WO2007041361A1 - Rnase anti-tumeur recombinante modifiee - Google Patents

Rnase anti-tumeur recombinante modifiee Download PDF

Info

Publication number
WO2007041361A1
WO2007041361A1 PCT/US2006/038180 US2006038180W WO2007041361A1 WO 2007041361 A1 WO2007041361 A1 WO 2007041361A1 US 2006038180 W US2006038180 W US 2006038180W WO 2007041361 A1 WO2007041361 A1 WO 2007041361A1
Authority
WO
WIPO (PCT)
Prior art keywords
cysteine
rnase
ribonuclease
modified
substituted
Prior art date
Application number
PCT/US2006/038180
Other languages
English (en)
Inventor
Dianne L. Newton
David F. Nellis
Susanna M. Rybak
Original Assignee
The Government Of The United States, As Represented By The Secretary Of Health And Human Services, National Institutes Of Health, Office Of Technology Transfer
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Government Of The United States, As Represented By The Secretary Of Health And Human Services, National Institutes Of Health, Office Of Technology Transfer filed Critical The Government Of The United States, As Represented By The Secretary Of Health And Human Services, National Institutes Of Health, Office Of Technology Transfer
Publication of WO2007041361A1 publication Critical patent/WO2007041361A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • RNase A family members such as human RNases, ONCONASE®, and other RNase A family member proteins from Ranapipiens, e.g., RapLRl (see, e.g., U.S. Patent No. 6,869,604), have been shown to exhibit both in vitro and in vivo cytotoxicity to tumor cells both alone and as the toxic component for targeted therapies.
  • Ligand binding moieties such as antibodies, are often used to target the RNase A family member to a particular cell or cell type that expresses a marker, e.g., a marker that is associated with cancer.
  • RNase-containing conjugates can be generated using a variety of methods including chemical conjugation methods.
  • the current invention is based on the discovery that certain sites in ribonucleases, e.g., the Ranapipiens RNase A family member RapLRl, can be mutated to cysteine residues, which provide free-thiol attachment sites suitable for conjugation to another moiety.
  • the invention provides cysteine-modified RNase family A polypeptides, conjugates comprising such modified RNase polypeptides, and polynucleotides that encode the modified proteins.
  • the ribonuclease has at least 60%, often at least 80%, 85%, or at least 90% identity to SEQ ID NO:1 and comprises a cysteine substitution at a position that corresponds to 61, 60, 40, or 80, i.e., at position 61, 60, 40, or 80 as determined with reference to SEQ K) NO:1.
  • the ribonuclease comprises the amino acid sequence of SEQ ID NO:1 with substitutions S61C, T60C, R40C, andK80C.
  • a cysteine-modified RNase family A polypeptide of the invention comprises a cysteine substitution in a position corresponding to the region in SEQ ID NO:1 defined by the positions 51 (isoleucine) through 61 (threonine).
  • the invention provides a conjugate comprising a cysteine- modified ribonuclease of the invention joined to a ligand binding moiety or a label.
  • the ligand binding moiety can be for, example, an antibody.
  • the invention provides an isolated nucleic acid encoding a cysteine-substituted ribonuclease of the invention, expression vectors comprising the nucleic acids, and host cells comprising such vectors.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a cytotoxic amount of a conjugate that comprises a cysteine-modified ribonuclease of the invention joined to a ligand binding moiety, e.g., an antibody, peptide, hormone, growth factor, and the like, and a pharmaceutically acceptable carrier.
  • a ligand binding moiety e.g., an antibody, peptide, hormone, growth factor, and the like
  • the invention also provides methods of selectively killing cells comprising contacting cells to be killed with a cysteine-modified ribonuclease joined to a ligand binding moiety, e.g., an antibody.
  • the invention provides a method of making a cysteine- substituted ribonuclease member suitable for conjugation to a molecule, the method comprising introducing a cysteine substitution into an RNase A family polypeptide having at least 60% identity, often at least 80%, 85%, or 90% identity, to SEQ ID NO:1, wherein the cysteine is substituted at position 61, 60, 40, or 80 as determined with reference to SEQ ID NO: 1.
  • the cysteine is introduced into a region of the RNase family A polypeptide that corresponds to positions 51 (isoleucine) to 61 (threonine) of SEQ ID NO:!.
  • Figure 1 provides cDNA (SEQ ID NO:6) and protein (SEQ ID NO: 1) sequences of native rapLRl.
  • Figures 2 A and 2B show a representation of rapLR-1 showing the location of cysteine insertion sites; S61, T60, R40, and K80; and native disulfide-forming cysteine residues (C90-C48, C104-C87, C68-C19, and C30-C75).
  • accession IONC native amphibian Ranpirnase
  • RasMOL RasMOL software
  • Figure 3 provides exemplary data showing RNase activity of native rapLRl and cysteine-modified rapLRl proteins ( ⁇ cys mutants).
  • Figure 4 provides exemplary data showing cytotoxic activity of native rapLRl and cysteine-modified rapLRl proteins ( ⁇ cys mutants).
  • Figure 5 provides exemplary data showing enzymatic activity of conventional RFB4-onc conjugates in comparison to RFB4-cysteine-modified rapLRl conjugates.
  • Figure 6 provides exemplary data showing binding to Daudi B cell lymphoma cells of conventional RFB4-onc conjugates in comparison to RFB4-cysteine-modified rapLRl conjugates.
  • Figure 7 provides exemplary data showing cytotoxicity towards Daudi B cell lymphoma cells of conventional RFB4-onc conjugates in comparison to RFB4-cysteine- modified rapLRl conjugates.
  • Figure 8 provides exemplary data showing RNase activity of conventional hHMFGl -rapLRl conjugates compared to hHMFGl -cysteine-modified rapLRl conjugates.
  • Figure 9 provides exemplary data showing the binding to MCF7 cells of HMFGl- cysteine-modified rapLRl conjugates compared to conventional HMFGl -rapLRl and to HMFGl IgG.
  • Figure 10 provides exemplary data showing the cytotoxicity toward MCF7 cells of conventional hHMFGl -rapLRl compared to cysteine-modified rapLRl conjugates.
  • cyste-substituted ribonuclease or “cysteine-modified ribonuclease” are used interchangeably to refer to an RNase that has at least 60% identity or greater amino 5 acid sequence identity to SEQ ID NO:1, typically at least 80 or 85% amino acid identity to SEQ ID NO:1, and most often at least 90% or 95% or greater identity to SEQ ID NO:1; and that comprises a cysteine that is substituted at position 61, 60, 40, or 80, as determined with reference to SEQ ID NO: 1.
