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WO2009111729A1 - Anticorps anti-dnp et procédés d’utilisation - Google Patents

Anticorps anti-dnp et procédés d’utilisation Download PDF

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Publication number
WO2009111729A1
WO2009111729A1 PCT/US2009/036383 US2009036383W WO2009111729A1 WO 2009111729 A1 WO2009111729 A1 WO 2009111729A1 US 2009036383 W US2009036383 W US 2009036383W WO 2009111729 A1 WO2009111729 A1 WO 2009111729A1
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Prior art keywords
antibody
polypeptide
sequences
seq
set forth
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Inventor
Xiao-Bo Chen
Michael Farrell
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Ventana Medical Systems Inc
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Ventana Medical Systems Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the present invention relates generally to diagnostic compositions and methods.
  • the invention is more specifically related to anti-DNP antibodies and their manufacture and use. Such antibodies are useful, for example, in the methods for the detection and/or visualization of target nucleic acid polymers.
  • ISH In situ hybridization
  • IHC immunohistochemistry
  • Signal detection and amplification techniques often rely upon pairs of binding agents such as streptavidin/biotin, avidin/biotin and antigen/antibody complexes. Additional techniques for signal amplification employ hapten conjugate/anti-hapten antibody complexes such as digoxygenin/anti-digoxygenin, and dinitrophenyl/anti-DNP, among others.
  • a DNP labeled nucleic acid probe is bound to a specific DNA or RNA target sequence in cells or tissues and rabbit anti-DNP monoclonal antibody is used to detect the DNP labeled probe bound to the target sequence.
  • This reaction is then amplified by recognition of the rabbit primary antibody with a mouse anti-rabbit antibody and the further binding of a biotinylated secondary antibody (goat anti-mouse IgG). Streptavidin conjugated alkaline phosphatase is then used to cleave a chromogenic substrate, which generates a visible blue nuclear signal where the DNP labeled probe is bound to target sequence.
  • DNA sequences encoding anti-DNP antibodies would also allow construction and use of antibody derivatives such as single-chain-variable-fragment antibody, epitope tagged antibody to facilitate purification and detection, fusion proteins with enzymes for detection, and the like.
  • antibody derivatives such as single-chain-variable-fragment antibody, epitope tagged antibody to facilitate purification and detection, fusion proteins with enzymes for detection, and the like.
  • the present invention addresses these needs and offers other related advantages.
  • the present invention provides anti-DNP antibody amino acid sequences, as well as polynucleotides encoding such antibodies.
  • the identification of the DNA sequences encoding anti-DNP antibodies, and the amino acid sequences of such antibodies, permits the production of recombinant anti-DNP antibody at high yields in common and easily maintained cell lines, and thus overcomes many of the difficulties associated with prior hybridoma cell lines used in the production of anti-DNP antibodies.
  • the present invention provides an isolated polynucleotide comprising a sequence provided in any one of SEQ ID NOs: 1 , 3, 5, 7, or 10 or a fragment thereof; a complement thereof; sequences that hybridize to a sequence set forth in any one of SEQ ID NOs: 1 , 3, 5, 7, or 10, under highly stringent conditions; sequences having at least 90% identity to a sequence set forth in any one of SEQ ID NOs: 1 , 3, 5, 7, or 10; degenerate variants of a sequence set forth in any one of SEQ ID NOs: 1 , 3, 5, 7, or 10; or sequences consisting of at least 15 contiguous residues of a sequence set forth in any one of SEQ ID NOs: 1 , 3, 5, 7, or 10.
  • the polynucleotide encodes a polypeptide, variable region, or functional fragment thereof, i.e., a leader, FR or CDR fragment, of an antibody described herein.
  • the present invention includes an isolated polypeptide comprising an amino acid sequence encoded by a polynucleotide of the present invention; sequences set forth in any one of SEQ ID NOs: 2, 4, 6, 8, or 11 ; sequences having at least 90% identity to a sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, or 11 ; and sequences consisting of at least 5 contiguous residues of a sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, or 11.
  • the polypeptide comprises or consists of a polypeptide or functional fragment thereof, i.e., a leader, FR or CDR fragment, of a polypeptide sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, or 11.
  • the present invention includes an expression vector comprising a polynucleotide of the invention operably linked to an expression control sequence.
  • the present invention includes a host cell transformed or transfected with an expression vector of the present invention.
  • the present invention includes an isolated antibody, or antigen-binding fragment thereof, comprising a polypeptide of the present invention.
  • the antibody is a monoclonal antibody.
  • the antibody is a rabbit anti-DNP monoclonal antibody or antigen-binding fragment thereof.
  • the antibody is a single chain variable fragment antibody.
  • the antibody is an IgG or IgE antibody.
  • the antibody is conjugated to an epitope tag selected from the group consisting of: HIS6, MYC, FLAG, V5, VSV-G, and HA.
  • the antibody is conjugated to a label.
  • an isolated antibody which comprises an amino acid sequence set forth in any one of SEQ ID NOs: 6, 8, or 11 , wherein the antibody specifically binds to DNP.
  • the amino acid sequence is encoded by a polynucleotide sequence as set forth in any one of SEQ ID NOs: 5, 7, or 10.
  • the antibody is a single chain variable fragment antibody.
  • the antibody is conjugated to an epitope tag.
  • the antibody is conjugated to a label.
  • recombinant polypeptide by culturing a host cell comprising at least one polynucleotide of the invention under conditions effective for synthesis of a recombinant polypeptide, and isolating the synthesized polypeptide therefrom, wherein the polypeptide is an antibody or a fragment or derivative thereof that specifically binds to DNP.
  • the recombinant polypeptide produced by the method comprises an amino acid sequence derived from one or all of SEQ ID NOs: 2, 4, 6, 8, or 11 , or encoded by one or all of SEQ ID NOs: 1 , 3, 5, 7, or 10.
  • the polynucleotide encoding the polypeptide is codon optimized for expression in a host cell of interest (e.g., Pichia pastoris). In some embodiments, the polynucleotide encoding the polypeptide is codon optimized for expression in mammalian cells, for example mammalian tissue culture cell lines (e.g., CHO, HEK293, 3T3, etc).
  • the present invention provides kits, e.g., diagnostic or molecular marker detection kits, comprising at least one polynucleotide, polypeptide or antibody of the present invention.
  • the kits may further comprise any of a number of auxiliary binding agents, or other desired agents, depending on the particular context and intended use of the kit and its contents, as further described herein.
  • Figure 1 shows the cDNA sequence of the kappa chain of a rabbit anti-DNP monoclonal antibody as set forth in SEQ ID NO: 1.
  • the leader sequence is shown in plaintext; the variable region is shown in bold-face type; and the constant region is italicized.
  • Figure 2 shows the amino acid sequence, as set forth in SEQ ID NO: 1.
  • FIG. 1 encoded by the cDNA sequence of the kappa chain of the rabbit anti- DNP monoclonal antibody as set forth in SEQ ID NO: 1.
  • the leader sequence is shown in plaintext; the variable region is shown in bold-face type; and the constant region is italicized. CDR regions are underlined.
  • Figure 3 shows the cDNA sequence of the heavy chain of a rabbit anti-DNP monoclonal antibody as set forth in SEQ ID NO: 3.
  • the leader sequence is shown in plaintext; the variable region is shown in bold-face type; and the constant region is italicized.
  • Figure 4 shows the amino acid sequence, as set forth in SEQ ID NO: 4, encoded by the cDNA sequence of the heavy chain of a rabbit anti-DNP monoclonal antibody as set forth in SEQ ID NO: 3.
  • the leader sequence is shown in plaintext; the variable region is shown in bold-face type; and the constant region is italicized. CDR regions are underlined.
  • Figure 5 shows an agarose gel analysis of the RT-PCR products generated in the cDNA synthesis of mRNA purified from a rabbit anti-DNP monoclonal antibody hybridoma cell line.
  • the gel shows PCR amplifications with primer sets directed against the kappa and lambda light chains of a rabbit anti-DNP monoclonal antibody.
  • Figure 6 shows the results of a screening assay for cDNAs representing the kappa chain of a rabbit anti-DNP monoclonal antibody.
  • Figure 7 shows an agarose gel analysis of the RT-PCR products generated in the cDNA synthesis of mRNA purified from a rabbit anti-DNP monoclonal antibody hybridoma cell line. The gel shows PCR amplifications with primer sets directed against the heavy chain of a rabbit anti-DNP monoclonal antibody.
  • Figure 8 shows the results of a screening assay for cDNAs representing the heavy chain of a rabbit anti-DNP monoclonal antibody.
  • Figure 9 shows the results of a screening assay for pCI vector constructs bearing the kappa and heavy chain cDNAs of a rabbit anti-DNP monoclonal antibody.
  • Figure 10 shows the results of a screening assay for pCMV vector constructs bearing the kappa and heavy chain cDNAs of a rabbit anti-DNP monoclonal antibody.
  • Figure 11 shows exemplary immunofluorescence staining for a rabbit anti-DNP monoclonal antibody in cell lines stably transfected with unligated or ligated anti-DNP kappa and heavy chain polynucleotide expression constructs.
