WO1998048837A1 - Polyalkylene oxide-modified single chain polypeptides - Google Patents

Polyalkylene oxide-modified single chain polypeptides Download PDF

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Publication number
WO1998048837A1
WO1998048837A1 PCT/US1998/008654 US9808654W WO9848837A1 WO 1998048837 A1 WO1998048837 A1 WO 1998048837A1 US 9808654 W US9808654 W US 9808654W WO 9848837 A1 WO9848837 A1 WO 9848837A1
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Prior art keywords
polypeptide
antigen
binding
chain
polyalkylene oxide
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PCT/US1998/008654
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English (en)
French (fr)
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WO1998048837A9 (en
Inventor
Marc D. Whitlow
Robert G. L. Shorr
David R. Filpula
Lihsyng S. Lee
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Enzon, Inc.
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Application filed by Enzon, Inc. filed Critical Enzon, Inc.
Priority to JP54734798A priority Critical patent/JP2002505574A/ja
Priority to CA2288994A priority patent/CA2288994C/en
Priority to EP98920001A priority patent/EP0979102A4/en
Priority to AU72666/98A priority patent/AU7266698A/en
Publication of WO1998048837A1 publication Critical patent/WO1998048837A1/en
Publication of WO1998048837A9 publication Critical patent/WO1998048837A9/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/6857—Antibody fragments
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the chemical modification of single chain polypeptides by means of covalent attachment of strands of poly(ethylene glycol) PEG and similar poly(alkylene oxides) to single chain polypeptide binding molecules that have the three dimensional folding and, thus, the binding ability and specificity, of the variable region of an antibody.
  • Such preparations of modified single chain polypeptide binding molecules have reduced immugenicity and antigenicity as well as having a longer halflife in the bloodstream as compared to the parent polypeptide. These beneficial properties of the modified single chain polypeptide binding molecules make them very useful in a variety of therapeutic applications.
  • the invention also relates to multivalent antigen-binding molecules capable of PEGylation. Compositions of, genetic constructions for, methods of use, and methods for producing PEGylated antigen-binding proteins are disclosed.
  • Antibodies are proteins generated by the immune system to provide a specific molecule capable of complexing with an invading molecule, termed an antigen. Natural antibodies have two identical antigen-binding sites, both of which are specific to a particular antigen. The antibody molecule "recognizes" the antigen by complexing its antigen-binding sites with areas of the antigen termed epitopes. The epitopes fit into the conformational architecture of the antigen- binding sites of the antibody, enabling the antibody to bind to the antigen.
  • the antibody molecule is composed of two identical heavy and two identical light polypeptide chains, held together by interchain disulfide bonds. The remainder of this discussion on antibodies will refer only to one pair of light/heavy chains, as each light/heavy pair is identical. Each individual light and heavy chain folds into regions of approximately 110 amino acids, assuming a conserved three- dimensional conformation.
  • the light chain comprises one variable region (V L ) and one constant region (C L ), while the heavy chain comprises one variable region (V H ) and three constant regions (C H 1, C H 2 and C H 3). Pairs of regions associate to form discrete structures.
  • the light and heavy chain variable regions associate to form an "Fv" area which contains the antigen-binding site.
  • the constant regions are not necessary for antigen binding and in some cases can be separated from the antibody molecule by proteolysis, yielding biologically active (i.e., binding) variable regions composed of half of a light chain and one quarter of a heavy chain.
  • a multivalent antigen-binding protein has more than one antigen-binding site.
  • a multivalent antigen-binding protein comprises two or more single-chain protein molecules.
  • Ladner et al. also discloses the use of the single chain antigen binding molecules in diagnostics, therapeutics, in vivo and in vitro imaging, purifications, and biosensors.
  • the use of the single chain antigen binding molecules in immobilized form, or in detectably labeled forms is also disclosed, as well as conjugates of the single chain antigen binding molecules with therapeutic agents, such as drugs or specific toxins, for delivery to a specific site in an animal, such as a human patient.
  • Huston et al. discloses a family of synthetic proteins having affinity for preselected antigens.
  • the contents of U.S.Patent 5,091,513 are incorporated by reference herein.
  • the proteins are characterized by one or more sequences of amino acids constituting a region that behaves as a biosynthetic antibody binding site (BABS).
  • the sites comprise (1) noncovalently associated or disulfide bonded synthetic V H and V L regions, (2) V H -V L or V L -V H single chains wherein the V H and V L are attached to a polypeptide linker, or (3) individual V H or V L domains.
  • the binding domains comprises complementarity determining regions (CDRs) linked to framework regions (FRs), which may be derived from separate immunoglobulins.
  • U.S. Patent 5,091,513 also discloses that three subregions (the CDRs) of the variable domain of each of the heavy and light chains of native immunoglobulin molecules collectively are responsible for antigen recognition and binding. These CDRs consist of one of the hypervariable regions or loops and of selected amino acids or amino acid sequences disposed in the framework regions that flank that particular hypervariable region. It is said that framework regions from diverse species are effective in maintaining CDRs from diverse other species in proper conformation so as to achieve true immunochemical binding properties in a biosynthetic protein.
  • U.S. Patent 5,091 ,513 includes a description of a chimeric polypeptide that is a single chain composite polypeptide comprising a complete antibody binding site.
  • This single chain composite polypeptide is described as having a structure patterned after tandem V H and V L domains, with a carboxyl terminal of one attached through an amino acid sequence to the amino terminal of the other. It thus comprises an amino acid sequence that is homologous to a portion of the variable region of an immunoglobulin heavy chain (V H ) peptide bonded to a second amino acid sequence that was homologous to a portion of the variable region of an immunoglobulin light chain (V L ).
  • amino acid sequences such as the single chain polypeptides described above, and fusion proteins thereof, have not been associated with significant antigenicity in mammals, it has been desirable to prolong the circulating life and even further reduce the possibility of an antigenic response.
  • the relatively small size of the polypeptides and their delicate structure/activity relationship have made polyethylene glycol modification difficult and unpredictable. Most importantly, it was unknown how to modulate retained activity of the polypeptides after conjugation with polymers, such as PEG.