  • the phrase "determined with reference to" in the context of identifying changes in .0 amino acid sequence means that the amino acid as indicated in the reference sequence at that position is changed to an alternative amino acid.
  • the serine at position 61 of SEQ ID NO:1 is changed to a cysteine (S 61C).
  • S 61C cysteine
  • a related sequence that has an S61C substitution has a cysteine introduced into the position that corresponds to serine 61 of SEQ ID NO:1 when the related sequence is .5 aligned for maximal correspondence to SEQ ID NO: 1.
  • the residue at the position of the related ribonuclease that corresponds to position 61 need not be a serine.
  • Nucleic acid and “polynucleotide” are used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or
  • JO modified backbone residues or linkages which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids
  • nucleic acid sequence can readily be determined from the sequence of the other strand.
  • any particular nucleic acid sequence set forth herein also discloses the complementary strand.
  • Polypeptide “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid 50 polymers, as well as, amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -
  • amino acid analogs refers to compounds that have the same fundamental chemical structure as a naturally occurring amino acid, i.e., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • R groups e.g., norleucine
  • modified peptide backbones but retain
  • amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the
  • Constantly modified variants applies to both nucleic acid and amino acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to
  • nucleic acid 5 variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid can be modified to yield a functionally 0 identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
  • amino acid sequences With respect to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologues, and alleles of the invention.
  • substitutions may be made wherein an aliphatic amino acid (G, A, I, L, or V) is substituted with another member of the group, or substitution such as the substitution of one polar residue for another, such as arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine.
  • substitutions may be made wherein an aliphatic amino acid (G, A, I, L, or V) is substituted with another member of the group, or substitution such as the substitution of one polar residue for another, such as arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine.
  • substitutions may be made wherein an aliphatic amino acid (G, A, I, L, or V) is substituted with another member of the group, or substitution such as the substitution of one polar residue for another, such as arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine.
  • the term “antibody” refers to an immunoglobulin molecule, or portion thereof, immunologically reactive with a particular antigen, and includes both polyclonal and monoclonal antibodies.
  • the term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), recombinant single chain Fv fragments (scFv), and disulfide stabilized (dsFv) Fv fragments.
  • the term “antibody” includes all antigen binding forms of antibodies (e.g., Fab', F(ab')2, Fab, Fv and rlgG.) as well as engineered forms of such binding forms.
  • An antibody immunologically reactive with a particular antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors or by immunizing an animal with the antigen or with DNA encoding the antigen.
  • nucleic acid or protein when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular 5 components with which it is associated in the natural state. It is preferably in a homogeneous state, although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein which is the predominant species present in a preparation is substantially purified.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., amino acid
  • ZO definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence algorithm program parameters Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence 0 comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local alignment algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm of Needleman & Wunsch, J. MoI. Biol.
  • BLAST and BLAST 2.0 are used, typically with the default parameters, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighborhood word score threshold (Altschul et al, supra).
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. ScL USA 89:10915)).
  • W wordlength
  • E expectation
  • BLOSUM62 scoring matrix see Henikoff & Henikoff (1989) Proc. Natl. Acad. ScL USA 89:10915.
  • the BLAST2.0 algorithm is used with the default parameters.
  • “Pharmacologically effective amount” refers to an amount of an agent effective to produce the intended pharmacological result.
  • “Pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, buffers, and excipients, such as a phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsions, such as an oil/water or water/oil emulsion, and various combinations thereof
  • Suitable pharmaceutical carriers and formulations are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co., Easton, 1995).
  • Preferred pharmaceutical carriers depend upon the intended mode of administration of the active agent. Typical modes of administration include enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous or intraperitoneal i0 injection; or topical, transdermal, or transmucosal administration).
  • a "pharmaceutically acceptable salt” is a salt that can be formulated into a compound for pharmaceutical use including, e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.
  • Anti-tumor RNases have clinical use either alone, combined with drugs, or as the toxic component of targeted therapies. In the latter case, the RNase is conjugated to a targeting moiety. Traditional chemical conjugation reactions typically treat the RNase to chemically introduce a cysteine residue that can be used to link the RNase to another molecule. These methods require a large excess of unmodified RNase. Furthermore, the
  • the current invention provides genetically modified thiol-containing RNase molecules that can be used in much lower amounts to generate chemical conjugate. These modified RNase molecules have an inserted cysteine, resulting in a thiol group that provides a specific attachment site for an RNase to a conjugate molecule, e.g., a targeting moiety such as an antibody. Because there is only one site of attachment per molecule of RNase, the side reactions, such as multimerization of the 5 conjugate and formation of RNase multimers, that occur with chemically modified RNase containing greater than one attachment site per RNase are eliminated.
  • Cysteine-modified RNases of the invention have cysteines substituted for particular residues. These residues provide sites for chemical linkages where the resulting chemical conjugate maintains RNase activity.
