid cell.
  • KLH keyhole limpet hemocyanin
  • Another method for preparing antibodies is by in vi tro immunization techniques, such as using spleen cells.
  • an antigen is injected into a culture of murine spleen cells, and then an antibody produced to the antigen is screened.
  • an antibody produced to the antigen is screened.
  • as little as 0.1 ⁇ g of antigen can be used, although it is preferred to use about 1 ⁇ g/ml of antigen.
  • spleen cells are harvested and incubated at the desired amount, e.g., 1 x 10 7 cells/mL, in medium plus the desired antigen at a concentration typically around 1 ⁇ g/mL. Thereafter, one of several adjuvants, depending upon the results of the filter immunoplaque assay, is added to the cell culture.
  • adjuvants include N-acetylmuramyl-L-alanyl-D-isoglutamine, or Salmonella typhimurium mytogen, or T-cell condition, which can be produced by conventional techniques or obtained commercially.
  • the spleen cells are incubated with the antigen for four days and then harvested.
  • Single cell suspension of the in vi tro immunized mouse spleen cells are then incubated, for example, on antigen-nitrocellulose membranes in microfilter plates.
  • the antibodies produced are detected by using a label from the antibodies, such as horseradish peroxidase-labelled second antibody, such as rabbit anti-mouse IgA, IgG and IgM.
  • a label from the antibodies such as horseradish peroxidase-labelled second antibody, such as rabbit anti-mouse IgA, IgG and IgM.
  • biotinylated rabbit anti-mouse heavy chain specific antibodies can be used, followed by a horseradish peroxidase- avidin reagent .
  • the insoluble products of the enzymatic reaction are visualized as blue plaques on the membrane. These plaques are counted, for example, by using 25 times magnification.
  • the nitrocellulose microfilter membrane readily absorbs a variety of antigens, and the filtration unit used for the washing step is preferred because it facilitates the plaque assay.
  • the antibodies are then screened by standard techniques to find the antibodies of interest. Cultures containing the antibodies of interest are grown and induced, and the supernatants are passed through a filter and then through a column, such as an antigen affinity column or an anti-tag peptide column. The binding affinity is tested using a mini-gel filtration technique. A radioimmunoassay can be used to screen the antibodies.
  • the reagent, a gene that encodes at least the antigen binding portion of the antibody, is synthesized, together with a nucleotide sequence encoding a stabilon sequence. The gene preferably does not contain the normal signal peptide sequences.
  • the antibody gene preferably also does not encode functional secretory sequences .
  • V H and V L genes such as by creating V H and V L libraries from murine spleen cells that have been immunized either by the above-described in vi tro immunization technique or by conventional in vivo immunization and from hybridoma cell lines that have already been produced or are commercially available.
  • V H and V L libraries One method involves upswing spleen cells to obtain mRNA which is used to synthesize cDNA. Double-stranded cDNA can be made using PCR to amplify the variable region with a derivative BN terminal V region primer and a J region primer or with V H family specific primers, e.g., mouse-12, human-7.
  • the genes of the V H and V L domains of a broadly neutralizing antibody to the envelope glycoprotein of HIV-1, such as F105 can be cloned and sequenced.
  • the first strand cDNA can be synthesized from total RNA by using oligo dT priming and the Moloney murine leukemia virus reverse transcriptase according to known procedures .
  • This first strand cDNA is then used to perform PCR reactions using typical PCR conditions to amplify the cDNA of the immunoglobulin genes. DNA sequence analysis is then performed.
  • Heavy chain primer pairs consist of a forward V H primer and a reverse J H primer, each containing convenient restriction sites for cloning.
  • the Kabat database on immunoglobulins could be used to analyze the amino acid and codon distribution found in the seven distinct human V H families. From this, the 35 base pair universal 5' V H primer is designed. One could use a primer, such as TTTGCGGCCGCTCAGGTGCA (G/A) CTGCTCGAGTC (T/C) GG (SEQ ID NO:3) which is degenerate for two different nucleotides at two positions and will anneal to the 5' end of FR1 sequences.
  • a restriction site such as the 5' Notl site (left-underlined) , can be introduced for cloning the amplified DNA and is located 5' to the first codon to the V H gene.