  • PEG polyethylene glycol
  • PEG polyalkalene oxide
  • the hydroxyl end groups of the polymer must first be converted into reactive functional groups. This process is frequently referred to as “activation” and the product is called “activated PEG” or activated polyalkylene oxide.
  • activated PEG Methoxy poly(ethylene glycol) (mPEG), capped on one end with a functional group, reactive towards amines on a protein molecule, is used in most cases.
  • the activated polymers are reacted with a therapeutic agent having nucleophilic functional groups that serve as attachment sites.
  • nucleophilic functional group commonly used as an attachment site is the e-amino groups of lysines.
  • Free carboxylic acid groups suitably activated carbonyl groups, oxidized carbohydrate moieties and mercapto groups have also been used as attachment sites.
  • PEGs such as succinimidyl succinate derivatives of PEG (“SS-PEG”)
  • SS-PEG succinimidyl succinate derivatives of PEG
  • Zalipsky in U.S. PatentNo.5,122,614, discloses poly(ethylene glycol)-N- succinimide carbonate and its preparation. This form of the polymer is said to react readily with the amino groups of proteins, as well as low molecular weight peptides and other materials that contain free amino groups.
  • linkages between the amino groups of the protein, and the PEG are also known in the art, such as urethane linkages (Veronese et al, Appl. Biochem. Biotechnol 77:141-152 (1985)), carbamate linkages (Beauchamp et al, Analyt. Biochem. 131:25-33 (1983)), and others.
  • IgG immunoglobulin G
  • poly(ethylene glycol) that has previously been activated by cyanuric chloride.
  • the coupled IgG was studied for physicochemical and biological properties such as molecular structure, size- exclusion chromatographic behavior, surface activity, interfacial aggregability, heat aggregability inducing nonspecific complement activation, and antigen- binding activity.
  • the poly(ethylene glycol) coupling to IgG increased the apparent Stokes' radius and the surface activity of IgG and stabilized IgG on heating and/or on exposure to interfaces, while no structural denaturation of IgG was observed.
  • the suppressed nonspecific aggregability was interpreted mainly by difficulty in association between the modified IgG molecules.
  • Tullis in U.S. PatentNo.4,904,582, describes oligonucleotide conjugates wherein the oligonucleotides are joined through a linking arm to a hydrophobic moiety, which could be a polyalkyleneoxy group.
  • the resulting conjugates are said to be more efficient in membrane transport, so as to be capable of crossing the membrane and effectively modulating a transcriptional system.
  • the compositions can be used in vitro and in vivo, for studying cellular processes, protecting mammalian hosts from pathogens, and the like.
  • the present invention relates to polyalkylene oxide/amino acid sequence conjugates and processes for preparing them.
  • Suitable amino acid sequences are peptides, such as, single chain polypeptides having binding affinity for an antigen, for example, those described by Ladner et al in U.S. Patent No. 4,946,778 and Huston et al. in U.S. Patent No. 5,091,513.
  • the present invention relates to a physiologically active, substantially non-immunogenic polypeptide conjugate containing at least one polyalkylene oxide strand coupled to a single chain polypeptide having binding affinity for an antigen.
  • the single chain polypeptide includes:
  • the present invention relates to a process for preparing physiologically active, substantially non-immunogenic polypeptide compositions.
  • the process includes coupling a polyalkylene oxide to a single chain polypeptide having the attributes described above.
  • the poly(alkylene oxides) used herein are poly(ethylene glycols) that have been activated for coupling to the target polypeptide.
  • the invention is also directed to a single-chain antigen-binding polypeptide
  • a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site, wherein the single-chain antigen-binding polypeptide-polyalkylene oxide conjugate has an antigen binding affinity within a range of about one-fold to about ten-fold of the antigen binding affinity of the native, unconjugated form of the single-chain antigen-binding polypeptide.
  • the invention is also directed to a single-chain antigen-binding polypeptide -polyalkylene oxide conjugate, comprising:
  • a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site, wherein the single-chain antigen-binding polypeptide-polyalkylene oxide conjugate has an antigen binding affinity within about ten-fold of the antigen binding affinity of the native, unconjugated form of the single-chain antigen-binding polypeptide.
  • the invention is also directed to a single-chain antigen-binding polypeptide -polyalkylene oxide conjugate, comprising:
  • the single-chain antigen-binding polypeptide-polyalkylene oxide conjugate has an antigen binding affinity within about five-fold of the antigen binding affinity of the native, unconjugated form of the single-chain antigen-binding polypeptide.
  • the invention is also directed to a single-chain antigen-binding polypeptide
  • a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site, wherein the single-chain antigen-binding polypeptide-polyalkylene oxide conjugate has an antigen binding affinity within about two-fold of the antigen binding affinity of the native, unconjugated form of the single-chain antigen-binding polypeptide.
  • the invention is also directed to a single-chain antigen-binding polypeptide capable of polyalkylene oxide conjugation, comprising:
  • a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site, wherein the single-chain antigen-binding polypeptide has at least one Cys residue wherein the Cys residue is capable of polyalkylene oxide conjugation and the Cys residue is located at a position selected from the group consisting of (i) the amino acid position 11 , 12, 13, 14 or 15 ofthe light chain variable region; (ii) the amino acid position 77, 78 or 79 ofthe light chain variable region; (iii) the amino acid position 11, 12, 13, 14 or 15 ofthe heavy chain variable region; (iv) the amino acid position 82B, 82C or 83 ofthe heavy chain variable region; (v) any amino acid position ofthe peptide linker; (vi) adjacent to the C-terminus of polypeptide
  • the invention is also directed to a single-chain antigen-binding polypeptide capable of polyalkylene oxide conjugation, comprising:
  • the invention is also directed to a single-chain antigen-binding polypeptide capable of polyalkylene oxide conjugation, comprising: (a) a first polypeptide comprising the antigen binding portion ofthe variable region of an antibody heavy or light chain;
  • a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site, wherein the single-chain antigen-binding polypeptide has at least two consecutive Cys residue wherein the consecutive Cys residues are capable of polyalkylene oxide conjugation and any one of the consecutive Cys residues is located at a position selected from the group consisting of (i) any amino acid position ofthe peptide linker; (ii) adjacent to the C-terminus of polypeptide (a) or (b); and (iii) combinations thereof, wherein the polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an antigen.