  • RNase ⁇ cys refers to .0 cysteine-modified RNases. Positions for cysteine substitution can be selected, e.g., based on their geometric distance from the naturally occurring cysteines such that they would not interfere with the proper folding of the molecule.
  • RNases that are modified in accordance with the invention are pancreatic RNase A '.5 family members, which are known to be cytotoxic when delivered to target cells. Comparisons of the ribonuclease sequences provided here can be made to described sequences in the pancreatic RNase A superfamily. Many of such members are known and include, but are not limited to, ONCONASE® (Ardelt, W. et al, J. Biol. Chem. 266:245 (1991)); eosinophil derived neurotoxin (EDN) and human eosinophil cationic protein (ECP) »0 (Rosenberg, et al, J. Exp. Med.
  • ONCONASE® &Rana RNase from liver, rapLRl
  • ONCONASE® and rapLRl native and variant nucleic acid and polypeptides sequences and methods of making such proteins are well known in the art (see, e.g., U.S. Patent No. 6,869,604; 6,045,793; and 6,395,276).
  • cysteine-modified RNases are described with reference to the amino acid sequence of native rapLRl (SEQ ID NO:1).
  • conservative variants of rapLRl can also be used.
  • Such variants typically comprise a polypeptide that has RNase activity, but comprises conservative modifications to regions outside of the active site. Such conservative modifications can be substitutions, deletions, or insertions.
  • the RNase molecules typically share at least 60% identity, more often at least 80%, 85%, or at least 90% or greater amino acid sequence identity to SEQ ID NO:1.
  • the 5 sequence identity can be over the entire molecules or can be over a segment, for example 90 contiguous amino acids of SEQ ID NO: 1.
  • residues corresponding to the active site residues, Pyrl, Lys9, HislO, Lys31, Thr35, His97, and Phe98 of SEQ ID NO:1 are conserved in variant RNase A family members of the invention.
  • the cysteine-modified RNase proteins of the invention comprise mutated RNase proteins in which cysteine is substituted for the naturally occurring residue at particular sites.
  • cysteines are substituted at positions 61, 60, 40, or 80, as determined with reference to SEQ ID NO: 1. It is understood that such position designations do not
  • L 5 indicate the absolute residue number in the claimed molecule per se, but indicate where in the claimed RNase proteins the residue occurs when the amino acid sequence of the RNase to be modified is maximally aligned with the rapLRl sequence of SEQ ID NO:1. Alignment can be performed either manually or using a sequence comparison algorithm described above. It is further understood that the parent RNase that is mutated to incorporate the cysteine residue
  • a cysteine-modified RNase of the invention includes an RNase with a cysteine introduced at position 61, position 60, position 40, or position 80, regardless of whether a serine, threonine, arginine, or lysine, respectively, is present at that position in the parent RNase prior to cysteine modification.
  • a site that corresponds to position 61, position 60, position 40, or position 80 of 25 SEQ ID NO:1 can often be determined by sequence alignments.
  • the position is determined by comparing the structure of a candidate RNase A family member protein to a structure of a rapLRl protein.
  • the structure of rapLRl can be determined, e.g., using co-ordinates archived in the Protein Databank, Accession IONC (native amphibian Ranprinase) and adapting the co-ordinate using molecular modeling software, e.g., RasMOL 0 software (RasWin Molecular Graphics, Version 2.7.2.1).
  • the cysteines are introduced into the region that is represented by T60 and S61 in SEQ ID NO:1.
  • the T60 and S61 residues are found near the apex of the connecting bridge between two three-stranded beta sheets.
  • Examination of spacefilling models for [1ONC], which is analogous to RapLRl indicate that T60 and S61 are at the edge of a slightly concave 'face' outlined by the residues isoleucine 51 through serine 61. This face is surrounded by the protruding side chains of lysine 49, lysine 55, lysine 85, and glutamine 62, as well as serine 61 or threonine 60 (depending on the mutant). Accordingly, cysteine substitutions can be introduced in the region corresponding to isoleucine 51 through serine 61 as determined by structural analysis or by sequence alignment.
  • R40C can be identified from its position within a distinct amino acid sequence motif. An alignment of several RNase superfamily members was described previously, e.g., in, Youle, et al, Crit. Rev. ofTher. Drug, Carrier Systems 10:1-28 (1993). On a linear basis, the RapLRl, R40, is located in a region between the second and third conserved cysteines. The R40C mutation is the eleventh residue following the second cysteine (defined in the following discussion as position 1).
  • This region contains highly conserved residues including an active site lysine residue (position 2), a conserved nucleobase-binding motif NTF (postions 5-7), several semi-conserved hydrophobic residues (V/I/L at positions 8, 15 and 18), and the last cysteine residue (position 19).
  • a position in a RNase superfamily member that corresponds to the R40C mutation site in SEQ ID NO:1 is within the pattern established by these conserved residues.
  • the position of the R40C mutation is well defined by the flanking pattern of conserved residues and is independent of changes outside this region.
  • the R40C position can also be identified from a structural viewpoint.
  • a structural analogy can be drawn between the known geometric coordinates of a related RNase A superfamily member, maintained in the Brookhaven Protein Database, (identified as [ 1 ONC] , Mosimann, S. C, et al., J. MoI. Biol. 236:1141-1153 (1994)) and the RapLRl sequence.
  • the front-side of the approximately hemispherical molecule is defined as the side containing the RNA-binding groove.
  • the R40C mutation site is one of two arginine residues located in the approximate middle of the curved, backside of the hemisphere.
  • R40 is uniquely located after a beta-strand in which R40 forms part of a two residue bridge connecting the beta strand to an 8-residue alpha-helical structure.
  • the apex of this bridge orients the side chain of R40 into the solvent accessible space surrounding the molecule.
  • S61C can be identified from its position within a distinct amino acid sequence motif. As noted above, an alignment of several RNase superfamily members was described previously, e.g., in, Youle, et ah, supra. On a linear basis, the RapLRl, S61C site, is located in a region between the third and fourth conserved cysteines. The S61C mutation site is the seventh residue prior to the fourth conserved cysteine. Thus, the position of the S61C mutation is well defined relative to the conserved fourth cysteine residue of the RNase A superfamily and is independent of changes outside this region.