  • Figure 12 shows exemplary results from an immunoblotting experiment for secreted rabbit anti-DNP monoclonal antibody in the culture media of cell lines stably transfected with unligated anti-DNP kappa and heavy chain polynucleotide expression constructs.
  • SEQ ID NO: 1 is the cDNA sequence encoding a kappa chain of a rabbit anti-DNP monoclonal antibody.
  • SEQ ID NO: 2 is the amino acid sequence of a kappa chain of a rabbit anti-DNP monoclonal antibody.
  • SEQ ID NO: 3 is the cDNA sequence encoding a heavy chain of a rabbit anti-DNP monoclonal antibody.
  • SEQ ID NO: 4 is the amino acid sequence of a heavy chain of a rabbit anti-DNP monoclonal antibody.
  • SEQ ID NO: 5 is the polynucleotide sequence encoding the kappa chain portion of a rabbit anti-DNP scFv.
  • SEQ ID NO: 6 is the amino acid sequence of the kappa chain portion of a rabbit anti-DNP scFv.
  • SEQ ID NO: 7 is the polynucleotide sequence encoding the heavy chain portion of a rabbit anti-DNP scFv.
  • SEQ ID NO: 8 is the amino acid sequence of the heavy chain portion of a rabbit anti-DNP scFv.
  • SEQ ID NO: 9 is amino acid sequence of the peptide spacer of a rabbit anti-DNP scFv.
  • SEQ ID NO: 10 is the Pichia pastoris codon-optimized polynucleotide sequence encoding a rabbit anti-DNP scFv.
  • SEQ ID NO: 11 is the amino acid sequence of a rabbit anti-DNP scFv.
  • SEQ ID NO: 12 is the polynucleotide sequence of a kappa chain forward primer used to amplify a rabbit anti-DNP kappa chain cDNA.
  • SEQ ID NO: 13 is the polynucleotide sequence of a kappa chain reverse primer used to amplify a rabbit anti-DNP kappa chain cDNA.
  • SEQ ID NO: 14 is the polynucleotide sequence of a lambda chain reverse primer used to amplify a rabbit anti-DNP lambda chain cDNA.
  • SEQ ID NO: 15 is the polynucleotide sequence of a lambda chain forward primer used to amplify a rabbit anti-DNP lambda chain cDNA.
  • SEQ ID NO: 16 is the polynucleotide sequence of a heavy chain forward primer used to amplify a rabbit anti-DNP heavy chain cDNA.
  • SEQ ID NO: 17 is the polynucleotide sequence of a heavy chain reverse primer used to amplify a rabbit anti-DNP heavy chain cDNA.
  • SEQ ID NO: 18 is the polynucleotide sequence of a heavy chain forward primer used to amplify a rabbit anti-DNP heavy chain cDNA for cloning into the pCI plasmid vector.
  • SEQ ID NO: 19 is the polynucleotide sequence of a kappa chain forward primer used to amplify a rabbit anti-DNP kappa chain cDNA for cloning into the pCI plasmid vector.
  • SEQ ID NO: 20 is the polynucleotide sequence of a heavy chain forward primer used to amplify a rabbit anti-DNP heavy chain cDNA for cloning into the pCMV plasmid vector.
  • SEQ ID NO: 21 is the polynucleotide sequence of a kappa chain forward primer used to amplify a rabbit anti-DNP kappa chain cDNA for cloning into the pCMV plasmid vector.
  • SEQ ID NO: 22 is the polynucleotide sequence of a universal reverse primer used to amplify rabbit anti-DNP heavy and kappa chain cDNAs for cloning into the pCI and pCMV plasmid vectors.
  • the present invention relates generally to polynucleotide and polypeptide sequences of both the light and heavy chains of a rabbit monoclonal anti-DNP antibody.
  • the present invention further provides antibody fragments thereof and derivatives of the anti-DNP antibody, such as a single- chain-variable-fragment antibody, an epitope tagged antibody to facilitate purification and detection, antibody fusion proteins with enzymes for detection, and the like, as further described herein.
  • the present invention provides polypeptide sequences corresponding to anti-DNP antibodies, subsequences, fragments and variants thereof, as well as polynucleotides encoding same.
  • the invention provides an anti-DNP monoclonal antibody comprising an amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, or 11.
  • the present invention provides a single chain variant fragment antibody comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 6, 8, or 11.
  • the present invention provides polypeptides comprising at least a variable region or functional domain of an anti-DNP antibody herein.
  • the present invention further provides anti-DNP antibodies having the same binding specificity and substantially the same polypeptide sequences of the complementarity determining regions (CDRs) of the antibodies herein.
  • CDRs complementarity determining regions
  • the present invention further provides polypeptides comprising at least a DNP-binding region of an anti-DNP antibody herein.
  • the present invention further includes polypeptides encoded by a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity or homology to a polynucleotide set forth in any one of SEQ ID NOs: 1 , 3, 5, 7, or 10, or a variable region or a functional domain encoded thereof.
  • polypeptides of the invention comprise amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to a polypeptide sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 11.
  • the present invention in another aspect, provides polypeptide fragments comprising or consisting of at least 5, 10, 15, 20, 25, 50, or 100 contiguous amino acids, or more, including all intermediate lengths, of a polypeptide set forth herein.
  • polypeptide fragments of the present invention comprise one or more hypervariable (HV) regions or CDRs of an antibody variable chain (V L or V H ) polypeptide.
  • HV hypervariable
  • a CDR is typically on the order of 5-20 amino acids in length, and thus, the present invention includes polypeptide fragments comprising or consisting of at least 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of a polypeptide composition set forth herein, such as those containing a polypeptide set forth in SEQ ID NO: 2, 4, 6, 8, or 11 , or those encoded by a polynucleotide sequence of SEQ ID NO: 1 , 3, 5, 7, or 10.
  • Polypeptide fragments and variants of the present invention preferably retain one more biological or binding activities of the polypeptide from which they are derived.
  • a polypeptide fragment or variant of an antibody herein preferably binds to the same epitope as the full length antibody in which it is present.
  • the polypeptide fragments and variants provided by the present invention exhibit a level of binding activity (with respect to DNP) of at least 50%, preferably at least 70%, and most preferably at least 90% or more of that exhibited by a full-length antibody sequence set forth herein.
  • polypeptides of the present invention comprise variable regions of antibody light chains and heavy chains (V L or V H ), full length antibody light chains and heavy chains, and single chain antibodies, each comprising at least one polypeptide sequence set forth herein, or those encoded by a polynucleotide sequence set forth herein.
  • the light chain of an antibody of the present invention is of the kappa isotype and the heavy chain may be any of gamma, alpha, delta, mu, or epsilon, and any subclass thereof.
  • a constant region may be any of IgG, IgA, IgD, IgM, or IgE, for example.
  • a polypeptide of the present invention is a component of an antibody fragment or variant thereof, as described in further detail infra.
  • a polypeptide (or antibody) of the present invention is epitope-tagged.
  • epitope tagged refers to a chimeric polypeptide comprising a polypeptide, such as an antibody or fragment of the present invention, fused to a "tag polypeptide.”
  • the tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused.
  • the tag polypeptide is also preferably fairly unique so that the antibody does not substantially cross-react with other epitopes.
  • Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).
  • the epitope tag as found in a chimeric polypeptide described herein is useful, for example, for identifying and isolating the tagged proteins.
  • Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (HIS6; poly-his) or poly- histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 (Field et al., MoI. Cell.
  • Protein containing the FLAG peptide can be performed by immunoaffinity chromatography using an affinity matrix comprising the anti-FLAG M2 monoclonal antibody covalently attached to agarose (Eastman Kodak Co., New Haven, CT).
  • affinity matrix comprising the anti-FLAG M2 monoclonal antibody covalently attached to agarose (Eastman Kodak Co., New Haven, CT).
  • tag polypeptides include the KT3 epitope peptide (Martin et al., Science, 255:192-194 (1992)); an ⁇ -tubulin epitope peptide (Skinner et al., J. Biol. Chem., 266:15163-15166 (1991 )); and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)).
  • Polypeptides of the invention may be prepared using any of a variety of known synthetic and/or recombinant techniques. Polypeptides, portions and other variants generally less than about 150 amino acids can be generated by synthetic means, using techniques well known to those of ordinary skill in the art. In one illustrative example, such polypeptides are synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain (See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963). Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Applied BioSystems Division (Foster City, CA), and may be operated according to the manufacturer's instructions.
  • polynucleotides encoding a polypeptide described herein, such as those encoding an antibody or binding fragment thereof having an amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 11 , as well as variable regions and functional domains thereof, or a sequence that hybridizes under moderately stringent conditions, or alternatively, under highly stringent conditions, to such a polynucleotide sequence.
  • antibody as used herein can include monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired binding activity.
  • immunoglobulin Ig
  • An “isolated antibody” is one that has been separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody is purified: (1 ) to greater than 95% by weight of antibody as determined by the Bradford method, and most preferably more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N- terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • the basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • An IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called J chain, and therefore contains 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain.