  • the invention is further directed to a genetic sequence encoding a single- chain antigen-binding polypeptide capable of polyalkylene oxide conjugation, comprising:
  • a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site, wherein the single-chain antigen-binding polypeptide has at least one Cys residue wherein the Cys residue is capable of polyalkylene oxide conjugation and the Cys residue is located at a position selected from the group consisting of (i) the amino acid position 11, 12, 13, 14 or 15 ofthe light chain variable region; (ii) the amino acid position 77, 78 or 79 ofthe light chain variable region; (iii) the amino acid position 11, 12, 13, 14 or 15 of the heavy chain variable region; (iv) the amino acid position 82B, 82C or 83 ofthe heavy chain variable region; (v) any amino acid position ofthe peptide linker; (vi) adjacent to the C-terminus of polypeptide
  • polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an antigen.
  • the invention is further directed to a genetic sequence encoding a single- chain antigen-binding polypeptide capable of polyalkylene oxide conjugation, comprising:
  • the invention is further directed to a genetic sequence encoding a single- chain antigen-binding polypeptide capable of polyalkylene oxide conjugation, comprising: (a) a first polypeptide comprising the antigen binding portion ofthe variable region of an antibody heavy or light chain;
  • the genetic sequence may be DNA or RNA.
  • the invention is directed to a replicable cloning or expression vehicle comprising the above described DNA sequence.
  • the invention is also directed to such vehicle which is a plasmid.
  • the invention is further directed to a host cell transformed with the above described DNA.
  • the host cell may be a bacterial cell, a yeast cell or other fungal cell, an insect cell or a mammalian cell line.
  • a preferred host is Pichia pastoris.
  • the invention is directed to a method of producing a single-chain antigen- binding polypeptide capable of polyalkylene oxide conjugation, comprising: (a) providing a first genetic sequence encoding a first polypeptide comprising the antigen binding portion ofthe variable region of an antibody heavy or light chain;
  • the invention is further directed to a multivalent single-chain antigen- binding protein, comprising two or more single-chain antigen-binding polypeptides, each single-chain antigen-binding polypeptide comprising:
  • a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site, wherein the single-chain antigen-binding polypeptide has at least one Cys residue wherein the Cys residue is capable of polyalkylene oxide conjugation and the Cys residue is located at a position selected from the group consisting of (i) the amino acid position 11, 12, 13, 14 or 15 ofthe light chain variable region; (ii) the amino acid position 77, 78 or 79 ofthe light chain variable region; (iii) the amino acid position 11, 12, 13, 14 or 15 of the heavy chain variable region; (iv) the amino acid position 82B, 82C or 83 ofthe heavy chain variable region; (v) any amino acid position ofthe peptide linker; (vi) adjacent to the C-terminus of polypeptide (a) or (b); and (vii) combinations thereof, wherein the polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an
  • the Cys polyalkylene oxide conjugation sequence may be capable of attaching a polyalkylene oxide moiety and the Cys residue is located at a position selected from the group consisting of (i ? ) the amino acid position 77 ofthe light chain variable region; (ii') the amino acid position 82B ofthe heavy chain variable region; (iii') the amino acid position 3 ofthe peptide linker; (iv') adjacent to the C-terminus of polypeptide (a) or (b); (v') N-terminus and C-terminus; and (vi') combinations thereof, wherein the polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an antigen.
  • the oligo-Lys polyalkylene oxide conjugation sequence may be capable of attaching a polyalkylene oxide moiety at the oligo-Lys residues located adjacent to the C- terminus ofthe protein, wherein the polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an antigen.
  • the C-terminus of the second polypeptide (b) may be the native C-terminus.
  • the C-terminus ofthe second polypeptide (b) may comprise a deletion of one or plurality of amino acid residue(s), such that the remaining N-terminus amino acid residues ofthe second polypeptide are sufficient for the polyalkylene oxide conjugated polypeptide to be capable of binding an antigen.
  • the C-terminus ofthe second polypeptide may comprise an addition of one or plurality of amino acid residue(s), such that the polyalkylene oxide conjugated polypeptide is capable of binding an antigen.
  • the first polypeptide (a) may comprise the antigen binding portion ofthe variable region of an antibody light chain and the second polypeptide (b) comprises the antigen binding portion ofthe variable region of an antibody heavy chain.
  • the invention is also directed to a method of detecting an antigen suspected of being in a sample, comprising:
  • the invention is further directed to a method of imaging the internal structure of an animal, comprising administering to the animal an effective amount of the polyalkylene oxide conjugated polypeptide or protein of the invention, wherein the polyalkylene oxide conjugated polypeptide is conjugated to one or plurality of detectable label or chelator molecule(s), or conjugated to a carrier having one or plurality of detectable label or chelator molecule(s) bound to the carrier, and measuring detectable radiation associated with the animal.
  • Animal includes human and nonhuman.
  • the invention is also directed to a method for treating a targeted disease, comprising administering an effective amount of a composition comprising the polyalkylene oxide conjugated polypeptide or protein of the invention and a pharmaceutically acceptable carrier vehicle, wherein the polyalkylene oxide conjugated polypeptide is conjugated to one or plurality of bioactive molecules, such as peptides, lipids, nucleic acids (i.e., phosphate-lysine complexes), drug, toxin, boron addend or radioisotope molecule(s), or conjugated to a carrier having one or plurality of peptides, lipids, nucleic acids (i.e., phosphate-lysine complexes), drug, toxin, boron addend or radioisotope molecule(s) bound to the carrier.
  • bioactive molecules such as peptides, lipids, nucleic acids (i.e., phosphate-lysine complexes), drug, toxin, boron addend or radio
  • Figure 1 is a graphical representation of three competition ELISA's in which unlabeled PEG modified CC49/212 SCA (closed squares), CC49/212 SCA (open squares), CC49 IgG (open circles), and MOPC-21 IgG (+) competed against a CC49 IgG radiolabeled with I25 I for binding to the TAG-72 antigen on a human breast carcinoma extract.