  • S61C can also be identified from a structural viewpoint. Based on the related RNase A superfamily member structure, supra, the front-side of the approximately hemispherical molecule is defined the side containing the RNA-binding groove. The S61C mutation site is located slightly off-center in the curved, backside of the hemisphere. S61 is located within a short four-residue bridge connecting two structural three-stranded, beta sheets. More specifically, the S61C site is located two residues before the first residue of third beta strand. Finally, S61C is located approximately opposite the R40C replacement site across the RapLRl long axis. This bridge is constrained by the beta stands to orient the side chain of S61C into the solvent accessible space surrounding the molecule.
  • T60C can be identified from its position within a distinct amino acid sequence motif. An alignment of several RNase superfamily members was described, supra.. On a linear basis, the RapLRl, T60C, is located in a region between the third and fourth conserved cysteines. The T60C mutation site as the eighth residue prior to the fourth conserved cysteine. Thus, the position of the T60C mutation is well defined relative to the conserved fourth cysteine residue of the RNase A superfamily and is independent of changes outside this region.
  • T60C can also be identified from a structural viewpoint. Ih the structure, the front- side of the approximately hemispherical molecule is defined herein as the side containing the RNA-binding groove.
  • the T60C mutation site is located slightly off-center in the curved, backside of the hemisphere. T60 is located within a short four-residue bridge connecting two structural three-stranded, beta sheets. More specifically, the T60C mutation site is located three residues before the first residue of third beta strand. Finally, the T60C mutation site is located approximately opposite the R40C replacement site across the RapLRl long axis. This bridge is constrained by the beta strands to orient the side chain of T60C into the solvent accessible space surrounding the molecule.
  • K80C can be identified from its position within a distinct amino acid sequence motif.
  • the RapLRl, K80C mutation site is located in a region between the last two highly conserved cysteine motifs respectively of the form CxY and VxC.
  • the K80C mutation site is the sixth residue following the cysteine residue of the CxY motif and the ninth residue prior to the cysteine residue of the VxC motif.
  • the K80C mutation is well defined relative to the fifth and sixth highly conserved cysteine residues of the RNase A superfamily and is independent of changes outside this region.
  • K80C can also be identified from a structural viewpoint.
  • the K80C mutation site is located mid-way between the front- and backsides of the molecule.
  • K80C is the fourth residue of the third beta strand. This highly constrained beta strand orients the side chain of K80C into the solvent accessible space surrounding the molecule.
  • a cysteine residue can be introduced using methodology well known in the art, e.g., various site-directed mutagenesis protocols. For example, often the cysteine is introduced by synthesizing an oligonucleotide primer containing the mutation. The primer containing the mutation is synthesized to only one strand of the double-stranded template. A polymerase such as PfuTurbo® extends the mutagenic primer with a high level of accuracy under non- strand displacing conditions, generating one strand bearing the mutation. The parental DNA template is digested away to enrich the mutated single-stranded DNA. The mixture is then transformed into competent cells in which the mutant single-stranded DNA is converted into duplex form in vivo.
  • PfuTurbo® a polymerase
  • Kits for performing site direct mutagenesis are commercially available, e.g., from Stratagene, La Jolla, California. Recombinant production
  • This invention relies on routine techniques in the field of recombinant genetics for the preparation of cysteine-modified RNase polypeptide. Basic texts disclosing the general methods of use in this invention include Sambrook & Russell, Molecular Cloning, A
  • RNase of SEQ ID NO:1 ,2, 3, 4, or 5, or a conservative variant thereof is typically expressed in a host cell using a nucleic acid that encodes an amino terminal methionine.
  • Expression of RNases is well known in the snt,(see, e.g., U.S. Patent Nos. 6,869,604 and 6,045,793).
  • Eukaryotic and prokaryotic host cells may be used such as animal cells, insect cells, bacteria, fungi, and yeasts. Methods for the use of host cells in expressing isolated nucleic acids are well known to those of skill and may be found, for example, in the general reference, supra. Accordingly, this invention also provides for host cells and expression vectors comprising the nucleic acid sequences described herein.
  • Nucleic acids encoding RNase proteins can be made using standard recombinant or synthetic techniques. Nucleic acids may be RNA, DNA, or hybrids thereof. Given the polypeptides of the present invention, one of skill can construct a variety of clones containing functionally equivalent nucleic acids, such as nucleic acids that encode the same polypeptide. Cloning methodologies to accomplish these ends, and sequencing methods to verify the sequence of nucleic acids are well known in the art.
  • the nucleic acid compositions of this invention are synthesized in vitro.
  • Deoxynucleotides may be synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetrahedron Letts. 22(20): 1859-1862 (1981), using an automated synthesizer, e.g., as described in Needham-VanDevanter, et al., Nucleic Acids Res. 12:6159-6168 (1984).
  • polypeptide sequences are altered by changing the corresponding nucleic acid sequence and expressing the polypeptide.
  • polypeptide sequences can also be generated synthetically using commercially available peptide synthesizers to produce any desired polypeptide.
  • ribonucleases see, e.g., U.S. Patent Nos. 6,869,604 and 6,045,793.
  • the physical characteristics and general properties of RNases are known to skilled practitioners.
  • the active site of RNase A family members has been identified and specific effects of mutations in RNases are known.
  • general knowledge regarding the nature of proteins and nucleic acids allows one of skill to select appropriate sequences with activity similar or
