Genencor International (Danisco A/S) in the US was developing I V, In addition, there was negligible IgG antibody response to the variant interferon-β. Herceptin IgG human antibody were made and transformed into Trichoderma reesei. Genencor International, a Danisco Company. Page. GENENCOR INTERNATIONAL PALO ALTO CA Initial genetic constructs for the Herceptin IgG human antibody were made and transformed into Trichoderma.
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This invention relates to the field of chimeric molecules. Frequently, one of the constituent molecules of a chimeric molecule is a “targeting molecule”. Another constituent of the chimeric molecule may ogg an “effector molecule”.
The effector molecule refers to a molecule that is to be specifically transported to the target to which the chimeric molecule is specifically directed. Chimeric molecules comprising a targeting moiety attached to an effector moiety have been used in a wide variety of contexts. Converselyangiogenesis inducers have been proposed for the treatment of atherosclerosis.
Typically, the target recognized by the targeting moiety is not the desired site of action of the effector molecule. ADP ribosylation in the case of Pseudomonas exotoxin. Similarly, targeted liposomes are typically internalized through a receptor- mediated process or through the action of the lipid.
Targeted intrabodies and gene therapy vectors are also internalized for expression within the cell. In addition, a common goal in the design of targeted chimeric molecules has been the increase of binding specificity and avidity. It is generally believed that, by increasing avidity and specificity the concentration of the chimeric molecule to achieve a given result will decrease. Thus, release of the chimeric molecule from its target is generally viewed as undesirable.
Because the chimeric molecule is typically internalized in the case of targeted cells and the activity of the effector molecule is directed to a molecule other than the specifically recognized target, chimeric molecules typically act in a “stoichiometric” manner. That is, each chimeric molecule is essentially consumed upon interaction with its “substrate” and activity of the chimeric molecule is unavailable for subsequent reactions.
This invention provides a novel approach to the design of chimeric molecules. In one embodiment, the molecules of this invention specifically bind to a target molecule and degrade that bound molecule. In preferred embodiments, this results in a loss of activity e. In this manner the chimeric molecule is “regenerated” and essentially catalytic. Because a single chimeric molecule can attack and degrade an essentially limitless number of targets, the so called “catalytic antagonists” of this invention are highly effective at relatively low dosages.
The antagonist comprises a targeting moiety that specifically binds to the target molecule and the targeting moiety is attached to an enzyme that degrades the target molecule to reduce binding of the target molecule to its cognate ligand. In particularly preferred embodiments, the degradation of the target molecule also reduces binding of the antagonist to the target molecule.
Thus, in these embodiments, the antagonist is released from the target thereby allowing the antagonist to bind and degrade another target molecule. In certain preferred embodiments, the cysteine is a cysteine that is substituted for a native amino acid other than cysteine in or near a subsite comprising a substrate binding site of the enzyme. In some embodiments, the cysteine is a cysteine that is substituted for an amino acid forming a substrate binding site.
Preferred enzymes include, but are not limited to a protease, an esterase, an amidase, a peptidase, a lactamase, a cellulase, an oxidase, an oxidoreductase, a reductase, a transferase, a hydrolase, an isomerase, a ligase, a lipase, a phospholipase, a phosphatase, a kinase, a sulfatase, a lysozyme, a glycosidase, a nuclease, an aldolase, a ketolase, a lyase, a cyclase, a reverse transcriptase, a hyaluronidase, an amylase, a cerebrosidase, and a chitinase.
In a particularly preferred embodiment, the enzyme is a serine hydrolase. In a particularly preferred embodiment the enzyme is a Bacillus lentus subtilisin.
Enhanced immunogenicity of a functional enzyme by T cell epitope modification
In preferred embodiments, the cysteine is substituted for an amino acid in a subtillisin, where the amino acid corresponds to a reference residue in a Bacillus lentus subtilisin, where the reference residue is at or near a residue selected from the group consisting of residueresidueresidueresidueresidue 62, residue 96, residueresidueresidueand residue In another embodiment the enzyme is a chymotrypsin-type serine protease and the cysteine is substituted for the amino acid corresponding to a reference residue in a mature trypsin Protein Data Bank entry 1TPPwherein said reference residue is at or near a residue selected from the group consisting of Tyr94, Leu99, Glnl75, Aspl89, Serl90, Glnl92, Phe41, Lys60, Tyrl51, Ser, and Lys In yet another embodiment the enzyme is an aspartyl protease.