  • Figure 2 shows the DNA and protein sequence of CC49/218 SCA which has four engineered cysteine residues at the positions indicated by the codons underlined and marked by an asterisk. Also highlighed are the CDR sequences (double underlined) and the 218 linker (underlined and labeled).
  • cysteine residues in the protein which are involved in two disulfide bonds. These are not underlined.
  • the four engineered cysteine residues occur independently in four different mutants currently, but may be combined in the exact four-mutant codon version shown in this figure.
  • Figure 3 shows the DNA and protein sequence of CC49/218 SCA with an engineered oligo-lysine C-terminal tail segment.
  • the eight new lysine residues were genetically engineered at a B stEII site and are shown underlined and marked with asterisks. Also highlighted are the CDR sequences (double underlined), the 218 linker (underlined and labeled) and selected restriction sites.
  • Figure 4 is a graphical representation of three competition ELISA's in which unlabeled SC-PEG unreacted CC49/218 SCA (closed squares), CC49/218 SCA (open squares), unlabeled XUS-PEG unreacted CC49/218 SCA (open circles), SC-PEG modified CC49/218 SCA (closed circles), XUS-PEG modified CC49/218 SCA (open triangles), CC49 IgG (closed triangles), an Anti-FITC SCA (dashed line) or BL-3 IgG (dotted line) were competed against a CC49 IgG radiolabeled with 125 I for binding to the TAG- 72 antigen on a human breast carcinoma extract.
  • Figure 5 shows the pharmacokinetics of plasma retention of SCA and PEG-SCA.
  • the details ofthe experiment are described in Example 13.
  • Figure 6 shows an SDS-PAGE of the purified CC49-multimers cross- linked by PEG5000 under reducing conditions. The details ofthe experiment are described in Example 14.
  • the lanes ofthe gel contain the following: 1) trimeric form; 2) dimeric form; 3) dimeric form; 4) mixed population; 5) native CC49; 6) PEG-CC49 monomer; 7) PEG-CC49 monomer; 8) empty; 9) empty; and 10) molecular weight standards.
  • Figure 7 shows the binding kinetics of Mono-, Di-, Tri-, -PEG-CC49. The details of the experiment are described in Example 14.
  • Native CC49 is represented by the solid box.
  • PEG-mono-CC49 is represented by the open box.
  • PEG-Di-CC49 is represented by the solid diamond.
  • PEG-Tri-CC49 is represented by the open diamond.
  • Figure 8 shows the results ofthe competition assay performed in Example 16.
  • Nat is native CC49-SCA
  • C2 is PEG SC2000-CC49-SCA
  • GC is glyco- CC49-SCA
  • B* is the biotinylated CC49-SCA
  • C 12 is the PEG-SC 12,000-CC49- SCA
  • F5 is PEG-Flan-5000-CC49-SCA
  • C20 is PEG-SC20000-CC49-SCA.
  • the present invention is directed to the novel combination of a polyalkylene glycol and a single chain polypeptide having binding affinity for an antigen, the polyalkylene glycol and polypeptide preferably being joined together by means of a coupling agent.
  • Single Chain Polypeptides Single Chain Polypeptides
  • the invention relates to the discovery that polyalkylene oxide conjugated single-chain antigen-binding proteins ("SCA") or single-chain variable fragments of antibodies (“sFv”), such as PEGylated SCA proteins, have significant utility beyond that of the nonPEGylated single-chain antigen-binding proteins.
  • SCA polyalkylene oxide conjugated single-chain antigen-binding proteins
  • sFv single-chain variable fragments of antibodies
  • PEGylated SCA protein has significant utility beyond that of the nonPEGylated single-chain antigen-binding proteins.
  • PEGylated SCA protein has a PEG moiety which reduces antigenicity and increases the halflife ofthe modified polypeptide in the bloodstream.
  • the invention is directed to monovalent and multivalent SCA proteins capable of PEGylation, compositions of monovalent and multivalent PEGylated SCA proteins, methods of making and purifying monovalent and multivalent PEGylated SCA proteins, and uses for PEGylated SCA proteins.
  • the invention is also directed to PEGylated SCA proteins having a diagnostic or therapeutic agent covalently attached to an Cys- linked PEGylated polypeptide or an oligo-Lys linked PEGylated polypeptide.
  • SCA single-chain antigen-binding molecule
  • SCA single-chain antigen-binding molecule
  • Fv Fv
  • sFv Fv
  • V L variable region of an antibody
  • V H variable region of an antibody
  • V L variable region of an antibody
  • V L variable region of an antibody
  • V L variable region of an antibody
  • V L variable region of an antibody
  • V L variable region of an antibody
  • V L variable region of an antibody
  • V L variable region of an antibody
  • V L variable region of an antibody
  • V L variable region of an antibody
  • the single polypeptide chain thus comprises a pair of variable regions connected by a polypeptide linker.
  • the regions may associate to form a functional antigen- binding site, as in the case wherein the regions comprise a light-chain and a heavy- chain variable region pair with appropriately paired complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the single-chain protein is referred to as a "single- chain antigen-binding protein" or "single-chain antigen-binding molecule.”
  • Single-chain Fvs can and have been constructed in several ways. Either V L is the N-terminal domain followed by the linker and V H (a V L -Linker- V H construction) or V H is the N-terminal domain followed by the linker and V L (V H -Linker- V L construction).
  • the preferred embodiment contains V L in the N- terminal domain (see, Anand, N.N., et al, J. Biol. Chem. 266:21874-21879 (1991)).
  • multiple linkers have also been used.
  • SCA sFv
  • the Fv domains have been selected from the group of monoclonal antibodies known by their abbreviations in the literature as 26-10, MOPC 315, 741F8, 520C9, McPC 603, D1.3, murine phOx, human phOx, RFL3.8 sTCR, 1 A6, Sel55-4, 18-2-3, 4-4-20, 7A4-1, B6.2, CC49, 3C2, 2c, MA- 15C5/K 12 G 0 , Ox, etc. (see, Huston, J.S. et al, Proc. Natl. Acad. Sci. USA £5:5879-5883 (1988); Huston, J.S.