  • an expression vector is constructed that includes such elements as a promoter to direct transcription, a transcription/translation terminator, a ribosome
  • Suitable bacterial promoters are well known in the art and described, e.g., in the references providing expression cloning methods and protocols cited hereinabove.
  • Bacterial expression systems for expressing ribonuclease are available in, e.g., E. coli, Bacillus sp., and Salmonella (see, also, Palva, et al, Gene 22:229-235 (1983); Mosbach, et al, Nature 302:543-545 (1983). Kits for such expression
  • .0 systems are commercially available.
  • Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.
  • the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the ribonuclease-encoding nucleic acid in host cells.
  • the 15 cassette thus contains a promoter operably linked to the nucleic acid sequence encoding ribonuclease and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination.
  • the nucleic acid sequence encoding ribonuclease may be linked to a cleavable signal peptide sequence to promote secretion of the encoded protein by the transformed cell.
  • signal peptides Such signal peptides
  • the signal peptides from human ribonucleases such as EDN, tissue plasminogen activator, insulin, and others.
  • Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.
  • the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination.
  • the termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
  • the particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as ⁇ BR322 based plasmids, pSKF, pET 15b, pET23D, pET-22b(+), and fusion expression systems such as GST and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., 6-his.
  • vectors comprise, in addition to the expression cassette containing the coding sequence, the T7 promoter, transcription initiator and terminator, the ⁇ BR322 ori site, a bla coding sequence and a lacl operator.
  • a cDNA encoding an RNase of this invention was inserted into pET-22b(+).
  • the vectors comprising the nucleic acid sequences encoding the RNase molecules or the fusion proteins may be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines.
  • vectors may be expressed by transgenic animals, preferably sheep, goats and cattle. Typically, in this expression system, the recombinant protein is expressed in the transgenic animal's milk.
  • the expression vectors or plasmids of the invention can be transferred into the chosen host cell by well-known methods such as calcium chloride transformation for E. coli and calcium phosphate treatment, liposomal fusion or electroporation for mammalian cells.
  • Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo and hyg genes.
  • the RNase protein can be purified according to standard procedures of the art, including ammonium sulfate precipitation, column chromatography (including affinity chromatography), gel electrophoresis and the like ⁇ see, generally, R. Scopes, Protein Purification, Springer-Verlag, N. Y. (1982), Guider, Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. N. Y. (1990); Sambrook and Ausubel, both supra; and U.S. Patent Nos. 6,869,604; 6,045,793; and 6,395,276).
  • an expressed RNase of the 5 invention that comprises a signal peptide is cleaved within the bacterial periplasm.
  • the protein is typically treated with a cleaving agent or a combination of cleaving agents to remove the methionine.
  • cleaving the amino terminal methionine is meant cleaving the amino 0 terminal methionine or amino terminal peptide from an expressed RNase polypeptide.
  • the cleaving agent may be a proteolytic enzyme such as an exopeptidase or endopeptidase (collectively, "peptidase") or a chemical cleaving agent.
  • Exopeptidases include aminopeptidase M (Pierce, Rockford, IL) which sequentially remove amino acids from the amino-terminus. Cleavage of the amino terminal methionine by exopeptidases may L 5 be controlled by modulating the enzyme concentration, temperature, or time under which the cleavage takes place. The resulting mixture may be purified for the desired protein by means well known to those of skill, for example, on the basis of length by electrophoresis.
  • the chemical cleaving agent, cyanogen bromide is conveniently employed to selectively cleave methionine residues.
  • the cleaving agent employed to cleave the amino terminal methionine will typically be chosen so as not to break a peptide bond within the RNase polypeptide.
  • use of a particular cleaving agent may guide the choice of conservative substitutions of the conservative variants of the polypeptides of the present invention.
  • the sequence of the native protein of SEQ ID NO:1 contains a methionine at position 23. This methionine
  • !5 can be changed, for example, to a leucine to prevent cleavage of the RNase polypeptide chain with a -1 methionine by CNBr.
  • RNases of the invention can be purified using standard protocols. An exemplary description of purification from bacterial cultures is provided below. Purification ofRNasefrom Bacterial Cultures
  • the KISf ases of this invention can be isolated and purified from the broth in which the expressing bacteria have been grown without having to resort to the cell lysis methods detailed below.
  • RNase expressed in E. coli may be exported into the periplasm of the bacteria.
  • the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to skill in the art (see Ausubel, and Trayer, H.R. & Buckley, III, C.E., J Biol. Chem. 245(18):4842 (1970)).
  • the proteins may form insoluble aggregates.
  • inclusion bodies Purification of aggregate proteins (hereinafter referred to as inclusion bodies) involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells.
  • the inclusion bodies are recovered and solubilized, typically by the addition of a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor (or a combination of solvents each having one of these properties); the proteins that formed the inclusion bodies may then be renatured by dilution or dialysis with a compatible buffer.
  • Suitable solvents include, but are not limited to urea, formamide, and guanidine hydrochloride.
  • the protein can be separated from other bacterial proteins by standard separation techniques.
  • cysteine-modified RNase proteins of the invention can be used in any applications in which an RNase such as ONCONASE® or rapLRl is used (see, e.g., 6,869,604; 6,045,793), they are typically used as components of conjugates in which the RNase is conjugated to another molecules (e.g., a cytotoxin, a label (directly or indirectly detectable), a ligand, or a drug or liposome).
  • the RNase is conjugated to a targeting moiety, e.g., an antibody or a ligand that binds a cell surface receptor.
  • the procedure for attaching the RNase to the other molecule can vary according to the nature of the molecule to which to RNase is to be attached.