  • the 4-chain unit is generally about 150,000 daltons.
  • Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the ⁇ and ⁇ chains and four CH domains for ⁇ and ⁇ isotypes.
  • Each L chain has at the N-terminus, a variable domain (V L ) followed by a constant domain (CL) at its other end.
  • the V L is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (C H 1 ). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • the pairing of a VH and V L together forms a single antigen-binding site.
  • immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) and mu ( ⁇ ), respectively.
  • the ⁇ and ⁇ classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgGI , lgG2, lgG3, lgG4, IgAI , and lgA2.
  • variable refers to the fact that certain segments of the V domains differ in sequence among antibodies.
  • the V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen.
  • variability is not evenly distributed across the 110-amino acid span of the variable domains.
  • the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by short regions of extreme variability called “hypervariable regions.”
  • FRs framework regions
  • hypervariable regions typically each 9-18 amino acids long. However, they have been found to range from 4-28 depending upon the particular antigen/epitope.
  • variable domains of native heavy and light chains each comprise four FRs, largely adopting a ⁇ -sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991 )).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
  • ADCC antibody dependent cellular cytotoxicity
  • hypervariable region when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding.
  • the hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., around about residues 24-34 (L1 ), 50-56 (L2) and 89-97 (L3) in the V L , and around about 1-35 (H1 ), 50-65 (H2) and 95-102 (H3) in the V H ; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.
  • CDR complementarity determining region
  • residues from a "hypervariable loop” e.g., residues 26-32 (L1 ), 50-52 (L2) and 91-96 (L3) in the VL, and 26-32 (H1 ), 53-55 (H2) and 96-101 (H3) in the VH ; Chothia and Lesk, J. MoI. Biol. 196:901-917 (1987)).
  • the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
  • CDRs complementarity determining regions
  • one or more of the CDRs may be inserted within framework regions, which may be naturally occurring or consensus framework regions.
  • Antibodies can be modified using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101 ; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology
  • One or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its binding partner. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Alterations of these and other types may be made at the polynucleotide level using methodologies well known and available in the art.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. However, in certain particular embodiments, the monoclonal antibodies are made using known recombinant DNA methodologies in bacterial, eukaryotic, animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).
  • Antibodies of the invention further include intact antibodies.
  • An "intact” antibody is one that comprises an antigen-binding site as well as a C L and at least heavy chain constant domains, CH 1 , CH 2 and CH 3.
  • the constant domains may be native sequence constant domains or amino acid sequence variants thereof.
  • Antibody fragments are also provided by the present invention.
  • An "antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; minibodies; diabodies; triabodies; tetrabodies; linear antibodies (see U.S. Pat. No. 5,641 ,870; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]; Hollinger and Hudson. Nature Biotechnology. Vol.23, No.9, Sept. 2005, pp.1126-1136); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen- binding fragments, called “Fab” fragments, and a residual "Fc” fragment, a designation reflecting the ability to crystallize readily.
  • the Fab fragment consists of an entire L chain along with the variable region domain of the H chain (V H ), and the first constant domain of one heavy chain (CH 1 ).
  • V H variable region domain of the H chain
  • CH 1 first constant domain of one heavy chain
  • Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.
  • Pepsin treatment of an antibody yields a single large F(ab') 2 fragment that roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen.
  • Fab' fragments differ from Fab fragments by having additional residues at the carboxy terminus of the C H 1 domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • the "Fc" fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.
  • Fv is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Single-chain Fv also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.
  • the sFv polypeptide further comprises a polypeptide linker/spacer between the V H and V L domains that enables the sFv to form the desired structure for antigen binding.
  • sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.
  • the present invention provides a scFv antibody comprising an amino acid sequence as set forth in any one of SEQ ID NO: 6, 8, or 11.
  • Anti-DNP diabodies are also provided by the present invention.
  • the term "diabodies” refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the VH and V L domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites.
  • Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161 ; and Hollinger ef a/., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
  • an antibody or fragment thereof is said to be “immunospecific,” “specific for” or to “specifically bind” an antigen if it reacts at a detectable level with the antigen, preferably with an affinity constant, Ka 1 of greater than or equal to about 10 4 M" 1 , or greater than or equal to about 10 5 M “ 1 , greater than or equal to about 10 6 IVH 1 greater than or equal to about 10 7 M" 1 , greater than or equal to 10 8 M “1 , greater than or equal to 10 9 M “1 , greater than or equal to 10 1 ° M "1 , greater than or equal to 10 11 M "1 , greater than or equal to 10 12 M “1 , or greater than or equal to 10 13 M "1 .
  • Affinity of an antibody for its cognate antigen is also commonly expressed as a dissociation constant K D , and in certain embodiments, an antibody specifically binds to it cognate antigen if it binds with a K 0 of less than or equal to 10 "4 M, less than or equal to about 10 "5 M, less than or equal to about 10 "6 M, less than or equal to 10 "7 M, , less than or equal to 10 "8 M , less than or equal to 10 "9 M , less than or equal to 10 "10 M , less than or equal to 10 "11 M , less than or equal to 10 "12 M or less than or equal to 10 "13 M.
  • Affinities of antibodies can be readily determined using conventional techniques, for example, those described by Scatchard et al. (Ann. N.Y. Acad. Sci. USA 51 :660 (1949)).
  • Binding properties of an antibody or fragment thereof to antigens, cells or tissues thereof may generally be determined and assessed using immunodetection methods including, for example, immunofluorescence-based assays, such as immunohistochemistry (IHC) and/or fluorescence-activated cell sorting (FACS).
  • immunodetection methods including, for example, immunofluorescence-based assays, such as immunohistochemistry (IHC) and/or fluorescence-activated cell sorting (FACS).
  • an antibody having a "biological characteristic" of a designated antibody is one that possesses one or more of the biological characteristics of that antibody which distinguish it from other antibodies.
  • an antibody with a biological characteristic of a designated antibody will bind the same epitope as that bound by the designated antibody.
  • the invention provides a single chain Fv fragment (scFv) (see, e.g., WO 93/16185; U.S. Pat. Nos. 5,571 ,894; and 5,587,458).
  • scFv single chain Fv fragment
  • Fv and scFv have intact combining sites that are devoid of constant regions. Thus, they are suitable for reduced nonspecific binding during in vivo use.
  • scFv fusion proteins may also be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv (e.g., Antibody Engineering, ed. Borrebaeck, supra.
  • the antibody fragment may also be a "linear antibody", and U.S. Pat. No. 5,641 ,870).
  • antibodies of the present invention are chimeric antibodies that comprise sequences derived from different sources and/or species.
  • "Chimeric" antibodies refer generally to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).
  • chimeric antibodies of certain interest herein include antibodies having one or more antigen binding sequences (e.g., CDRs) as described herein and containing one or more sequences derived from an antibody of another species, e.g., an FR or C region sequence.
  • chimeric antibodies of the invention further include those comprising a rabbit variable domain antigen binding sequence of one antibody class or subclass and another sequence, e.g., FR or C region sequence, derived from another antibody class or subclass.
  • Chimeric antibodies of the invention also include those containing variable domain antigen-binding sequences related to those described herein and/or derived from a different species. Chimeric antibodies also include primatized and humanized antibodies.
  • chimeric antibodies may comprise residues that are not found in the antibody sequences provided herein, such as modifications made to further refine antibody performance. For further details, see Jones et a/., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
  • chimeric antibodies retain high binding affinity for the antigen and other favorable biological properties.
  • chimeric antibodies are prepared by a process of analysis of the parental sequences and various conceptual chimeric products using three-dimensional models of the parental and non-parental sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • antibodies of the present invention are bispecific or multi-specific.
  • Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes.
  • Exemplary bispecific antibodies may bind to two different epitopes of a single antigen.
  • Other such antibodies may combine a first antigen binding site with a binding site for a second antigen.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab') 2 bispecific antibodies).
  • bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the co- expression of two immunoglobulin heavy chain-light chain pairs in hybridoma cells, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J., 10:3655-3659 (1991 ).
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, C H 2, and C H 3 regions. It is preferred to have the first heavy-chain constant region (C H 1 ) containing the site necessary for light chain bonding, present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host cell.
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121 :210 (1986).
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH 3 domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).
  • Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
  • bispecific antibodies can be prepared using chemical linkage.
  • Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
  • the Fab 1 fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the "diabody” technology described by Hollinger et a/., Proc. Natl. Acad. Sci.
  • the fragments comprise a VH connected to a VL by a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).
  • amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.
  • amino acid sequence variants of an antibody may be prepared by introducing appropriate nucleotide changes into a polynucleotide that encodes the antibody, or a chain thereof, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution may be made to arrive at the final antibody, provided that the final construct possesses the desired characteristics.
  • the amino acid changes also may alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites. Any of the variations and modifications described above for polypeptides of the present invention may be included in antibodies of the present invention.
  • a useful method for identification of certain residues or regions of an antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells in Science, 244:1081-1085 (1989).