  • Linkers of the invention used to construct sFv (SCA) polypeptides are designed to span the C-terminus of V L (or neighboring site thereof) and the ⁇ - terminus of V H (or neighboring site thereof) or between the C-terminus of V H and the ⁇ -terminus of V L .
  • the preferred length ofthe peptide linker should be from 2 to about 50 amino acids. In each particular case, the preferred length will depend upon the nature ofthe polypeptides to be linked and the desired activity ofthe linked fusion polypeptide resulting from the linkage. Generally, the linker should be long enough to allow the resulting linked fusion polypeptide to properly fold into a conformation providing the desired biological activity.
  • the appropriate linker length may be estimated by consideration ofthe 3-dimensional conformation ofthe substituent polypeptides and the desired conformation ofthe resulting linked fusion polypeptide. Where such information is not available, the appropriate linker length may be empirically determined by testing a series of linked fusion polypeptides with linkers of varying lengths for the desired biological activity. Such linkers are described in detail in WO 94/12520, incorporated herein by reference.
  • Preferred linkers used to construct sFv (SCA) polypeptides have between 10 and 30 amino acid residues.
  • the linkers are designed to be flexible, and it is recommended that an underlying sequence of alternating Gly and Ser residues be used.
  • three charged residues may be included, two positively charged lysine residues (K) and one negatively charged glutamic acid residue (E).
  • K positively charged lysine residues
  • E negatively charged glutamic acid residue
  • one ofthe lysine residues is placed close to the N-terminus of V H , to replace the positive charge lost when forming the peptide bond ofthe linker and the V H .Such linkers are described in detail in U.S. Patent Application S. N.
  • An object ofthe present invention is to produce a single-chain antigen- binding polypeptide-polyalkylene oxide conjugate which retains antigen binding affinity within a range of about two-fold to about ten-fold ofthe antigen binding affinity ofthe native single-chain antigen-binding polypeptide.
  • Another object ofthe present invention is to produce an sFv (SCA) having one or more Cys residues such that the Cys residue is capable of being conjugated with PEG and the PEGylated polypeptide is capable of binding an antigen (i.e., the PEGylated polypeptide's ability to bind an antigen is not disrupted).
  • a further object ofthe present invention is to produce an sFv (SCA) having three or more consecutive Lys residues such that the Lys residues are capable of being conjugated with PEG and the PEGylated polypeptide is capable of binding an antigen (i.e., the PEGylated polypeptide's ability to bind an antigen is not disrupted).
  • SCA novel sFv
  • PEG polyethylene glycol
  • the activated PEG molecules would be thiol-reactive or amine-reactive polymers such as are well known in the art.
  • the designed changes correspond to amino acid residues on the sFv (SCA) surface which are well separated spatially from the antigen-binding site as deduced from known three-dimentional models ofthe antibody Fv domain.
  • a further object ofthe invention is to produce monovalent and multivalent sFvs (SCA) having one or more Cys PEG conjugation sequence(s).
  • a further object of the invention is to produce monovalent and multivalent sFvs (SCA) having three or more consecutive Lys (i.e., oligo-Lys) PEG conjugation sequence(s).
  • SCA monovalent and multivalent sFv
  • SC As the association of two or more sFvs
  • multivalent sFvs (SCAs) may be generated by chemically crosslinking two sFvs (SCAs) with C-terminal cysteine residues (Cumber et al. , J. Immunol.
  • Multivalent antigen-binding fusion proteins of the invention can be made by any process, but preferably according to the process formaking multivalent antigen-binding proteins set forth in WO 93/11161, incorporated herein by reference.
  • Cys PEGylation sites may occur in the V L and V H regions, adjacent to the C-terminus ofthe polypeptide (V L , V H or neighboring site thereof), the N-terminus ofthe polypeptide (V L , V H or neighboring site thereof), the linker region between the first and second polypeptide regions, or occur in a combination of these regions.
  • oligo-Lys PEGylation sites may occur in the polypeptide linker or in the C-terminus or adjacent to the C-terminus of the polypeptide.
  • the Cys or the oligo-Lys PEGylation site may occur in (1) the native C- terminus of V L (or V H ), (2) the C-terminus of V L (or V H ) wherein the C-terminus has a deletion of one or plurality of amino acid residue(s), such that the remaining N-terminus amino acid residues ofthe peptide are sufficient for the PEGylated polypeptide to be capable of binding an antigen or (3) the C-terminus of V L (or V H ) wherein the C-terminus has an addition of one or plurality of amino acid residue(s), such that the remaining N-terminus amino acid residues ofthe peptide are sufficient for the PEGylated polypeptide to be capable of binding an antigen.
  • C-terminus By “native” is intended the naturally occurring C-terminus ofthe immunoglobulin (first or second polypeptide).
  • C-terminus it is well understood in the art as intending the C-terminal amino acid residue or the C-terminal region of the polypeptide, which could include up to all of the amino acid residues of the polypeptide excluding the first N-terminal amino acid residue ofthe polypeptide.
  • C-terminus is intended as the C-terminal amino acid residue ofthe above mentioned three types of C-terminus ( 1 , 2, or 3), unless otherwise indicated or intended.
  • PEGylation sites were identified and engineered at residues within loop sites in regions of the sFv (SCA) that are diametrically opposed to the antigen binding site.
  • SCA sFv
  • the five loop regions and C-terminal extension chosen as preferred sites of glycosylation are among the most distant regions spatially removed from the binding site.
  • SCA sFv
  • the nucleic and amino acid sequences of each loop are examined for possible Cys PEGylation sites that may be engineered into the loop region.
  • the engineered placement ofthe Cys residue anywhere in these six identified regions can generate a preferred site for sFv (SCA) PEGylation.