  • the cysteine-modified RNase is attached to the other agent through a disulfide bond formed with the cysteine that has been introduced into the RNase.
  • the free thiol group can also be used in other types of linkages such as thioether linkages.
  • the moiety to which the RNase is joined can be chemically treated to provide a proper residue to form a bond with the introduced cysteine in the RNase.
  • Many procedures and linker molecules for attachment of various compounds including radionuclides, metal chelates, toxins and drugs to proteins such as RNases are known.
  • the cysteine modified RNase can be directly joined to the other molecule or can be joined through a linker.
  • a "linker”, as used herein, is a molecule that is used to join the RNase to the second molecule in the conjugate.
  • the linker is typically capable of forming covalent bonds to both molecules.
  • Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where both molecules are polypeptides, the linkers may be joined to the constituent amino acids through their side groups(for example through a disulfide linkage to cysteine).
  • chimeric conjugates comprising linkages that are cleavable in the vicinity of the target site may be used when the effector moiety is to be released at the target site. Cleaving of the linkage to release the agent from the ligand may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site.
  • a linker which is cleavable under conditions present at the tumor site (e.g., when exposed to tumor-associated enzymes or acidic pH) may be used.
  • cleavable linkers are known to those of skill in the art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014.
  • the mechanisms for release of an agent from these linker groups include, for example, irradiation of a photolabile bond and acid- catalyzed hydrolysis.
  • U.S. Pat. No. 4,671,958, for example includes a description of immunoconjugates comprising linkers that are cleaved at the target site in vivo by the proteolytic enzymes of the patient's complement system.
  • the cysteine-modified RNase conjugates of the invention are employed as the toxic moiety in therapeutic targeted toxin conjugates that selectively kill target cells, e.g., tumor cells that overexpress a protein to which the targeting moiety of the conjugate binds.
  • the targeting moiety can be any ligand binding moiety, including a hormone or growth factor that binds a cell surface receptor, a peptide that binds to the target protein of interest, or an antibody.
  • the target to which the ligand binding moiety binds is preferentially overexpressed on the cell type to be killed, e.g., on tumor cells.
  • targeting moieties include, but are not limited to, monoclonal antibodies directed against tumor cell markers such as heregulin, CD22, prostate-specific antigen, etc.; cytokines that target tumor cells, such as tumor necrosis factor; and other tumor cell binding proteins, including hCG.
  • cytotoxic factors can be joined to one ligand binding moiety.
  • the RNases of this invention can be joined to a monoclonal antibody directed against a tumor cell marker that is also joined to a synthetic drug with cytotoxic activity, such as paclitaxel or methotrexate.
  • Conjugates that comprise the cysteine-modified RNases of the invention also find use as cytotoxic agents against cells other than tumor cells.
  • the RNases of this invention can be joined to ligand binding moieties that specifically target B cells that secrete antibodies directed against self.
  • the RNases of this invention are useful in the treatment of autoimmune diseases.
  • Conjugates comprising cysteine-modified RNases of the invention can be administered for the treatment of disease, e.g., cancer, where it is desirable to target a cell with a cytotoxic conjugate.
  • the compositions for administration will commonly comprise a solution of the conjugate dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier.
  • a pharmaceutically acceptable carrier preferably an aqueous carrier.
  • aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of cysteine-modified RNase conjugate in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.
  • a typical pharmaceutical composition of the present invention for intravenous administration would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100 mg per patient per day may be used. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as REMINGTON'S PHARMACEUTICAL SCIENCE, 19TH ED., Mack Publishing Company, Easton, Pennsylvania (1995).
  • compositions of this invention can be administered for therapeutic treatments.
  • compositions are administered to a patient suffering from a disease, in an amount sufficient to cure or at least partially arrest the disease and its complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health.
  • An effective amount of the composition is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.
  • compositions Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the patient.
  • the composition should provide a sufficient quantity of cysteine-modified RNase conjugate protein to effectively treat the patient.
  • the dosage is administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy.
  • the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.
  • the conjugate proteins of this invention are useful for parenteral, topical, oral, or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment.
  • the pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration.
  • unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and lozenges.
  • the subject conjugate proteins and pharmaceutical compositions of this invention when administered orally, must be protected from digestion. This is typically accomplished either by complexing the protein with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the protein in an appropriately resistant carrier such as a liposome. Means of protecting proteins from digestion are well known in the art.
  • compositions of this invention are particularly useful for parenteral administration, such as intravenous administration or administration into a body cavity or lumen of an organ.
  • Controlled release parenteral formulations of the conjugate compositions of the present invention can be made as implants, oily injections, or as particulate systems.
  • Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles.
  • Microcapsules contain the therapeutic cysteine-modified RNase conjugate as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 ⁇ m are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 ⁇ m so that only nanoparticles are administered intravenously. Microparticles are typically around 100 ⁇ m in diameter and are administered subcutaneously or intramuscularly. See, e.g., Kreuter, J., COLLOIDAL DRUG DELIVERY SYSTEMS, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp.