  • a second restriction site such as an internal Xhol site, can be introduced as well (right-underlined) .
  • a 66 -base pair J H region oligonucleotide can be designed for reverse priming at the 3' end of the heavy chain variable gene, e.g., AGATCCGCCGCCACCGTCCCACCACCTCCGGAGC- CACCGCCACCTGAGGTGACCGTGACC (A/G) (G/T) GGT (SEQ ID NO:4) .
  • This primer additionally contains a 45 nucleotide sequence that encodes a linker, such as the (Gly-Gly-Gly-Gly-Ser) 3 (SEQ ID NO: 2) interchange linker.
  • This primer contains two degenerate positions with two nucleotides at each position based on the nucleotide sequence of the six human J H region minigenes .
  • Restriction sites can be used, for example, a BspEl site (left-underlined) is introduced into the interchange linker for cohesive end ligation with the overlapping forward V kappa primer. An internal BsTEII site (right-underlined) is introduced as well for further linker exchange procedures .
  • V kappa primer A similar strategy using a 45 nucleotide interchange linker is incorporated into the design of the 69 nucleotide human V kappa primer.
  • TCTGAGCTC (G/C) (T/A)G (A/C) TGACCCACTCTCCA (SEQ ID NO:5), which will anneal to the 5' end of the FR1 sequence, is degenerate at three positions (two nucleotides each) .
  • the interchange linker portion can contain a BspEI site for cohesive end cloning with the reverse J H primer, although other restriction sites can also be used.
  • An internal Sad site (right-underlined) can be introduced as well to permit further linker exchange procedures.
  • the reverse 47 nucleotide C kappa primer (Kabat positions 109-113) GGGTCTAGACTCGAGGATCCTTATTAACGCGTTGGTGCAGC- CACAGT (SEQ ID NO: 6) is designed to be complementary to the constant regions of kappa chains (Kabat positions 109-113).
  • This primer anneals to the 5' most end of the kappa constant region.
  • the primer contains an internal Mlul site (right- underlined) preceding two stop codons.
  • multiple restriction sites such as BamHI Xhol/Xbal (left-underlined) , can be introduced after the tandem stop codons .
  • a similar reverse nucleotide C kappa primer such as a 59 nucleotide primer can also be designed that contains a signal for a particular intracellular site, such as a carboxy terminal endoplasmic reticulum retention signal, Ser-Glu-Lys-Asp-Glu- Leu (SEQ ID NO: 7) ,
  • GGGTCTAGACTCGAGGATCCTTATTACAGCTCGTCCTTTTCGCTTGGTGCAG- CCACAGT SEQ ID NO : 8
  • Similar multiple restriction sites can be introduced after the tandem stop codons .
  • a PCR primer can then be designed, based on the leader sequence of the V H 71-4 germ line gene.
  • the V H 71-4 leader primer TTTACCATGGAACATCTGTGGTTC (SEQ ID NO: 9) contains a 5'NcoI site (underlined) .
  • This leader primer (P-L) is used in conjunction with a second J H primer for PCR amplification experiments.
  • the 35 base pair J H region oligonucleotide is designed to contain the same sequence for reverse priming at the 3' end of the heavy chain variable gene.
  • This primer contains two degenerate positions with two nucleotides at each position.
  • a BssHII site (left-underlined) 3' to and immediately adjacent to the codon determining the last amino acid of the J region, allows convenient cloning at the 3' end of the V H gene.
  • An internal BstEII site (right-underlined) is introduced as well. This sequence is used to amplify the V L sequence.
  • the fragments amplified by the P-L (leader primer) and P linker (reverse primer) and P-K (V2 primer) and P-CK primers (reverse CK primer) are then cloned into an expression vector, such as the pRc/CMV (Invitrogen) , and the resultant recombinant contains a signal peptide V H interchain linker and V L sequences under the control of a promoter, such as the CMV promoter. To this is then added the corresponding sequence for the stabilizing moiety (stabilon) .
  • a promoter such as the CMV promoter.
  • stabilizing moiety stabilon
  • the skilled artisan can readily choose other promoters that express the gene in the cell system of choice, e.g., a mammalian cell, preferably a human cell.
  • This single-chain antibody can be prepared based upon the present disclosure by any of a number of known means.