  • the engineered placement of the oligo-Lys residues in the linker, the C-terminus ofthe sFv (SCA)and/ or adjacent to the C-terminus of the sFv (SCA) can generate a preferred site for sFv PEGylation.
  • Figure 2 shows the following resulting designs: designed PEGylation site no. 1 in the light chain ofthe CC49/218 SCA; designed PEGylation site no.2 in the N-terminal end ofthe linker in CC49/218 SCA; designed PEGylation site no. 3 in the heavy chain ofthe CC49/218 SCA; designed PEGylation site no.4 at the C-terminus of the CC49/218 SCA.
  • Figure 3 shows the following resulting designs: designed oligo-Lys "hot spot" PEGylation sites at the C-terminus ofthe CC49/218 SCA. Any combination of these sites could be used.
  • nucleotide sequence which is used to introduce a Cys or oligo-Lys PEGylation site into the various positions will depend upon the naturally-occurring nucleotide sequence.
  • the most preferred sites are those in which it takes a minimum number of changes to generate the PEGylation site.
  • a particular amino acid may be encoded by multiple nucleotide sequences.
  • Site-directed mutagenesis is used to change the native protein sequence to one that incorporates the Cys residue or oligo-Lys residues for PEGylation.
  • the mutant protein gene is placed in an expression system, such as bacterial cells, yeast or other fungal cells, insect cells or mammalian cells.
  • the mutant protein can be purified by standard purification methods.
  • Oligonucleotide-directed mutagenesis methods for generating the Cys or oligo-Lys PEGylation sites and related techniques for mutagenesis of cloned DNA are well known in the art. See, Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. ( 1989); Ausubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons ( 1987), both incorporated herein by reference.
  • a preferred oligonucleotide-directed mutagenesis method for the present invention is according to Ho etal, Gene 77:51-59 (1989), incorporated herein by reference. Hosts and Vectors
  • the mutated DNA can be inserted into a cloning vector for further analysis, such as for confirmation ofthe DNA sequence.
  • the DNA sequence is operably linked to regulatory sequences controlling transcriptional expression and introduced into either a prokaryotic or eukaryotic host cell.
  • sFvs are typically produced by prokaryotic host cells
  • eukaryotic host cells are the preferred host cells.
  • Preferred host cells include yeast or other fungal cells, insect cells or mammalian cells. Standard protein purification methods may be used to purify these mutant proteins. Only minor modification to the native protein's purification scheme may be required.
  • DNA molecules such as purified genetic sequences or plasmids or vectors encoding the sFv (SCA) ofthe invention that have engineered Cys residues and/or oligo-Lys residues capable of PEG conjugation.
  • the DNA sequence for the PEGylated sFv (SCA) polypeptide can be chosen so as to optimize production in organisms such as prokaryotes, yeast or other fungal cells, insect cells or mammalian cells.
  • the DNA molecule encoding an sFv (SCA) having Cys residues and/or oligo-Lys residues for PEG conj ugation can be operably linked into an expression vector and introduced into a host cell to enable the expression ofthe engineered sFv (SCA) protein by that cell.
  • a DNA sequence encoding an sFv (SCA) having Cys and/or oligo-Lys PEGylation sites may be recombined with vector DNA in accordance with conventional techniques. Recombinant hosts as well as methods of using them to produce single chain proteins ofthe invention are also provided herein.
  • sFv (SCA) proteins of the invention can be accomplished in procaryotic cells.
  • Preferred prokaryotic hosts include, but are not limited to, bacteria such as Neisseria, Mycobacteria, Streptococci, Chlamydia and E coli.
  • Eukaryotic hosts for cloning and expression of such sFv (SCA) proteins ofthe invention include insect cells, yeast, fungi, and mammalian cells (such as, for example, human or primate cells) either in vivo, or in tissue culture.
  • a preferred host for the invention is Pichia pastoris.
  • the sFv (SCA) encoding sequence having Cys residues and/or oligo-Lys residues for PEG conjugation and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a non-replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the desired sFv (SCA) protein may occur through the transient expression ofthe introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced sFv (SCA) sequence into the host chromosome.
  • the sFv (SCA) sequence can be integrated into the host cell chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the sFv (SCA) sequence and marker.
  • the marker may complement an auxotrophy in the host (such as his4, leu2, or ura3, which are common yeast auxotrophic markers), biocide resistance, e.g., antibiotics, or resistance to heavy metals, such as copper, or the like.
  • the selectable marker gene can either be directly linked to the sFv (SCA) DNA sequence to be expressed, or introduced into the same cell by co-transfection.
  • the introduced sequence will be inco ⁇ orated into a plasmid vector capable of autonomous replication in the recipient host cell.
  • a plasmid vector capable of autonomous replication in the recipient host cell.
  • Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies ofthe vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • yeast vector systems Any of a series of yeast vector systems can be utilized. Examples of such expression vectors include the yeast 2-micron circle, the expression plasmids YEP13, YCP and YRP, etc., or their derivatives. Such plasmids are well known in the art (Botstein et al, Miami Wntr. Symp. 7P:265-274 (1982); Broach, J.R., In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445- 470 (1981); Broach, J.R., Cell 25:203-204 (1982)).
  • vectors For a mammalian host, several possible vector systems are available for expression.
  • One class of vectors utilize DNA elements which provide autonomously replicating extra-chromosomal plasmids, derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, or S V40 virus.
  • a second class of vectors relies upon the integration ofthe desired gene sequences into the host chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes may be selected by also introducing one or more markers which allow selection of host cells which contain the expression vector.
  • the marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or resistance to heavy metals, such as copper or the like.
  • the selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by co-transformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.
  • the cDNA expression vectors incorporating such elements include those described by Okayama, H., Mol Cell. Biol. 3:280 (1983), and others.
  • vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNHl ⁇ a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
  • Preferred vectors for expression in Pichia are pHIL-S 1 (Invitrogen Corp.) and pPIC9 (Invitrogen Corp.). Other suitable vectors will be readily apparent to the skilled artisan.
  • the DNA constructs may be introduced or transformed into an appropriate host.