  • Polymers can be used for ion-controlled release of conjugate compositions of the present invention.
  • Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, R., Accounts Chem. Res. 26:537-542 (1993)).
  • the block copolymer, polaxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston, et al, Pharm. Res. 9:425-434 (1992); and Pec, et al, J. Parent. ScL Tech.
  • hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema, et al, Int. J. Pharm. 112:215-224 (1994)).
  • liposomes are used for controlled release as well as drug targeting of the lipid- capsulated drug (Betageri, et al, LIPOSOME DRUG DELIVERY SYSTEMS, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known. See, e.g., U.S. Pat. No.
  • cysteine-modified RNase conjugate of the present invention Among various uses of the cysteine-modified RNase conjugate of the present invention are included a " variety of disease conditions caused by specific human cells that may be eliminated by the toxic action of the conjugate protein.
  • Cysteine substitutions were introduced into the parent rapLRl nucleic acid sequence shown in Figure 1. The substitutions were introduced into the sites as shown in Figures 2, 3, 4, and 5, resulting in the introduction of cysteine residues at position 61, 60, 40, and 80, respectively. Methods are available in the art for introducing site-directed cysteine f ⁇ ee-thiol attachment sites into proteins to facilitate conjugation of the proteins to another moiety (see, e.g., Chilkoti et al., Bioconj. Chem. 5:504-507, 1994). Commercial kits are also available. [0095] The following primers were made (each was prepared 5 ' phosphorylated, bold, italicized type indicates those nucleotides coding for the introduced cysteine):
  • K80C 5'-CCTTGCAAGTATAAATTArCrCAAATCAACTAATACATTTTG-S' (SEQ ID NO: 11)
  • R40C 5 '- AACACTTTTATCTATTCATYrCCCTGAGCCAGTGAAGGCC-3 ' (SEQ ID NO: 12)
  • T60C 5 '-TCCAAAAATGTGTTAACT ⁇ CCTCTGAGTTTTATCTCTCTCTG-S' (SEQ ID NO: 14)
  • the QuikChange Multi Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) was used for site-directed mutagenesis of rapLRl DNA (100 ng ) following the manufacturer's instructions using 100 ng of the respective 5 '-phosphorylated primers noted above. Each of the respective clones was sequenced to verify the mutations.
  • mut#l is S61C
  • mut#2 is T60C
  • mut#3 is R40C
  • mut#5 is K80C.
  • ribonuclease activity using yeast transfer RNA was determined at 37°C by monitoring the formation of perchloric acid-soluble nucleotides. Activity was measured in a final volume of 0.3 mL containing 0.32 mg/mL yeast tRNA, 130 mM MES 5 pH 6.0, 170 ⁇ g/ml human serum albumin and the appropriate concentrations of RNase (lO ⁇ L additions, dilutions made in 0.5 mg/mL human serum albumin).
  • the mixtures were incubated for 2 h at 37 0 C before termination with 0.75 mL of 3.4% ice-cold perchloric acid.
  • the stopped reactions were incubated on ice for 10 min before centrifugation (10 min) at top speed in an Eppendorf centrifuge. Absorbance of the supernatant was determined at OD 26 o nm - Replicate values were averaged, the blank (those tubes with dilution buffer and no enzyme) subtracted, and the data plotted versus the concentration of the RNase. Each assay was performed at least twice and the data averaged.
  • Figure 3 shows the RNase activity of the modified and parent (“std") rapLRl proteins. The results indicate that the cysteine modified proteins had RNase activity.
  • test samples (10 ⁇ L) were added to the appropriate well, and the cells incubated for 3 days at 37°C in a humidified CO 2 incubator. After 3 days the media was replaced with 100 ⁇ L of serum- and leucine- free RPMI- 1640 and 0.1 mCi of [ 14 C]leucine (lO ⁇ L) was added. The cells were incubated for an additional 2-4 hrs before being harvested onto glass fiber filters using a PHD cell harvester, washed with
  • ICs 0 the concentration of test sample that inhibits protein synthesis by 50%, was determined from semilogarithmic plots in which protein synthesis as a percentage of control (buffer-treated cells) was plotted versus test protein concentration.
  • IC 50 the concentration of test sample that inhibited protein synthesis by 50% as determined from semilogarithmic plots in which protein synthesis as a percentage of control (buffer- treated samples) was plotted versus test protein concentration.
  • the cysteine-modified rapLRl proteins were employed as conjugates to antibodies.
  • the modified rapLRl proteins were conjugated to the antibody RFB4 or hHMGl.
  • the conjugates were prepared using standard techniques.
  • the cysteine modified rapLRl was incubated for 1 h at room temperature with 2 niM DTT to free the thiol group blocked during the renaturation procedure. Following removal of excess DTT from the rapLRl solution by PD-10 chromatography, the thiol modified rapLRl was incubated overnight at room temperature with 2IT/DTNB modified antibody (1.5-2 fold molar excess of thiol rapLRl over the number of available coupling sites on the antibody).
  • the reaction between the thiol rapLRl and antibody was monitored spectrophotometrically by the appearance of thionitrobenzoate ion, a by-product released as the disulfide bonds between the RNase and antibody are formed.
  • the conjugate was separated from unconjugated RNase by chromatography on a Toyo Soda TSK3000 SW column (Toso Haas, Montgomeryville, PA) as described in Newton and Rybak (Newton & Rybak, Construction of R ⁇ bonuclease- Antibody Conjugates for Selective Cytotoxicity. Methods in Molecular Medicine. 25: Drug Targeting: Strategies, Principles, and Applications, G.E. Francis and C. Delgado (ed.), Humana Press, Inc., Totowa, NJ, pp 27-35.)
  • cysteine insertion sites S61C and T60C were particularly useful when a high substitution of rapLRl to antibody is required. All four sites, S61C, T60C, R40C and K80C sites are very effective for use in those conjugates requiring a lower substitution (less than 3 mols rapLRl/mol antibody).
  • the antibody-rapLRl conjugates were evaluated for RNase activity, the ability to bind to the target cell and for cytotoxic activity.
  • a summary of activities of rapLRl antibody conjugates is provided in Table 2.
  • Table 2 Summary of activities of rapLRl ⁇ cys antibody conjugates compared to conjugated made with standard rapLRl .
  • SEQ ID NO: 7 nucleic acid sequence encoding cysteine-modified RNase, modification at position 61 (S61C)
  • SEQ ID NO:8 nucleic acid sequence encoding cysteine-modified RNase, modification at position 60 (T60C)
  • SEQ ID NO: 9 nucleic acid sequence encoding cysteine-modified RNase, modification at position 40 (R40C)