  • the V H /J H -ICL and ICL-V kappa /C kappa PCR fragments are digested with Notl/BNspEI and BspEI/Xbal, respectively, and cloned into a plasmid, such as pSL1180, using SURE bacteria as hosts.
  • the resulting sFGV is restriction enzyme digested and the Notl/Bglll fragment is cloned into the Notl/BamHI site that is located 3' to the pelB signal peptide in a pET expression vector.
  • Plasmid fragments are obtained after suitable times, for example, 2 to 4 hours after induction at 24° with 0.2 mM IPTG and tested for its ability to bind its target, e.g., gpl20 binding activity, by standard techniques, such as ELISA.
  • V H 71-4 leader and a J H -BssHII primer are used to PCR amplify an intronless fragment containing the leader peptide and rearranged heavy chain gene.
  • the fragment is bound and cloned in the forward direction into an EcoRV site in a plasmid, for example, pSLll ⁇ O.
  • a NcoI/BstEII fragment is obtained and combined with the BstEII/Sph I fragment of, e.g., F105sFv from pSL1180, in a three-piece ligation with Ncol/SpHI digested pSL1180 to produce the V H 71-4/SCA.
  • a V H 71-4 SCA containing the carboxyl-terminated SEKDEL sequence can be constructed using an ICL-V kappa -SEKDEL PCR product that is blunt and cloned in the forward direction into an EcoRV site in pSL1180.
  • the fragment is removed by BspoEI/Xbal digestion and combined with the Ncol/BspEI fragment of V H 71-4/SCA in a three-part ligation with Ncol/Xbal digested pSL1180 to produce V H 71- 4/KDEL.
  • an EcoRI to Hindlll conversion linker is introduced into EcoRI digested pSL1180 containing the two single-chain antibodies.
  • a Hindlll/Xbal fragment from both single-chain antibodies is obtained and cloned into Hindlll/Xbal digested pRC/CMV to produce pRC/SCA and pRC/KDEL.
  • V H and V L domains can be used to construct sFv, Fv or Fab fragments .
  • a preferred target is one processed by the endoplasmic reticulum, where proteins are typically made.
  • RNA, DNA, or cellular proteins, or nucleic acids that are subsequently processed.
  • virally-encoded proteins such as lentiviruses
  • structural proteins are typically cytoplasmically expressed, whereas regulatory proteins can be expressed in or near the nucleus.
  • localization sequences for such targets.
  • the stabilized antibodies of the present invention can be delivered intracellularly and can be expressed there and bind to a target protein. Localization sequences have been divided into routing signals, sorting signals, retention or salvage signals, and membrane topology-stop transfer signals (Pugsley, 1989) .
  • Myristolation sequences can be used to direct the antibody to the plasma membrane.
  • Table 2 sets forth the amino- terminal sequences for known N-myristoyl proteins and their subcellular location.
  • myristoylation sequences can be used to direct the antibodies to different subcellular locations, such as the nuclear region.
  • Localization sequence may also be used to direct antibodies to organelles, such as the mitochondria and the Golgi apparatus.
  • the sequences Met-Leu-Phe-Asn-Leu-Arg- Xaa-Leu-Asn-Asn-Ala-Ala-Phe-Arg-His-Gly-His-Asn-Phe-Met-Val- Arg-Asn-Phe-Arg-Cys-Gly-Gly-Pro-Leu-Xaa can be used to direct the antibody to the mitochondrial matrix. See, e.g., Tang et al for localization of proteins to the Golgi apparatus .
  • PM Plasma Membrane
  • G Golgi
  • N Nuclear
  • C Cytoskeleton
  • s cytoplasm (soluble)
  • M Membrane
  • tat is located in subnuclear and subnucleolar regions of infected cells.
  • the tat antibody target the nuclear and/or nucleolar regions of the cell. Since this antibody is to be synthesized in the cytoplasm, it does not have a leader sequence. To target the nuclear and/or nucleolar regions, the antibody does need a localization sequence.
  • Preferred nuclear targeting sequences are SV40 and preferred nucleolar targeting regions are tat nucleolar signals.
  • a tat antibody a single-chain antibody, with SV40 nuclear localization signal
  • tat will bind to tat and can reduce tat activity by over 80% when compared to the antibody with an immunoglobulin leader sequence, which directs the antibody to a different cellular compartment, such as the ER.