More preferably the enzyme is a pepsin-type protease and the cysteine is substituted for the amino acid corresponding to a reference residue in the mature human pepsin Protein Data Bank entry 1PSNwhere the reference residue is at or near a residue selected from the group consisting of Tyr9, Metl2, Glul3, Gly76, Thr77, Phel 11, Phel 17, Ilel28, Serl30, Tyrl89, Ile, Glu, Met, Gln, Met, Leu, and Glu In still yet another embodiment the enzyme is a cysteine protease.
More preferably the enzyme is a papain and the cysteine is substituted for the amino acid corresponding to a reference residue in a mature papain Protein Data Bank entry 1BQIwhere the reference residue is at or near a residue selected from the group consisting of Asnl8, Ser21, Asn64, Tyr67, Trp69, Glnl 12, Glnl42, Aspl58, Trpl77, and Phe In certain embodiments the enzyme is a metalloprotease and the cysteine is substituted for the amino acid corresponding to a reference residue in the mature human matrix metalloprotease Protein Data Bank entry Cwhere the reference residue is at or near a residue selected from the group consisting of Leul 11, Phel75, Tyrl76, Serl82, Leul84, Phel89, Tyr, Asp, Lys, and Ile In certain embodiments the targeting moiety includes, but is not limited to an antigena carbohydrate, a nucleic acid, a lipid, a coordination complex, a sugar, a vitamin, a dendrimer, and a crown ether.
In a particularly preferred embodiment the targeting moiety is a cognate ligand for a receptor or an enzyme. In another particularly preferred embodiment the targeting moiety is an inhibitor for a receptor or an enzyme. In another embodiment the enzyme is a protease and said targeting moiety is a receptor. In one particularly preferred embodiment the targeting moiety specifically binds to a soil and the enzyme degrades a component of the soil.
In another embodiment this invention provides a method of degrading a target molecule. The method involves contacting the target molecule with a catalytic antagonist comprising a targeting moiety that specifically binds to the target molecule the targeting moiety being attached to an enzyme that degrades the target molecule.
In a preferred embodiment the degradation of the target molecule releases the antagonist thereby allowing the antagonist to bind and degrade another target molecule.
Enhanced immunogenicity of a functional enzyme by T cell epitope modification
gnencor In preferred embodiments, the targeting moiety is joined to the enzyme through the sulfur group on a cysteine. Preferred antagonist molecules include, but are not limited to the catalytic antagonist molecules described above. The enzyme preferably comprises a targeting moiety attached genendor a subsite comprising the substrate binding site of said enzyme.
In preferred embodiments, the targeting moiety is coupled to said enzyme through to a sulfur of a cysteine in said subsite of said enzyme. The cysteine may be a native cysteine or a cysteine is substituted for a native amino acid that is not cysteine in the subsite of the enzyme. Preferred enzymes include, but are not limited to a protease, an esterase, an amidase, a peptidase, a lactamase, a cellulase, an oxidase, an oxidoreductase, a reductase, a transferase, a hydrolase, an isomerase, a ligase, a lipase, a phospholipase, a phosphatase, a kinase, a sulfatase, a lysozyme, a glycosidase, a glycosyltransferase, a nuclease, an aldolase, a ketolase, a lyase, a cyclase, a reverse transcriptase, a hyaluronidase, an amylase, a cerebrosidase and a chitinase.
In a subtilisin, the cysteine is preferably subsitited for amino acids at or near a subsite selected from the group consisting of an SI subsite, an ST subsite, and an S2 subsite. Particularly preferred sites for substitution of the cysteine in various enzymes include, but are not limited to those igy above. Fenencor, particularly preferred targets and targeting moieties include those identified above.
In certain embodiments the targeting moiety is an inhibitor for a receptor or an enzyme, in other embodiments the targeting moiety is selected from the group consisting of a growth factor, a cytokine, and a receptor ligand. In certain embodiments, the enzyme is a protease and the targeting moiety is a ligand selected from the group consisting of a carbohydrate, a iggg or vitamin analog, an enzyme inhibitor, a peptide, a pharmaceutical that is a small organic molecule, genencoor biotin.
In one embodiment the gebencor moiety specifically binds to a soil and said enzyme degrades a component of the soil. In still yet another embodiment this invention provides methods of directing the activity of an enzyme to a specific target. The methods comprise providing an enzyme having altered substrate specificity said enzyme comprising a targeting moiety attached to a subsite egnencor the substrate binding region of said enzyme; and contacting the target with the enzyme, whereby the enzyme specifically binds to the target thereby localizing the activity of the enzyme at the target.