Abstract

L'invention concerne des molécules d'RNases modifiées au moyen de la cystéine et des séquences d'acides nucléiques codant de telles molécules. Les RNases modifiées au moyen de la cystéine peuvent être utilisées pour fabriquer des conjugués chimiques en autres fractions, telles que des anticorps. L'invention concerne également des conjugués de RNases modifiées au moyen de la cystéine et des procédés d'utilisation de ces conjuguées.
PCT/US2006/038180 2005-09-30 2006-09-29 Rnase anti-tumeur recombinante modifiee WO2007041361A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US72229505P 2005-09-30 2005-09-30
US60/722,295 2005-09-30

Publications (1)

Publication Number Publication Date
WO2007041361A1 true WO2007041361A1 (fr) 2007-04-12

Family

ID=37758635

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2006/038180 WO2007041361A1 (fr) 2005-09-30 2006-09-29 Rnase anti-tumeur recombinante modifiee

Country Status (1)

Country Link
WO (1) WO2007041361A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013079924A3 (fr) * 2011-11-29 2013-08-01 The University Of Sheffield Exonucléase modifiée

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997031116A2 (fr) * 1996-02-21 1997-08-28 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, represented by THE SECRETARY OF THE DEPARTMENT OF H EALTH AND HUMAN SERVICES Proteines de ribonuclease de recombinaison
US6175003B1 (en) * 1999-09-10 2001-01-16 Alfacell Corporation Nucleic acids encoding ribonucleases and methods of making them
US6280991B1 (en) * 1997-10-15 2001-08-28 Wisconsin Alumni Research Foundation Engineered cytotoxic ribonclease

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997031116A2 (fr) * 1996-02-21 1997-08-28 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, represented by THE SECRETARY OF THE DEPARTMENT OF H EALTH AND HUMAN SERVICES Proteines de ribonuclease de recombinaison
US6280991B1 (en) * 1997-10-15 2001-08-28 Wisconsin Alumni Research Foundation Engineered cytotoxic ribonclease
US6175003B1 (en) * 1999-09-10 2001-01-16 Alfacell Corporation Nucleic acids encoding ribonucleases and methods of making them

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BACKER MARINA V ET AL: "Engineering S-protein fragments of bovine ribonuclease A for targeted drug delivery.", PROTEIN EXPRESSION AND PURIFICATION, vol. 26, no. 3, December 2002 (2002-12-01), pages 455 - 461, XP002422205, ISSN: 1046-5928 *
BERNHARD SUSAN L ET AL: "Cysteine analogs of recombinant barley ribosome inactivating protein form antibody conjugates with enhanced stability and potency in vitro", BIOCONJUGATE CHEMISTRY, vol. 5, no. 2, 1994, pages 126 - 132, XP002422207, ISSN: 1043-1802 *
KREITMAN R J ET AL: "Site-Specific Conjugation to Interleukin 4 Containing Mutated Cysteine Residues Produces Interleukin-4-Toxin Conjugates with Improved Binding and Activity", BIOCHEMISTRY, AMERICAN CHEMICAL SOCIETY. EASTON, PA, US, vol. 33, no. 38, 1994, pages 11637 - 11644, XP001206219, ISSN: 0006-2960 *
MOSIMANN S C ET AL: "Comparative Molecular Modeling and Crystallization of P-30 Protein: A Novel Antitumor Protein of Rana pipiens Oocytes and Early Embryos", PROTEINS: STRUCTURE, FUNCTION AND GENETICS, ALAN R. LISS, US, vol. 14, no. 3, 1 November 1992 (1992-11-01), pages 392 - 400, XP000675464, ISSN: 0887-3585 *
MOSIMANN S C ET AL: "Refined 1.7 A X-ray Crystallographic Structure of P-30 Protein, an Amphibian Ribonuclease with Anti-Tumor Activity", JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB, vol. 236, no. 4, 1994, pages 1141 - 1153, XP000675664, ISSN: 0022-2836 *
NEWTON D L ET AL: "Cytotoxic Ribonuclease Chimeras. Targeted Tumoricidal Activity in vitro and in vivo", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM,, US, vol. 267, no. 27, 25 September 1992 (1992-09-25), pages 19572 - 19578, XP002235500, ISSN: 0021-9258 *
RYBAK S M ET AL: "Rational Immunotherapy with Ribonuclease Chimeras: An Approach Towards Humanizing Immunotoxins", CELL BIOPHYSICS, vol. 21, no. 1-3, August 1992 (1992-08-01), pages 121 - 138, XP009008218, ISSN: 0163-4992 *
RYBAK S N ET AL: "Cytotoxic Potential of Ribonuclease and Ribonuclease Hybrid Proteins", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM,, US, vol. 266, no. 31, 5 November 1991 (1991-11-05), pages 21202 - 21207, XP002007717, ISSN: 0021-9258 *
STIMMEL JULIE B ET AL: "Site-specific conjugation on serine-cysteine variant monoclonal antibodies", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 275, no. 39, 29 September 2000 (2000-09-29), pages 30445 - 30450, XP002422206, ISSN: 0021-9258 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013079924A3 (fr) * 2011-11-29 2013-08-01 The University Of Sheffield Exonucléase modifiée

Similar Documents

Publication Publication Date Title
Boix et al. Role of the N terminus in RNase A homologues: differences in catalytic activity, ribonuclease inhibitor interaction and cytotoxicity
CA2325341A1 (fr) Molecules chimeres mutagenisees a base d'il-13
JP3469247B2 (ja) 組換えリボヌクレアーゼタンパク質
AU707004B2 (en) Modified proteins
JP4214397B2 (ja) 変異ベロ毒素を用いた抗癌剤
AU752370B2 (en) Utilization of wolinella succinogenes asparaginase to treat diseases associated with asparagine dependence
US6045793A (en) Recombinant ribonuclease proteins
AU727310B2 (en) Human DNASE resistant to actin inhibition
US20030099629A1 (en) Recombinant onconase and chemical conjugates and fusion proteins of recombinant onconase
WO2007041361A1 (fr) Rnase anti-tumeur recombinante modifiee
KR19990087571A (ko) 하향 조절 내성 c3 컨버타제
US6869604B1 (en) Recombinant anti-tumor RNAse
US6569429B1 (en) Human DNase II
ES2241268T3 (es) Onconasa recombinante y proteinas de fusion de onconasa recombinante.
EP1068332B1 (fr) Ribonuclease antitumorale recombinee
RU2811464C2 (ru) Новые варианты гиалуронидазы с улучшенной стабильностью и содержащая их фармацевтическая композиция
CN110461354A (zh) 蛋白质缀合物
AU773347B2 (en) Recombinant anti-tumor RNASE
EP1219706A2 (fr) Procédé de préparation d' asparaginase recombinée à partir de Wolinella succinogenes
EP1277838A1 (fr) RNAse anti tumorale recombinante
CA2401916A1 (fr) Ribonuclease antitumorale recombinee
WO2010038010A1 (fr) Analogues de somatostatine
MXPA97001783A (es) Proteinas c3 humanas modificadas

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06836159

Country of ref document: EP

Kind code of ref document: A1