  • the structural proteins are targeted in the cytoplasm, such as envelop and gag, whereas the regulatory proteins, such as tat and rev, are targeted in the nucleus and nucleolar regions.
  • the tax protein of HTLV-1 or HTLV-2 is also preferably searched for in the nucleus or nucleolus . If possible, it is preferable to use the localization signals of the target protein to direct the antibody to the desired location.
  • HIV-12 tat protein has a nucleolar localization signal, which is preferably used.
  • the gene for the antibody can encompass genes for the heavy chain and light chain regions, including a sequence for imparting a stabilizing amino acid at the N-terminal end of the antibody expressed.
  • the gene is operably linked to a promoter or promoters which result in its expression. Promoters that permit expression in mammalian cells are well known and include a CMV, a viral LTR, such as the rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the SV40 early promoter, E. coli lac UV5 promoter, and the herpes simplex tk promoter.
  • This DNA sequence is referred to as the antibody cassette.
  • the antibody cassette is delivered to the cell by any known means.
  • a cassette containing these antibody genes such as the sFv gene
  • a mammalian expression vector such as a herpes vector, an adenovirus vector or a pox vector, a retroviral vector, a plasmid bound to an antibody, etc.
  • cells can be transduced by standard techniques well known to the skilled artisan.
  • this cassette is introduced in the cell using an HIV viral vector, which is defective in packaging HIV sequences, but will preferentially target HIV susceptible cells.
  • a promoter that will differentially express the gene in the desired target cell.
  • the HIV viral proteins in the cell can result in enhanced expression of the antibody when compared to uninfected cells.
  • Intracellular expression of the antibody permits it to bind the target. This disrupts the function of the target, e.g., a protein, including the undesired function.
  • expressing the sFv of a broadly neutralizing antibody to envelope glycoprotein can intracellularly block the transport and interaction with the CD4 molecules of the HIV-1 glycoprotein, as well as cleavage of the protein.
  • Another example uses an antibody specific for one which is targeted to breast tissue to keep the neu protein of the cell.
  • target protein also assists the antibody in folding to the correct conformational state.
  • antibody-ligand complexes prevent the target from operating in its typical manner. For instance, cytopathic fusion mediated by the HIV-1 gp 120/41 is inhibited in the cells. This is shown by co-transfecting CD4 + Hela cells with the HIV-1 glycoprotein expressing pSVIII env and sFv or sFv-
  • KDEL plasmid DNAs at a ratio of 1:5 or transfecting the transformed cells with pSVIII.
  • a viral infection such as an HIV-1 infection to alleviate the undesirable effect of the virus.
  • a viral infection such as an HIV-1 infection
  • other viral and metabolic diseases such as infections by DNA viruses, such as herpes simplex, and RNA viruses, such as HTLV-1 and 2.
  • Antibodies against reverse transcriptase can interfere with template binding functions of the protein.
  • Antibodies to this protein are known and include C2003, which binds to a sequence in the C-terminal portion of the p66 component. This antibody also binds to HIV-2. Such antibodies can be screened for from patient sera and antibodies cloned as described above.
  • the TAR element which is responsive to tat, is located at the 5' end of messenger viral RNA. Tat binding to this TAR element has been shown to result in a depression of tar inhibitor of translation in vi tro .
  • the tar element increases transcription, initiation, and also acts as an anti- attenuator of transcription elongation. By directing an antibody against the tar sequence, inhibition of tat binding will occur and there will be a dramatic decrease in transcription efficiency. This will ultimately result in an inhibition or reduction of virus production.
  • a similar approach can be used to produce antibodies against the rev responsive element.
  • Rev controls the synthesis of viral structural proteins, including the capsid protein, replicative enzymes and the envelope glycoprotein.
  • the rev protein controls virion protein expression by controlling the cytoplasmic accumulation of RNA species. In the absence of rev activity, small multiply- spliced viral RNA species accumulate; in the presence of rev, full-length and partially-spliced envelope glycoprotein messenger RNAs accumulate. Antibodies directed against the rev responsive element should inhibit rev binding to the rev responsive element and, therefore, inhibit the major biological effect of rev.