  • a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with PSCA antigen.
  • Those amino acid locations demonstrating functional sensitivity to the substitutions are refined by introducing further or other variants at, or for, the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed anti- antibody variants are screened for the desired activity.
  • Amino acid sequence insertions include amino- and/or carboxy- terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide.
  • Other insertional variants of an antibody include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody.
  • variants are an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue.
  • the sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative and non-conservative substitutions are contemplated. Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
  • cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody.
  • the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
  • a convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site.
  • the antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed.
  • alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding.
  • Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein.
  • Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody.
  • N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • X is any amino acid except proline
  • O-linked glycosylation refers to the attachment of one of the sugars N- acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
  • glycosylation sites is accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites).
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O- linked glycosylation sites).
  • ADCC antigen-dependent cell- mediated cyotoxicity
  • CDC complement dependent cytotoxicity
  • This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody.
  • cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J.
  • Homodimeric antibodies with enhanced anti-infection activity may also be prepared using heterobifunctional cross- linkers as described in Wolff et a/., Cancer Research 53:2560-2565 (1993).
  • an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989).
  • antibodies of the present invention may also be modified to include an epitope tag or label, e.g., for use in purification or diagnostic applications.
  • an epitope tag or label e.g., for use in purification or diagnostic applications.
  • linking groups include disufide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents, disulfide and thioether groups being preferred.
  • Immunoconjugates may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1 -carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p- azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6- diisocyanate), and bis-active fluorine compounds (such as 1 ,5-difluoro-2,4-
  • Particular coupling agents include N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 [1978]) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a disulfide linkage.
  • the linker may be a "cleavable linker" facilitating release of one or more cleavable components.
  • an acid-labile linker may be used (Cancer Research 52: 127-131 (1992); U.S. Pat. No. 5,208,020).
  • the antibody may be linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
  • nonproteinaceous polymers e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
  • the antibody also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacylate)microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • macroemulsions for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Carriers as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.
  • physiologically acceptable carrier is an aqueous pH buffered solution.
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as polysorbate 20 (TWEEN TM) polyethylene glycol (PEG), and poloxamers (PLURONICSTM), and the like.
  • buffers such as phosphate, citrate, and other organic acids
  • nucleic acid and “polynucleotide” are used interchangeably herein to refer to single- or double-stranded RNA, DNA, or mixed polymers.
  • Polynucleotides may include genomic sequences, extra- genomic and plasmid sequences, nucleic acid vectors, and smaller engineered gene segments that express, or may be adapted to express polypeptides.
  • isolated nucleic acid is a nucleic acid that is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence.
  • the term embraces a nucleic acid sequence that has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • a substantially pure nucleic acid includes isolated forms of the nucleic acid. Of course, this refers to the nucleic acid as originally isolated and does not exclude genes or sequences later added to the isolated nucleic acid by the hand of man.
  • a “native sequence" polynucleotide is one that has the same nucleotide sequence as a polynucleotide derived from nature (e.g., encoding an antibody herein).
  • Such native sequence polypeptides can be isolated from nature ⁇ e.g., from a desired species) or can be produced by recombinant or synthetic means.
  • the present invention encompasses polynucleotide variants of a polynucleotide sequence set forth herein, such as a polynucleotide sequence set forth in SEQ ID NOs: 1 , 3, 5, 7, and 10.
  • a polynucleotide "variant,” as the term is used herein, is a polynucleotide that typically differs from a polynucleotide disclosed herein, or encoding an antibody sequence disclosed herein, in one or more substitutions, deletions, additions and/or insertions.
  • Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the polynucleotide sequences of the invention and evaluating one or more biological activities (e.g., binding specificity) of the encoded polypeptide as described herein and/or using any of a number of techniques well known in the art.
  • biological activities e.g., binding specificity
  • polynucleotide sequences are provided that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to a polynucleotide sequence herein or to a polynucleotide encoding a polypeptide herein.
  • the present invention provides polynucleotide fragments comprising various lengths of contiguous stretches of sequence identical to or complementary to one or more of the sequences disclosed herein.
  • polynucleotides are provided by this invention that comprise at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides of one or more of the sequences disclosed herein as well as all intermediate lengths there between.
  • intermediate lengths means any length between the quoted values, such as 16, 17, 18, 19, etc.; 21 , 22, 23, etc.; 30, 31 , 32, etc.; 50, 51 , 52, 53, etc.; 100, 101 , 102, 103, etc.; 150, 151 , 152, 153, efc.; including all integers through 200-500; 500-1 ,000, and the like.
  • polynucleotide compositions are provided that are capable of hybridizing under moderate to high stringency conditions to a polynucleotide sequence provided herein, or a fragment thereof, or a complementary sequence thereof.
  • Hybridization techniques are well known in the art of molecular biology.
  • suitable moderately stringent conditions for testing the hybridization of a polynucleotide of this invention with other polynucleotides include prewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C-60°C, 5 X SSC, overnight; followed by washing twice at 65 0 C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1 % SDS.
  • the stringency of hybridization can be readily manipulated, such as by altering the salt content of the hybridization solution and/or the temperature at which the hybridization is performed.
  • suitable highly stringent hybridization conditions include those described above, with the exception that the temperature of hybridization is increased, e.g., to 60-65 0 C or 65-7O 0 C.
  • the polypeptide encoded by the polynucleotide variant or fragment has the same or similar binding specificity (i.e., specifically or preferentially binds to DNP) as the polypeptide encoded by the native polynucleotide.
  • polynucleotides described above e.g., polynucleotide variants, fragments and hybridizing sequences, encode polypeptides that have a level of binding activity of at least about 50%, preferably at least about 70%, and more preferably at least about 90% of that for a polypeptide sequence specifically set forth herein.
  • polynucleotides of the present invention may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • illustrative polynucleotide segments with total lengths of about 10,000, about 5000, about 3000, about 2,000, about 1 ,000, about 500, about 200, about 100, about 50 base pairs in length, and the like, (including all intermediate lengths) are contemplated to be useful in many implementations of this invention.
  • alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides.
  • the resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).
  • the inventors contemplate the mutagenesis of the disclosed polynucleotide sequences to alter one or more properties of the encoded polypeptide, such as its binding specificity or binding strength.
  • Techniques for mutagenesis are well-known in the art, and are widely used to create variants of both polypeptides and polynucleotides.
  • a mutagenesis approach such as site-specific mutagenesis, may be employed for the preparation of variants and/or derivatives of the polypeptides described herein.
  • site-specific mutagenesis may be employed for the preparation of variants and/or derivatives of the polypeptides described herein.
  • specific modifications in a polypeptide sequence can be made through mutagenesis of the underlying polynucleotides that encode them.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences including the nucleotide sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Mutations may be employed in a selected polynucleotide sequence to improve, alter, decrease, modify, or otherwise change the properties of the polynucleotide itself, and/or alter the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.
  • the polynucleotide sequences provided herein can be used as probes or primers for nucleic acid hybridization, e.g., as PCR primers.
  • the ability of such nucleic acid probes to specifically hybridize to a sequence of interest will enable them to be of use in detecting the presence of complementary sequences in a given sample.
  • other uses are also envisioned, such as the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
  • nucleic acid segments that comprise a sequence region of at least about 15 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 15 nucleotide long contiguous sequence disclosed herein will find particular utility.
  • Longer contiguous identical or complementary sequences e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) and even up to full length sequences will also be of use in certain embodiments.
  • Polynucleotide molecules having sequence regions consisting of contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200 nucleotides or so (including intermediate lengths as well), identical or complementary to a polynucleotide sequence disclosed herein, are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting, and/or primers for use in, e.g., polymerase chain reaction (PCR).
  • the total size of the fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment.
  • hybridization probe of about 15-25 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than 12 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to 25 contiguous nucleotides, or even longer where desired. Hybridization probes may be selected from any portion of any of the sequences disclosed herein.
  • probe and primer sequences are governed by various factors. For example, one may wish to employ primers directed towards the termini of the total sequence.
  • Polynucleotides of the present invention may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCRTM technology of U. S. Patent 4,683,202, by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
  • polypeptide is used in its conventional meaning, i.e., as a sequence of amino acids.
  • the polypeptides are not limited to a specific length of the product.
  • Peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise.
  • This term also does not exclude post-expression modifications of the polypeptide, for example, glycosylates, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • a polypeptide may be an entire protein, or a subsequence thereof.
  • polypeptides of interest in the context of this invention are anti-DNP antibody amino acid sequences, subsequences and variants, e.g., comprising CDRs and being capable of binding DNP with desired affinity.
  • anti-DNP antibody amino acid sequences, subsequences and variants e.g., comprising CDRs and being capable of binding DNP with desired affinity.
  • polynucleotide sequences encoding such anti- DNP antibody amino acid sequences, subsequences and variants are also of interest in the context of the invention.
  • An "isolated polypeptide" is one that has been identified and separated and/or recovered from a component of its natural environment.