  • Various techniques may be employed, such as transformation, transfection, protoplast fusion, calcium phosphate precipitation, electroporation, or other conventional techniques.
  • the cells After the cells have been transformed with the recombinant DNA (or RNA) molecule, the cells are grown in media and screened for appropriate activities. Expression of the sequence results in the production of the mutant sFv (SCA) for PEG conjugation of the present invention.
  • SCA mutant sFv
  • the straight chain polyalkylene glycols employed in the practice of the present invention are ofthe structural formula
  • R is selected from the group consisting of hydrogen, lower alkyl, and mixtures thereof
  • R 1 is selected from the group consisting of hydrogen and lower alkyl
  • n is a positive integer.
  • lower alkyl is meant an alkyl group having from one to four carbon atoms, i.e., methyl, ethyl, propyl, butyl, and isomers of the foregoing.
  • R is preferably selected from the group consisting of hydrogen, methyl, and mixtures thereof
  • R 1 is preferably selected from the group consisting of hydrogen and methyl
  • n is preferably a positive integer of 500 or less.
  • R is most preferably hydrogen
  • R 1 is most preferably methyl
  • n is most preferably an integer of 7 to 150.
  • poly(alkylene glycols) employed in the practice ofthe present invention are poly(ethylene glycol), poly(propylene glycol), mixtures thereof, and copolymers of poly(ethylene glycol) and poly(propylene glycol), wherein one of the terminal hydroxyl groups ofthe polymer may be substituted with a lower alkyl group.
  • a preferred polyalkylene glycol for use in the present invention is poly(ethylene glycol)-hydrazine.
  • the most preferred polyalkylene glycol for use in the present invention is methoxy polyethylene glycol).
  • the polyalkylene glycol employed in the practice ofthe present invention will be designated PAG, which term is intended to include both compounds wherein R 1 is hydrogen and compounds wherein R 1 is alkyl.
  • PEG refers to poly(ethylene glycol) and mPEG refers to methoxy poly(ethylene glycol).
  • the PAG does not have to be of a particular molecular weight, but it is preferred that the molecular weight be between about 500 and about 40,000; more preferably, between about 2,000 and about 20,000.
  • the choice of molecular weight of PAG is made based on the nature of the particular polypeptide employed, for example, the number of amino or other groups available on the polypeptide for modification. Molecular weights of about 10,000 and about 20,000 are most preferred.
  • PAGs that contain two terminal hydroxyl groups per moiety are capable of crosslinking other polymers, e.g. proteins. Where, as is often the case, crosslinking would be deemed undesirable, such crosslinking can be minimized or prevented by means known in the art.
  • a preferred means for preventing crosslinking is to preblock one end ofthe PAG, such as is done in the commercially available methoxy poly(ethylene glycol).
  • the P AGs employed in the practice ofthe present invention are preferably coupled to polypeptides by means of suitable coupling agents .
  • suitable coupling agents A useful review of a number of coupling agents that can be employed in the practice ofthe present invention appears in Dreborg etal, Critical Reviews in Therapeutic Drug Carrier
  • the terminal hydroxyl groups of PEG can be transformed into amine, carboxyl, or hexamethyl isocyanate groups. See, for example, Zalipsky et al, 1983, supra.
  • a mixed anhydride derivative of carboxylated mPEG can be prepared in the presence of triethylamine and then reacted with proteins:
  • Carboxylated mPEG can also be reacted with hydroxysuccinimide in the presence of dicyclohexylcarbodumide and dimethyl formamide for reaction with protein:
  • Veronese et al, Appl. Biochem. & Biotechnol 77:141-152 (1985) describe the activation of methoxy poly(ethylene glycol) with phenylchloroformates, e.g., 2,4,5-trichlorophenylchloroformate or p -nitrophenylchloroformate. These derivatives are linked to peptides by urethane linkages:
  • Zalipsky in U.S. Patent No. 5,122,614, describes the activation of PEG by conversion into its N-succinimide carbonate derivative (“SC-PEG”): o o
  • BSC-PEG polyethylene glycol-bis-succinidyl carbonate
  • activated P AGs can also be employed in the practice ofthe present invention.
  • the preferred activated PAG for use in the practice ofthe present invention is selected from the group consisting of SS-PEG and SC-PEG.
  • SC-PEG is most preferred.
  • the invention further provides for the use of branched, substantially non- antigenic polymers for polyalkylene oxide conjugation ofthe sFv (SCA) proteins corresponding to the formula:
  • (A) represents an activated functional group capable of undergoing nucleophilic substitution.
  • (A) can be a group which is capable of bonding with biologically active nucleophiles or moieties capable of doing the same.
  • R includes a poly(alkylene oxide) PAO such as poly(ethylene glycol) PEG or mPEG. It is preferred that each chain have a molecular weight of between about 200 and about 12,000 daltons and preferably between about 1,000 and about 10,000 daltons. Molecular weights of about 5,000 daltons are most preferred.
  • PAO poly(alkylene oxide) PAO
  • PAO poly(ethylene glycol) PEG or mPEG. It is preferred that each chain have a molecular weight of between about 200 and about 12,000 daltons and preferably between about 1,000 and about 10,000 daltons. Molecular weights of about 5,000 daltons are most preferred.
  • 2 or 3 polymer chains are joined to the alphatic linking moiety (L).
  • Suitable aliphatics included substituted alkyl diamines and triamines, lysine esters and malonic ester derivatives.
  • the linking moieties are perferably non-planar, so that the polymer chains are not rigidly fixed.
  • the linking moiety (L) is also a means for attaching the mulitple polymer chains or "branches" to (A), the moeity through which the polymer attaches to the sFv (SCA) protein.
  • (L) preferably includes a multiply-functionalized alykyl group containing up to 18, and more preferably between 1-10 carbon atoms.
  • a heteroatom such as nitrogen, oxygen or sulfur may be included within the alkyl chain.
  • the alkyl chain may also be branched at a carbon or nitrogen atom.
  • (L) is a single nitrogen atom.