Preferred enzymes include, but are not limited to, the “redirected” enzymes described above. This invention also provides methods of enhancing the activity of a drug that acts as an inhibitor of a receptor or genencro enzyme. The methods involve coupling a hydrolase to said drug such that when said drug binds said receptor or enzyme, the hydrolase degrades the receptor or enzyme.
In preferred embodiments, the method increases the dosage therapeutic window of said drug. In certain preferred embodiments, the hydrolase is a metalloprotease, a geenencor protease, an aspartyl protease, and the like.
This invention also provides a method of inhibiting an enzyme or a receptor. The method comprises contacting the enzyme or receptor with a chimeric molecule comprising a ligand that binds the enzyme or receptor attached to an enzyme that degrades the cognate ligand of the enzyme or receptor.
The enzyme thus becomes linked to the enzyme or receptor where it is free to degrade the cognate ligand thereby ifg the cognate ligand from activating the receptor or acting as a substrate for the enzyme. Preferred hydrolases include, but are not limited to a serine protease, a cysteine protease, an aspartyl protease, a pepsin-type protease, and a metalloprotease.
In certain embodiments, this invention does not include catalytic lgg, e. Bioscience and Bioengineering, The inhibition can be a blocking or destroying of the function of the “target” molecule. In preferred embodiments, the inhibition or blockage is genncor partial or complete degradation of the target molecule. Thus, in preferred embodiments, the degradation of the target molecule ultimately results in the release of the catalytic antagonist so that it is free to attack another target molecule.
The reaction is preferably sub-stoichiometric ratio of catalytic antagonist to target is less than 1 and a single catalytic antagonist is free to degrade any number of target molecules.
A “target molecule” refers to a molecule that is specifically bound by the catalytic antagonist or specifically directed enzymes described herein. Where a catalytic antagonist is employed gejencor target molecule is partially or completely degraded by that antagonist.
Genfncor “targeting moiety” refers to a moiety in the chimeric molecule that that specifically binds to the target molecule. Prior to coupling the targeting moiety to the enzyme, the targeting moiety is a targeting molecule.
In preferred embodiments, the targeting moiety is one of a pair of cognate binding genenor. The binding may be by one or more of a variety of mechanisms including, but not limited to ionic interactions, covalent interactions, hydrophobic interactions, van der Waals interactions, etc. The term ligand is used to refer to a molecule that specifically binds to another molecule. Commonly a ligand is a soluble molecule, e.
In these cases, typically the smaller of the two members of the binding pair is called the ligand. Thus, in a lectin-sugar interaction, the sugar would be the ligand even iggg it is attached to a much larger molecule, venencor is of the saccharide.
The terms “polypeptide”, “ohgopeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The term also includes variants on the traditional peptide linkage joining the amino acids making up the polypeptide.
The terms “nucleic acid” or “oligonucleotide” or grammatical equivalents herein refer to at least two nucleotides covalently linked together. A nucleic acid of the present invention is preferably single-stranded or double stranded and will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide Beaucage et al. Other analog nucleic acids include those with positive backbones Denpcy et al.
Dan Cook; Mesmaeker et al. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments.
The term “residue” as used herein refers to natural, synthetic, or modified amino acids. The term enzyme includes proteins that are capable of catalyzing chemical changes in other substances without being permanently changed themselves. The enzymes can be wild-type enzymes or variant enzymes. Enzymes within the scope of the present invention include, but are not limited to, proteases, esterases, amidases, peptidases, lactamases, cellulases, oxidases, oxidoreductases, reductases, transferases, hydrolases, isomerases, ligases, upases, phospholipases, phosphatases, kinases, sulfatases, lysozymes, glycosidases, glycosyltransferases, nucleases, aldolases, ketolases, lyases, cyclases, reverse transcriptases, hyaluronidases, amylases, cerebrosidases, chitinases, and the like.
A “mutant enzyme” is an enzyme that has been changed by replacing an amino acid residue with a cysteine or other residue.
A “chemically modified” enzyme is an enzyme that has been derivatized to bear a substituent not normally found at that location in the enzyme. The derivatization typically is of a post translational modification, occasionally performed in vivo, but more typically performed ex vivo. A “chemically modified mutant enzyme” or “CMM” is an enzyme in which an amino acid residue has been replaced with another amino acid residue preferably a cysteine and the replacement residue is chemically derivatized to bear a substituent not normally found on that residue.
The term “thiol side chain group”, “thiol containing group”, and “thiol side chain” are terms that can be used interchangeably and include groups that are used to replace the thiol hydrogen of a cysteine.