  • the isolated polypeptide is purified (1 ) to greater than 95% by weight of polypeptide, e.g., as determined by the Lowry method, and preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, and/or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue, silver staining or the like.
  • Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, an isolated polypeptide is prepared by at least one purification step.
  • a “native sequence” polypeptide is one that has the same amino acid sequence as a polypeptide (e.g., antibody) derived from nature (e.g., from a desired species). Such native sequence polypeptides are isolated from nature or are produced by recombinant or synthetic means.
  • a polypeptide "variant,” as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions.
  • Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the polypeptide sequences of the invention and evaluating one or more biological activities of the polypeptide (e.g., binding specificity) as described herein and/or using any of a number of techniques well known in the art.
  • polypeptide sequences are provided that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to a polypeptide sequence herein.
  • Modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics.
  • a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics.
  • one skilled in the art will typically change one or more of the codons of the encoding DNA sequence.
  • amino acids may be substituted for other amino acids in an antibody amino acid sequence without appreciable loss of its ability to bind antigen. Since it is the binding capacity and nature of an antibody that largely defines its desired functional activity, certain amino acid sequence substitutions can be made in the antibody sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain an antibody with like binding properties and specificity. It is thus contemplated that various changes may be made in the polypeptide sequences herein, or corresponding DNA sequences that encode said polypeptides, without appreciable loss of their biological utility or activity.
  • a polypeptide variant will contain one or more conservative substitutions.
  • a "conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of an amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, nucleic acids, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982).
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1 ); glutamate (+3.0 ⁇ 1 ); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1 ); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (- 3.4).
  • amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.
  • variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer.
  • Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the secondary structure, hydropathic nature and/or binding specificity of the polypeptide.
  • Polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein.
  • the polypeptide may also be fused in-frame or conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support.
  • a peptide linker/spacer sequence may also be employed to separate multiple polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and/or tertiary structures, if desired.
  • Such a peptide linker sequence can be incorporated into a fusion polypeptide using standard techniques well known in the art.
  • Certain peptide spacer sequences may be chosen, for example, based on: (1 ) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and/or (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
  • peptide spacer sequences contain, for example, GIy, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala, may also be included in the spacer sequence.
  • amino acid sequences which may be usefully employed as spacers include those disclosed in Maratea et al., Gene 40:39 46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751 ,180.
  • spacers may include, for example, Glu-Gly-Lys- Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp (Chaudhary et a!., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:1066-1070) and Lys-Glu-Ser-Gly-Ser-Val-Ser-Ser- Glu-Gln-Leu-Ala-Gln-Phe-Arg-Ser-Leu-Asp (Bird et al., 1988, Science 242:423- 426).
  • spacer sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
  • Two coding sequences can be fused directly without any spacer or by using a flexible polylinker composed, for example, of the pentamer GIy- Gly-Gly-Gly-Ser repeated 1 to 3 times.
  • a spacer has been used in constructing single chain antibodies (scFv) by being inserted between VH and VL (Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5979-5883).
  • a peptide spacer in certain embodiments, is designed to enable the correct interaction between two beta-sheets forming the variable region of the single chain antibody.
  • a peptide spacer is between 1 to 5 amino acids, between 5 to 10 amino acids, between 5 to 25 amino acids, between 5 to 50 amino acids, between 10 to 25 amino acids, between 10 to 50 amino acids, between 10 to 100 amino acids, or any intervening range of amino acids.
  • a peptide spacer comprises about 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids in length.
  • the peptide spacer comprises the amino acid sequence set forth in SEQ ID NO: 9.
  • two sequences are said to be “identical” if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity.
  • a “comparison window” as used herein refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wl), using default parameters.
  • This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol.
  • optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981 ) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. MoI. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wl), or by inspection.
  • BLAST and BLAST 2.0 are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. MoI. Biol. 215:403-410, respectively.
  • BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website.
  • cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). 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.
  • a scoring matrix can be 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 "percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
  • the invention further provides vectors and host cells comprising a nucleic acid of the present invention, as well as recombinant techniques for the production of a polypeptide of the present invention.
  • Vectors of the invention include those capable of replication in any type of cell or organism, including, e.g., plasmids, phage, cosmids, and mini chromosomes.
  • vectors comprising a polynucleotide of the present invention are vectors suitable for propagation or replication of the polynucleotide, or vectors suitable for expressing a polypeptide of the present invention, for example pCI and pCIneo vectors (Promega, Madison, Wl), pCMV vectors, pRSV vectors, and the like.
  • pCI and pCIneo vectors Promega, Madison, Wl
  • pCMV vectors pRSV vectors
  • polypeptide of the present invention are known in the art and commercially available.
  • Polynucleotides of the present invention may be synthesized, whole or in parts that are then combined, and inserted into a vector using routine molecular and cell biology techniques, including, e.g., subcloning the polynucleotide into a linearized vector using appropriate restriction sites and restriction enzymes.
  • Polynucleotides of the present invention may be amplified by polymerase chain reaction using oligonucleotide primers complementary to each strand of the polynucleotide. These primers may also include restriction enzyme cleavage sites to facilitate subcloning into a vector.
  • the replicable vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, and one or more marker or selectable genes.
  • the nucleotide sequences encoding the polypeptide, or functional equivalents may be inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • an appropriate expression vector i.e., a vector that contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook and Russell (2001 ) Molecular Cloning, A Laboratory Manual, 3 rd edition (Cold Spring Harbor Press, Plainview, N.Y.).
  • a variety of expression vector/host systems may be utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculovirus)
  • plant cell systems transformed with virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
  • variable regions of a gene expressing a monoclonal antibody of interest are amplified from a hybridoma cell using nucleotide primers.
  • primers may be synthesized by one of ordinary skill in the art, or may be purchased from commercially available sources (see, e.g., Stratagene (La JoIIa, California), which sell primers for amplifying mouse and human variable regions.
  • the primers may be used to amplify heavy or light chain variable regions, which may then be inserted into vectors such as ImmunoZAPTM H or ImmunoZAPTM L (Stratagene), respectively. These vectors may then be introduced into E. coli, yeast, or mammalian-based systems for expression.
  • control elements or "regulatory sequences" present in an expression vector are those non-translated regions of the vector, e.g., enhancers, promoters, 5' and 3' untranslated regions, that interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used.
  • promoters suitable for use with prokaryotic hosts include the phoa promoter, ⁇ -lactamase and lactose promoter systems, alkaline phosphatase promoter, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter.
  • phoa promoter ⁇ -lactamase and lactose promoter systems
  • alkaline phosphatase promoter alkaline phosphatase promoter
  • trp tryptophan
  • hybrid promoters such as the tac promoter.
  • Promoters for use in bacterial systems also usually contain a Shine-Dalgarno sequence operably linked to the DNA encoding the polypeptide.
  • Inducible promoters such as the hybrid lacZ promoter of the pBLUESCRIPT phagemid (Stratagene, La JoIIa, Calif.) or pSPORTI plasmid (Gibco BRL, Gaithersburg, MD) and the like may be used.
  • a variety of promoter sequences are known for eukaryotes and any may be used according to the present invention. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide.
  • AATAAA sequence that may be the signal for addition of the poly A tail to the 3 1 end of the coding sequence. All of these sequences may be suitably inserted into eukaryotic expression vectors. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are generally preferred.
  • Polypeptide expression from vectors in mammalian host cells may be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (e.g., Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, fowlpox virus, adenovirus (e.g., Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus, hepatitis-
  • vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker.
  • suitable expression vectors are pcDNA3.1 (Invitrogen, Carlsbad, CA) and pCI (Promega, Madison, Wl), both of which include a CMV promoter.
  • inducible promoter systems for regulating the expression of polypeptides of the present invention in mammalian cells are also contemplated, for example by cloning nucleotide sequences encoding polypeptides as described herein into vectors such as those found in the Tet-Off® ® and/or Tet-On® tetracycline responsive gene expression system (Clontech Laboratories, Mountain View, CA).
  • vectors such as those found in the Tet-Off® ® and/or Tet-On® tetracycline responsive gene expression system (Clontech Laboratories, Mountain View, CA).
  • a number of viral-based expression systems are available for mammalian expression of polypeptides.
  • sequences encoding a polypeptide of interest may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence.
  • Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain a viable virus that is capable of expressing the polypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81 :3655-3659).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • any of a number of expression vectors may be selected depending upon the use intended for the expressed polypeptide.
  • vectors that direct high level expression of fusion proteins that are readily purified may be used.
  • Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as pET vector systems and variants thereof (available from Novagen, Stratagene, and Invitrogen), in which the sequence encoding the polypeptide of interest may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ - galactosidase, so that a hybrid protein is produced; plN vectors (Van Heeke, G. and S. M.
  • pGEX Vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems may be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used.
  • suitable promoter sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • yeast promoters that are inducible promoters having the additional advantage of transcription controlled by growth conditions include the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde- 3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast enhancers also are advantageously used with yeast promoters.
  • sequences encoding polypeptides may be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311.