  • the rev protein regulates the synthesis of capsid, replicative enzyme and envelope glycoprotein production by regulating the accumulation of messenger RNA species from which they are made.
  • Structural protein messenger RNAs require binding of rev protein to the folded RNA structure called rev responsive element for translocation from the nucleus to the cytoplasm. Inhibition of rev binding by an anti-rev antibody should prevent virus expression from infected cells.
  • Such antibodies which have sufficient life in the body to inhibit rev binding can be synthesized using known technology based upon the present disclosure. For example, one can screen an RNA library with an antibody to obtain the desired antibody.
  • tumor formation and metastasis is dependent upon angiogenesis (Folkman et al, 1991) .
  • human melanoma has been found to produce several proteins with angiogenic activity, including fibroblast growth factor (gFGF) , transforming growth factor alpha (TGFa) , and transforming growth factor beta (TFGb) .
  • gFGF fibroblast growth factor
  • TGFa transforming growth factor alpha
  • TGFb transforming growth factor beta
  • Antibodies capable of such specific binding are known in the art .
  • a "cocktail” approach i.e., a mixture of antibodies
  • a cocktail of antibodies to at least envelope glycoprotein and tat is preferred.
  • Other cocktails include antibodies to reverse transcriptase, TAR, RRE, etc. These cocktails can be administered together or by co-transfections . It is preferred that no more than about three proteins in this intracellular region are targeted, preferably no more than about two. For example, gpl60 and gp41 can be targeted at the endoplasmic reticulum.
  • Another intracellular target is in a different cellular region, i.e., nucleus vs, endoplasmic reticulum, it can also be targeted without having a detrimental effect on antibody production.
  • One preferred cocktail of antibodies would be antibodies for at least one structural viral protein, such as capsid or envelope, and one for regulatory proteins, such as HIV rev, tat, HTLV-1 or tax, or for a nucleic acid sequence, such as TAR or RRE.
  • Another preferred cocktail is of antibodies to the same target but at various intracellular locations, which can be done using different localization sequences. Thus, if some target is not bound to the antibody at one location and, for instance, is further processed, it can be targeted at a subsequent location.
  • envelope glycoprotein one could use localization sequences to target the protein at a number of points in its processing path.
  • one preferred vector is to have at least two antibodies to capsid or envelope proteins and at least one antibody to a regulatory protein, such as to gpl60, gp41, tat and rev.
  • Another cocktail can include antibodies to both the viral mRNA and the protein it encodes.
  • HIV-encoded target proteins are nef, vpr and, for HIV-1, vpu, and, for HIV-2, vpx, more preferably, nef and vpu.
  • the nef protein exists in the cytoplasm, as well as being attached to the inner surface of the plasma membrane.
  • the protein is modified co- translationally by addition of myristic acid to the penultimate glycine residue of the amino terminus.
  • the vpr protein has been found incorporated into the capsid of the virus.
  • the vpu protein is located within the cytoplasm of cells and may be associated with sub-cellular organelles. Antibodies to these proteins can be made by the methodology described herein. Further, these proteins can be more specifically targeted by the skilled artisan based upon this disclosure by selection of appropriate localization sequences .
  • This method can be used to treat viral and metabolic disease, such as HIV, HTLV-1, HTLV-2 and herpes.
  • individuals having malignant tumors or who are susceptible to malignant cellular transformation caused by a high level of a protein or proteins, and altered protein or proteins or a combination thereof can be treated.
  • This method can be used as a prophylactic treatment to prevent or make it more difficult for such cells to be adversely affected by the undesired antigen by preventing processing of the protein, interaction by the undesired protein with other proteins, integration by the virus into the host cells, etc.
  • one preferred target is proteins that are processed by the endoplasmic reticulum.
  • Intracellular delivery of any of the antibody genes can be accomplished by using gene therapy techniques, such as described above.
  • the antibody can be any of the antibodies as discussed above.
  • HIV infects CD4 positive human lymphocytes and other immune cells.
  • an antibody that binds to at least one HIV encoded target molecule e.g., a protein
  • any of the known forms of gene therapy can be used to deliver genes to the target.