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J., et al. (1991 ) Results Probl. Cell Differ.
  • constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, e.g., Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).
  • An insect system may also be used to express a polypeptide of interest.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the sequences encoding the polypeptide may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the polypeptide-encoding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses may then be used to infect, for example, S.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon are provided. Furthermore, the initiation codon is in the correct reading frame to ensure correct translation of the inserted polynucleotide. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic.
  • Enhancer sequences are known, including, e.g., those identified in genes encoding globin, elastase, albumin, ⁇ -fetoprotein, and insulin.
  • an enhancer from a eukaryotic cell virus is used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the vector at a position 5' or 3 1 to the polypeptide-encoding sequence, but is preferably located at a site 5' from the promoter.
  • Expression vectors used in eukaryotic host cells will typically also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs.
  • These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding anti-PSCA antibody.
  • One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vector disclosed therein.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, plant or higher eukaryote cells described above.
  • suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B.
  • E. coli cloning host is E coli 294 (ATCC 31 ,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31 ,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • K lactis K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K.
  • a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing that cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding and/or function.
  • Different host cells such as CHO, COS, HeLa, MDCK, HEK293, and WI38, which have specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.
  • antibody heavy and light chains, or fragments thereof are expressed from the same or separate expression vectors. In one embodiment, both chains are expressed in the same cell, thereby facilitating the formation of a functional antibody or fragment thereof.
  • Full length antibody, antibody fragments, and antibody fusion proteins can be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos.
  • the antibody is isolated from the E. coli cell paste in a soluble fraction and can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out using a process similar to that used for purifying antibody expressed e.g., in CHO cells.
  • Suitable host cells for the expression of glycosylated polypeptides and antibodies include those derived from multicellular organisms. Examples of invertebrate cells include plant, e.g., Lemna (duckweed), and insect cells.
  • baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopicius (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified.
  • a variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
  • Propagation of antibody polypeptides and fragments thereof in vertebrate cells in culture has become a routine procedure.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod.
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • Host cells are transformed with the above-described expression or cloning vectors for polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • stable expression is generally preferred.
  • cell lines that stably express a polynucleotide of interest may be transformed using expression vectors that may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
  • any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) genes that can be employed in tk " or aprt " cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
  • npt which confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin, F. et a/.(1981 ) J. MoI. Biol. -/50:1-14); and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc.
  • recombinant cells containing sequences can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a polypeptide-encoding sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain and express a desired polynucleotide sequence may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA- DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include, for example, membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein.
  • a variety of protocols for detecting and measuring the expression of polynucleotide-encoded products, using either polyclonal or monoclonal antibodies specific for the product are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on a given polypeptide may be preferred for some applications, but a competitive binding assay may also be employed. These and other assays are described, among other places, in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul. Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
  • the sequences, or any portions thereof may be cloned into a vector for the production of an mRNA probe.
  • Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • reporter molecules or labels include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • the polypeptide produced by a recombinant cell may be secreted or contained intracellular ⁇ depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides of the invention may be designed to contain signal sequences that direct secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane.
  • a polypeptide of the present invention is produced as a fusion polypeptide further comprising a polypeptide domain that will facilitate purification of soluble proteins.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Amgen, Seattle, WA).
  • the inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen. San Diego, CA) between the purification domain and the encoded polypeptide may be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing a polypeptide of interest and a nucleic acid encoding 6 or more histidine residues preceding a thioredoxin or an enterokinase cleavage site.
  • the histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography) as described in Porath, J. et a/. (1992, Prot. Exp. Purif. 3:263-281 ) while the enterokinase cleavage site provides a means for purifying the desired polypeptide from the fusion protein.
  • IMIAC immobilized metal ion affinity chromatography
  • a polypeptide of the present invention is fused with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.
  • the signal sequence may be selected, for example, from the group of the alkaline phosphatase, penicillinase, 1 pp, or heat-stable enterotoxin Il leaders.
  • the signal sequence may be selected from, e.g., the yeast invertase leader, ⁇ factor leader (including Saccharomyces and Kluyveromyces ⁇ factor leaders), or acid phosphatase leader, the C. albicans glucoamylase leader, or the signal described in WO 90/13646.
  • yeast invertase leader e.g., the yeast invertase leader, ⁇ factor leader (including Saccharomyces and Kluyveromyces ⁇ factor leaders), or acid phosphatase leader, the C. albicans glucoamylase leader, or the signal described in WO 90/13646.
  • mammalian signal sequences as well as viral secretory leaders for example, the herpes simplex gD signal, are available.
  • the polypeptide or antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the polypeptide or antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies that are secreted to the periplasiic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
  • sodium acetate pH 3.5
  • EDTA EDTA
  • PMSF phenylmethylsulfonylfluoride
  • supematants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the polypeptide or antibody composition prepared from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • affinity chromatography is the preferred purification technique.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the polypeptide or antibody. Protein A can be used to purify antibodies or fragments thereof that are based on human ⁇ -i, 7 2 , or ⁇ 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)).
  • Protein G is recommended for all mouse isotypes and for human ⁇ 3 (Guss et al., EMBO J. 5:15671575 (1986)).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the polypeptide or antibody comprises a CH 3 domain
  • the Bakerbond ABXTM resin J. T. Baker, Phillipsburg, N.J.
  • the mixture comprising the polypeptide or antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).
  • the compositions of the present invention e.g., antibodies, polypeptide fragments and variants thereof, as well as polynucleotides encoding same
  • the compositions of the invention are useful in the context of essentially any biological or other assays or analysis wherein DNP binding specificity is desired.
  • the compositions of the invention are useful in any of a number of assays involving the detection, monitoring, and/or diagnosis of various diseases, disorders, and conditions that may be manifested by abnormal gene expression, protein expression and/or genetic rearrangements.
  • Various embodiments of the present invention relate, in part, to methods of detecting biological molecules in a sample, such as a cell or tissue. Such methods can be applied in a variety of known detection formats, including, but not limited to immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization (ISH), whole-mount in situ hybridization (WISH), fluorescent DNA in situ hybridization (FISH), flow cytometry, enzyme immuno-assay (EIA), and enzyme linked immuno-assay (ELISA).
  • IHC immunohistochemistry
  • ICC immunocytochemistry
  • ISH in situ hybridization
  • WISH whole-mount in situ hybridization
  • FISH fluorescent DNA in situ hybridization
  • flow cytometry enzyme immuno-assay
  • EIA enzyme immuno-assay
  • ELISA enzyme linked immuno-assay
  • ISH is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., primary binding agent) to localize a specific DNA or RNA sequence in a portion or section of a cell or tissue (in situ), or if the tissue is small enough, the entire tissue (whole mount ISH).
  • primary binding agent i.e., primary binding agent
  • DNA ISH can be used on genomic DNA to determine the structure of chromosomes.
  • Fluorescent DNA ISH FISH
  • FISH Fluorescent DNA ISH
  • RNA ISH hybridization histochemistry
  • the primary binding agent is conjugated to a detectable label that may be detected directly or indirectly.
  • a "conjugate” refers to any binding agent that is covalently linked to a detectable label.
  • DNA probes, RNA probes, monoclonal antibodies, fragments thereof, and antibody derivatives thereof, such as a single-chain-variable-fragment antibody or an epitope tagged antibody may all be covalently linked to a detectable label.
  • direct detection only one detectable binding agent is used, i.e., a primary detectable binding agent.
  • direct detection means that the binding agent that is conjugated to a detectable label may be detected, per se, without the need for the addition of a second binding agent.
  • a “detectable label” is a molecule or material that can produce a detectable (such as visually, electronically or otherwise) signal that indicates the presence and/or concentration of the label in a sample.
  • the detectable label can be used to locate and/or quantify the target to which the specific binding agent is directed. Thereby, the presence and/or concentration of the target in a sample can be detected by detecting the signal produced by the detectable label.
  • a detectable label can be detected directly or indirectly, and several different detectable labels conjugated to different specific-binding agents can be used in combination to detect one or more targets.
  • detectable labels which may be detected directly, include fluorescent dyes and radioactive substances and metal particles.
  • indirect detection requires the application of one or more additional binding agents, i.e., secondary binding agents, after application of the primary binding agent.
  • additional binding agents i.e., secondary binding agents
  • the detection is performed by the detection of the binding of the secondary binding agent to the primary detectable binding agent.
  • primary detectable binding agents requiring addition of a secondary binding agent include enzymatic detectable binding agents and hapten detectable binding agents (e.g., a DNP labeled DNA or antibody).
  • the detectable label is conjugated to a nucleic acid polymer which comprises the first binding agent (e.g., in an ISH, WISH, or FISH process).
  • the detectable label is conjugated to an antibody which comprises the first binding agent (e.g., in an IHC process).
  • detectable labels which may be conjugated to binding agents used in the methods of the present invention include fluorescent labels, enzyme labels, radioisotopes, chemiluminescent labels, electrochemiluminescent labels, bioluminescent labels, polymers, polymer particles, metal particles, haptens, and dyes.
  • fluorescent labels include 5-(and 6)- carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)- carboxamido hexanoic acid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, and dyes such as Cy2, Cy3, and Cy5, optionally substituted coumarin including AMCA, PerCP, phycobiliproteins including R- phycoerythrin (RPE) and allophycoerythrin (APC), Texas Red, Princeton Red, green fluorescent protein (GFP) and analogues thereof, and conjugates of R- phycoerythrin or allophycoerythrin, inorganic fluorescent labels such as particles based on semiconductor material like coated CdSe nanocrystallites.
  • RPE R- phycoerythrin
  • APC allophycoerythrin
  • GFP green fluorescent protein
  • polymer particle labels include micro particles or latex particles of polystyrene, PMMA or silica, which can be embedded with fluorescent dyes, or polymer micelles or capsules which contain dyes, enzymes or substrates.
  • metal particle labels include gold particles and coated gold particles, which can be converted by silver stains.
  • haptens examples include DNP, fluorescein isothiocyanate (FITC), biotin, and digoxigenin.
  • Examples of enzymatic labels include horseradish peroxidase (HRP), alkaline phosphatase (ALP or AP), ⁇ -galactosidase (GAL), glucose-6- phosphate dehydrogenase, ⁇ -N-acetylglucosamimidase, ⁇ -glucuronidase, invertase, Xanthine Oxidase, firefly luciferase and glucose oxidase (GO).
  • HRP horseradish peroxidase
  • ALP or AP alkaline phosphatase
  • GAL ⁇ -galactosidase
  • glucose-6- phosphate dehydrogenase ⁇ -N-acetylglucosamimidase
  • ⁇ -glucuronidase invertase
  • Xanthine Oxidase firefly luciferase
  • glucose oxidase GO
  • Examples of commonly used substrates for horseradishperoxidase include 3,3'-diaminobenzidine (DAB), diaminobenzidine with nickel enhancement, 3-amino-9-ethylcarbazole (AEC), Benzidine dihydrochloride (BDHC), Hanker- Yates reagent (HYR), lndophane blue (IB), tetramethylbenzidine (TMB), 4-chloro-1-naphtol (CN), .alpha.-naphtol pyronin (.alpha.-NP), o-dianisidine (OD), 5-bromo-4-chloro-3-indolylphosp- hate (BCIP), Nitro blue tetrazolium (NBT), 2-(p-iodophenyl)-3-p-nitropheny- l-5-phenyl tetrazolium chloride (INT), tetranitro blue tetrazolium (TNBT), 5-bromo-4-chloro-
  • Examples of commonly used substrates for Alkaline Phosphatase include Naphthol-AS-B 1 -phosphate/fast red TR (NABP/FR), Naphthol-AS-MX- phosphate/fast red TR (NAMP/FR), Naphthol-AS-B1 -phosphate/- fast red TR (NABP/FR), Naphthol-AS-MX-phosphate/fast red TR (NAMP/FR), Naphthol-AS- B1 -phosphate/new fuschin (NABP/NF), bromochloroindolyl phosphate/nitroblue tetrazolium (BCIP/NBT), 5-Bromo-4-chloro-3-indolyl-b ⁇ d-galactopyranoside (BCIG).
  • NABP/FR Naphthol-AS-B 1 -phosphate/fast red TR
  • NAMP/FR Naphthol-AS-MX- phosphate/fast red TR
  • luminescent labels include luminol, isoluminol, acridinium esters, 1 ,2-dioxetanes and pyridopyridazines.
  • electrochemiluminescent labels include ruthenium derivatives.
  • radioactive labels examples include radioactive isotopes of iodide, cobalt, selenium, tritium, carbon, sulfur and phosphorous.
  • Detectable labels may be linked to any binding agent that specifically binds to a biological marker of interest, e.g., an antibody, a nucleic acid probe, or a polymer.
  • detectable labels can also be conjugated to second, and/or third, and/or fourth, and/or fifth binding agents, etc.
  • each additional binding agent used to characterize a biological marker of interest may serve as a signal amplification step.
  • the biological marker may be detected visually using, e.g., light microscopy, fluorescent microscopy, electron microscopy where the detectable substance is for example a dye, a colloidal gold particle, a luminescent reagent.
  • Visually detectable substances bound to a biological marker may also be detected using a spectrophotometer.
  • the detectable substance is a radioactive isotope detection can be visually by autoradiography, or non- visually using a scintillation counter. See, e.g., Larsson, 1988, Immunocytochemistry: Theory and Practice, (CRC Press, Boca Raton, FIa.); Methods in Molecular Biology, vol. 80 1998, John D. Pound (ed.) (Humana Press, Totowa, N.J.).
  • the invention provides a first binding agent that is a DNP-labeled nucleic acid polymer or protein, which is used to contact a cell or tissue sample, and a second binding agent that is an antibody or other DNP-binding polypeptide herein.
  • the primary binding agent is a DNP labeled antibody that specifically recognizes a protein of interest in the cell or tissue, and a second binding agent that is an antibody or other DNP-binding polypeptide herein.
  • binding agents may be labeled with a directly or indirectly detectable label in order to detect the presence, amount, and/or localization of a biological marker of interest.
  • additional binding agents may be used, such as a third binding agent that binds the second binding agent, a fourth binding agent that binds the third binding agent, etc.
  • the primary binding agent is a DNP labeled nucleic acid polymer that is designed to specifically hybridize to the target DNA or RNA molecule(s) of interest.
  • the primary binding agent is a DNP labeled antibody that specifically recognizes a protein of interest in the cell or tissue.
  • the secondary binding agent is a rabbit anti-DNP monoclonal antibody of the invention, or a fragment or derivative or variant thereof.
  • the third binding agent is a mouse anti-rabbit antibody and the fourth binding agent is a biotin-labeled goat anti- mouse antibody.
  • a streptavidin or avidin labeled enzyme then contacts the fourth binding agent and serves to indicate the presence, amount, and/or localization of the biological marker of interest in the presence of a suitable chromogenic or fluorogenic substrate.
  • a directly detectable colloidal gold/streptavidin or avidin conjugate is used to visually indicate the presence, amount, and/or localization of the biological marker of interest.
  • the primary binding agent is a DNP labeled nucleic acid polymer that is designed to specifically hybridize to the target DNA or RNA molecule(s) of interest.
  • the primary binding agent is a DNP labeled antibody that specifically recognizes a protein of interest in the cell or tissue.
  • the secondary binding agent is a rabbit anti-DNP monoclonal antibody, or a fragment or derivative or variant thereof.
  • the third binding agent is mouse anti-rabbit antibody that is conjugated to a detectable label (e.g., alkaline phosphatase), which serves to indicate the presence, amount, and/or localization of the biological marker of interest in the presence of a suitable chromogenic or fluorogenic substrate.
  • the third binding agent is a mouse anti-rabbit antibody that is labeled with directly detectable colloidal gold particles in order to visually indicate the presence, amount, and/or localization of the biological marker of interest.
  • the primary binding agent is a DNP labeled nucleic acid polymer that is designed to specifically hybridize to the target DNA or RNA molecule(s) of interest.
  • the primary binding agent is a DNP labeled antibody that specifically recognizes a protein of interest in the cell or tissue.
  • the secondary binding agent is a rabbit anti-DNP monoclonal antibody, or a fragment, derivative or variant thereof, that is conjugated to a detectable label (e.g., alkaline phosphatase), which serves to indicate the presence, amount, and/or localization of the biological marker of interest in the presence of a suitable chromogenic or fluorogenic substrate.
  • the second binding agent is a rabbit anti-DNP monoclonal antibody, fragment, or derivative thereof that is labeled with directly detectable colloidal gold particles in order to visually indicate the presence, amount, and/or localization of the biological marker of interest.
  • automated staining devices may be used in various embodiments of the invention, including embodiments which provide methods of detecting multiple biological markers of interest. Detection of multiple markers frequently requires balancing of the signals emanating from the different detectable substances. When multiple markers are to be detected it may thus be advantageous to provide different amplification conditions (i.e., varying numbers of binding reagents).
  • kits for detecting a biological marker of interest in a sample wherein the kits contain at least one antibody, polypeptide, polynucleotide, vector or host cell as described herein.
  • the kit comprises at least one binding agent, which is a DNP-labeled nucleic acid polymer or protein, or a means for attaching DNP to a nucleic acid polymer or protein of interest, wherein the first binding agent is used to contact the cell or tissue sample.
  • the kit may further comprise a second binding agent which is a monoclonal anti-DNP antibody of the invention, fragment, derivative or variant thereof.
  • the kit may further comprise a third binding agent wherein the third binding agent may be any molecule that binds the second binding agent.
  • the kit may also further comprise a fourth binding agent wherein the fourth binding agent may be any molecule that binds the third binding agent.
  • a kit may comprise a primary binding agent that is a DNP labeled antibody that specifically recognizes a protein of interest in a cell or tissue.
  • the kit may further comprise a secondary binding agent which is a rabbit anti-DNP monoclonal antibody of the invention, or a fragment, derivative or variant thereof.