  • a cell-specific gene transfer mechanism which uses receptor- mediated endocytosis to carry RNA or DNA molecules into cells
  • a protein acting as a ligand is coupled to a poly-L- lysine, which then combines with RNA or DNA (the gene) to form soluble complexes by strong electrostatic interaction, whereby one can deliver the genes to the cells of interest.
  • a protein acting as a ligand is coupled to a poly-L- lysine, which then combines with RNA or DNA (the gene) to form soluble complexes by strong electrostatic interaction, whereby one can deliver the genes to the cells of interest.
  • an antibody against gpl20 or CD4 as the ligand one can specifically target such cells.
  • Antibodies used to target the cells can be coupled to the polylysme to form an antibody-polylysine conjugate by ligation through disulfide bonds after modification with a reagent, such as succinimidyl-3- (2-pyridyldithio) propionate (SPDP) .
  • SPDP succinimidyl-3- (2-pyridyldithio) propionate
  • the antibody-polylysine-gene complexes are produced by mixing the antibody polylysme conjugates with a moiety carrying the antibody cassette.
  • the polylysme has an average chain length of about 60 to about 500 lysine monomers .
  • Conjugation with the antibodies can be accomplished using SPDP.
  • First dithiopyridine groups are introduced into both antibody or polylysine by means of SPDP, and then the groups in the polylysme are reduced to give free sulfhydryl compounds, which upon mixing with the antibodies modified as described above, react to give the desired disulfide bond conjugates.
  • These conjugates can be purified by conventional techniques, such as using cation exchange chromatography . These conjugates are then mixed with the antibody cassette under conditions that permit binding.
  • the vectors can be introduced to the cells in vi tro with the transduced cells injected into the mammalian host wherein it will bind with the CD4 cell and be taken up.
  • the antibody cassette can be part of an episomal mammalian expression vector, for example, a vector which contains the human Pappova virus (BK) origin of replication and the BK large T antigen for extra- chromosomal replication in mammalian cells, a vector which contains an Epstein-Barr (EB) virus origin of replication, and nuclear antigen (EBNA01) to allow high copy episomal replication.
  • BK human Pappova virus
  • EB Epstein-Barr
  • EBNA01 nuclear antigen
  • mammalian expression vectors such as herpes virus expression vectors, or pox virus expression vectors, can also be used. These vectors are commercially available.
  • the antibody cassette is inserted into the expression vectors using standard techniques. These expression vectors can be mixed with the antibody-polylysine conjugates and the resulting antibody-polylysine-expression vector containing antibody cassette complexes can readily be made based upon the present disclosure. Sufficient amounts of these vectors are injected to obtain a serum concentration ranging between about 0.01 ⁇ g/ml to 30 ⁇ g/ml of antibody conjugate. Because the stabilized antibodies of the present invention last longer in the body than conventionally prepared antibodies, the amount of antibody used in some instances can be reduced.
  • the vectors according to the present invention can be administered by any conventional means, including parenteral injection, intraperitoneal injection, intravenous injection, intracranial injection or subcutaneous injection. Oral or other routes of administration can also be used.
  • the materials can be administered in any convenient form, such as mixed with an inert carrier, such as sucrose, lactose or starch.
  • the material can be in the form of tablets, capsules or pills.
  • parenteral administration the material is typically injected in a sterile aqueous or non-aqueous solution, suspension or emulsion in association with a pharmaceutically-acceptable parenteral carrier, such as physiological saline.

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Abstract

L'invention concerne la production de protéines, par exemple des anticorps à chaîne unique, qui sont stables dans leur forme libre. On produit ces protéines de manière intracellulaire en exprimant lesdits anticorps à chaîne unique et en stabilisant les résidus, les protéines étant ainsi stabilisées contre toute protéolyse in vivo.
PCT/US2000/000558 1999-01-11 2000-01-11 Methodes pour reguler la stabilite de proteines recombinantes, et produits utilises dans ces methodes WO2000042185A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2000050089A2 (fr) * 1999-02-26 2000-08-31 Mindset Biopharmaceuticals (Usa) Ltd. Regulation de la stabilite de proteines de recombinaison, anticorps et produits convenant pour cette regulation
WO2000050089A3 (fr) * 1999-02-26 2001-03-29 Mindset Biopharmaceuticals Usa Regulation de la stabilite de proteines de recombinaison, anticorps et produits convenant pour cette regulation

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