  • the kit may further comprise a third binding agent which is a mouse anti-rabbit antibody; and may further comprise a fourth binding agent which is a biotin-labeled goat anti-mouse antibody.
  • the kit may further comprise a streptavidin or avidin labeled enzyme, which contacts the fourth binding agent and serves to indicate the presence, amount, and/or localization of the biological marker of interest in the presence of a suitable chromogenic or fluorogenic substrate, as described herein throughout.
  • the kit may further comprise a directly detectable colloidal gold/streptavidin or avidin conjugate that is used to visually indicate the presence, amount, and/or localization of the biological marker of interest.
  • Rabbit monoclonal anti-DNP antibody has been produced by certain hybridoma cell lines. These cell lines, however, are unstable and the yield of anti-DNP antibody produced is undesirably very low.
  • the cDNAs encoding a rabbit monoclonal anti-DNP antibody have been isolated and cloned in order to generate improved cell culture systems for producing the anti-DNP antibody in high yields.
  • the 16 reverse primers for the Heavy chains were 35 T's followed by all possible dinucleotides. They were used in 8 reactions each containing a set of 2 reverse primers.
  • RNA was purified from a rabbit hybridoma cell line that produces anti-DNP antibody.
  • a cDNA synthesis reaction using AMV reverse transcriptase was carried out on 1 ⁇ g of the total RNA using standard protocols known in the art.
  • the heavy and light chains were amplified by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • a standard two temperature PCR amplification program e.g., 94 0 C denature, 68 0 C extension
  • the analysis of kappa and lambda light chain PCR amplification products demonstrated that the rabbit anti-DNP monoclonal antibody contained a kappa light chain, because only the kappa light chain primer set generated a PCR product (see, Figure 5).
  • PCR amplified kappa light chains were subsequently isolated by gel electrophoresis, blunted, and cloned into the pUC19 cloning vector and screened using EcoRI/Hindlll restriction enzyme digests.
  • Figure 6 shows that 11/18 clones contained only the predicted fragment.
  • the cDNA sequence of the kappa light chain of the rabbit anti-DNP monoclonal antibody was determined and is set forth in SEQ ID NO: 1 and Figure 1.
  • the encoded amino acid of the kappa chain is set forth in SEQ ID NO: 2 and Figure 2.
  • a standard two temperature PCR amplification program (e.g., 94 0 C denature, 70 0 C extension) was used with the primers described above to amplify rabbit anti-DNP monoclonal antibody heavy chain cDNAs.
  • the analysis of heavy chain PCR amplification products demonstrated that the rabbit anti- DNP monoclonal antibody was amplified by three different sets of primers (see, Figure 7).
  • PCR amplified heavy chains were subsequently cloned into the pUC19 cloning vector and screened using an EcoRI/Hindlll restriction enzyme digests.
  • Figure 8 shows that 7/9 clones contained the predicted fragment.
  • the cDNA sequence of the heavy chain of the rabbit anti-DNP monoclonal antibody was determined and is set forth in SEQ ID NO: 3 and Figure 3.
  • the encoded amino acid of the heavy chain is set forth in SEQ ID NO: 4 and Figure 4.
  • DNP monoclonal antibody were determined by comparing the FR regions among other rabbit monoclonal antibodies.
  • the CDR regions are underlined in Figures 1-4.
  • the availability of the cDNA sequences for the rabbit monoclonal anti-DNP antibody permits production of large amounts of recombinant antibody in common and easily maintained cell lines, such as CHO and HEK293 cells, among others.
  • These rabbit anti-DNP monoclonal antibody sequences also allow construction of antibody fragments, derivatives, and the like, such as single-chain-variable-fragment antibodies, epitope tagged antibodies and antibody-enzymes fusion proteins.
  • pUC19 constructs containing kappa and heavy chain of rabbit anti-DNP monoclonal antibody cDNAs were used as PCR templates to generate kappa and heavy chain ORFs that contained the appropriate restriction enzyme recognition sites.
  • the forward primers used to make the pCI constructs contained a Kpnl site and a Kozak sequence upstream of the start codon.
  • the forward primers used to make pCMV constructs contained an EcoRV site and a Kozak sequence upstream of the start codon.
  • a pUC19 vector specific sequence was used as a reverse primer for both pCI and pCMV constructs. The primers are summarized in Table 2.
  • Transient expression of rabbit monoclonal anti-DNP antibody was achieved by co-transfecting pCI and pCMV kappa and heavy chain constructs into HEK293 cells (ATCC# CRL-1573) and PEAKrapid cells (ATCC# CRL- 2828) using FuGENE HD transfection reagent (Roche, Cat# 04709705001 ) and standard protocols.
  • HEK293 and PEAKrapid cells were split into 6 well plates at a density of 5X10 5 cells/2 mL/well the day before transfection.
  • the transfection complex prepared for each well contained 1 ⁇ g of kappa chain plasmids, 1 ⁇ g of heavy chain plasmids, and 12 ⁇ l of FuGENE HD reagent. 5 ml of fresh medium was added into each well for the extended expression of the rabbit monoclonal anti-DNP antibody.
  • the culture supernatant was collected 7 days after transfection and the levels of antibody expression were assayed.
  • Stable mammalian cell lines were constructed to express recombinant rabbit monoclonal anti-DNP antibody.
  • the heavy chain of a rabbit monoclonal anti-DNP antibody (SEQ ID NO: 3) was cloned into an expression vector that confers methotrexate resistance, and the kappa chain of the rabbit monoclonal anti-DNP antibody (SEQ ID NO: 1 ) was cloned into another expression vector that confers neomycin resistance.
  • Both heavy chain and kappa chain constructs were linearized with the restriction enzyme Sfil before transfection. Two separate transfections were performed on DG44 cells. One transfection used an unligated DNA mixture (1 :1 ratio of heavy to light chain DNA).
  • the second transfection used ligated DNA (1 :1 ratio of heavy to light chain DNA). 48 hours after the transfection, cells were selected in selection medium for DHFR-positive cells (selection for heavy chain). Two weeks later, when cell viability exceeded 70%, the cells were switched into medium containing 500 ⁇ g/mL Geneticin® for the selection of neo-positive cells (selection for kappa chain). Cells were subjected to two rounds of selection for genomic amplification of DHFR gene using 0.5 ⁇ M and 5 ⁇ M MTX. Expression of anti-DNP antibody was confirmed in the stably transfected cell population using immunofluorescence staining and immunoblotting, the results of which are shown in Figures 11 and 12, respectively. EXAMPLE 4 ANTI-DNP SCFV ANTIBODY
  • a rabbit anti-DNP scFv was designed comprising amino acids A- 106 of the kappa chain variable region (amino acids 21-126 of SEQ ID NO: 2) and amino acids 1-9 of the adjacent kappa chain constant region (amino acids 127-135 of SEQ ID NO: 2), linked at the C-terminus via a 19 amino acid peptide spacer to the N-terminus of amino acids 3-127 of the heavy chain variable region (amino acids 20-144 of SEQ ID NO: 4) and amino acids 1-10 of the adjacent kappa chain constant region (amino acids 145-154 of SEQ ID NO: 4).
  • the amino acid sequence of the kappa chain polypeptide portion of the anti- DNP scFv is set forth in SEQ ID NO: 6.
  • the amino acid sequence of the peptide spacer of the anti-DNP scFv is set forth in SEQ ID NO: 9.
  • the amino acid sequence of the heavy chain polypeptide portion of the anti-DNP scFv is set forth in SEQ ID NO: 8.
  • the complete amino acid sequence of the anti-DNP scFv is set forth in SEQ ID NO: 11.
  • the genetic code uses 61 nucleotide triplets (codons) to encode 20 amino acids and three to terminate translation. Each amino acid is therefore encoded by between one and six synonymous codons. These codons are "read" in the ribosome by complementary tRNAs which have been charged with the appropriate amino acid. It is well known that the frequencies with which different codons are used can vary significantly between different organisms, between proteins expressed at high or low levels within the same organism, and sometimes within the same operon. Thus, codons of a particular sequence to be recombinantly expressed can be optimized for efficient expression in an organism of interest.
  • a codon optimized version of the recombinant rabbit monoclonal anti-DNP scFv antibody was designed for protein expression in Pichia pastoris based upon knowledge of Pichia pastoris codon usage.
  • the Pichia pastoris codon-optimized polynucleotide sequence encoding a rabbit anti-DNP scFv is set forth in SEQ ID NO: 10 and the amino acid sequence of the polypeptide is set forth in SEQ ID NO: 11.

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Abstract

L’invention concerne les anticorps monoclonaux anti-DNP et les compositions et procédés associés, qui peuvent être utilisés dans l’une quelconque d’une variété de procédés de diagnostic et/ou de détection afin de mesurer ou de contrôler l’expression génique, l’expression protéinique, la structure chromosomique, et similaires.
PCT/US2009/036383 2008-03-06 2009-03-06 Anticorps anti-dnp et procédés d’utilisation Ceased WO2009111729A1 (fr)

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