  • (L) and (R) are preferably joined by a reaction between nucleophilic functional groups on both (R) and (L). Each (R) is suitably functionalized to undergo nucleophilic substitution and bond with (L). Such functionalization of polymers is readily apparent to those of ordinary skill in the art. A wide variety of linkages are contemplated between (R) and (L).
  • Urethane (carbamate) linkages are preferred.
  • the bond can be formed, for example, by reacting an amino group such as l,3-diamino-2-propanol with methoxypolyethylene glycol succinimidyl carbonate as described in U.S. Patent No. 5,122,614.
  • Amide linkages which can be formed by reacting an amino- terminated non-antigenic polymer suchas methoxypolyethylene glycol-amine (mPEG amine) with an acyl chloride functional group. Examples of other such linkages include ether, amine, urea, and thio and thiol analogs thereof, as well as the thio and thiol analogs ofthe urethane and amide linkages discussed supra.
  • the moiety (A) of Formula II represents groups that "activate" the branched polymers ofthe present invention for conjugation with biologically active materials.
  • (A) can be a moiety selected from:
  • Functional groups capable of reacting with an amino group such as: a) carbonates such as the p-nitrophenyl or succinimidyl; b) carbonyl imidazole; c) azlactones; d) cyclic imide thiones; or e) isocyanates or isothiocyanates.
  • Functional groups capable of reacting with carboxylic acid groups and reactive with carbonyl groups such as: a) primary amines; or b) hydrazine and hydrazide functional groups such as the acyl hydrazides, carbazates, semicarbamates, thiocarbazates, etc.
  • Functional groups capable of reacting with mercapto or sulfhydryl groups such as phenyl glyoxals; see, for example, U.S. Patent No. 5,093,531.
  • the moiety (A) can also include a spacer moiety located proximal to the aliphatic linking moiety (L).
  • the spacer moiety may be a heteroalkyl, alkoxyl, alkyl containing up to 18 carbon atoms or even an additional polymer chain.
  • the spacer moieties can be added using standard synthesis techniques.
  • the branched polymers generally, U-PAO's or U-PEG's, are formed using conventional reaction techniques known to those of ordinary skill in the art.
  • umbrella-like branched polymers ofthe present invention react with biologically active nucleophiles to form conjugates.
  • the point of polymer attachment depends upon the functional group (A). For example,
  • (A) can be a succinimidyl succinate or carbonate and react with e-amino lysines.
  • the branched polymers can also be activated to link with any primary or secondary amino group, mercapto group, carboxylic acid group, reactive carbonyl group or the like found on biologically active polypeptides. Other groups are apparent to those of ordinary skill in the art.
  • branched polymers One ofthe main advantages ofthe use ofthe branched polymers is that the branching imparts an umbrella-like three dimensional protective covering to the materials they are conjugated with. This contrasts with the string-like structure ofthe straight chain polymers discussed, supra.
  • An additional advantage of the branched ploymers is that they provide the benefits associated with attaching several strands of polymers to a sFv protein but require substantially fewer conjugation sites. The desired properties of PEGylation are realized and the loss of bioactivity is minimized.
  • One or more of the activated branched polymers can be attached to a biologically active nucleophile, such as an sFv protein, by standard chemical reactions.
  • a biologically active nucleophile such as an sFv protein
  • the upper limit for (z) will be determined by the number of available nucleophilic attachment sites and the degree of polymer attachment sought by the artisan.
  • the degree of conjugation can be modified by varying the reaction stoichimetry using well-known techniques. More than one polymer conjugated to the nucleophile can be obtained by reacting a stoichimetric excess ofthe activated polymer with the nucleophile.
  • SCAs isolated sFvs
  • the fermentation of the sFv-producing E coli strains are performed at 32 °C using a casein digest-glucose-salts medium. At an optical density of 18 to 20 at 600 nm, sFv expression is induced by a 42° C temperature shock for one hour. After the fermentation is cooled to 10°C, the cells are harvested by centrifugation at 7000g for ten minutes. The wet cell paste is then stored frozen at -20 °C. Approximately 200 to 300 g of wet cell paste is normally recovered from one 10-liter fermentation.
  • the cell paste from three 10-liter fermentations (600- 900 g) is thawed overnight at 4°C and gently resuspended at 4°C in 50 mM Tris- HC1, 1.0 mM ⁇ DTA, 100 mM KC1, 0.1 mM phenylmethylsulfonyl chloride (PMSF), pH 8.0 (lysis buffer), using 10 liters of lysis buffer for every kilogram of wet cell paste.
  • PMSF 0.1 mM phenylmethylsulfonyl chloride
  • pH 8.0 pH 8.0
  • the chilled mixture is passed three times through a Manton-Gaulin cell homogenizer to fully lyse the cells.
  • the cell homogenizer raises the temperature ofthe cell lysate to 25 ⁇ 5 °C
  • the cell lysate is cooled to 5 ⁇ 2°C with a Lauda/Brinkman chilling coil after each pass. Complete lysis is verified by visual inspection under a microscope.
  • the cell lysate is centrifuged at 24,3 OOg for thirty minutes at 6 °C using a Sorvall RC-5B centrifuge.
  • the pellet contains the insoluble sFv and the supernatant is discarded.
  • the pellet is washed by gently scraping it from the centrifuge bottles and resuspending it in 5 liters of lysis buffer/kg of wet cell paste.
  • the resulting 3.0-4.5-liter suspension is again centrifuged at 24,300g for 30 min at 6 °C, and the supernatant is discarded.
  • This washing ofthe cell pellet removes soluble E. coli proteins and can be repeated as many as five times. At any time during this washing procedure the material can be stored as a frozen pellet at -20 °C.
  • a substantial time saving in the washing steps can be accomplished by utilizing a Pellicon tangential flow apparatus equipped with 0.22- ⁇ m microporous filters.
  • the washed cell pellet is solubilized at 4°C in freshly prepared 6 M guanidine hydrochloride, 50 mM Tris-HCl, 10 mM CaCl 2 , 50 mM KC1, pH 8.0 (denaturing buffer), using 6 ml/g of pellet. If necessary, a few quick pulses from a Heat Systems Ultrasonics tissue homogenizer can be used to complete the solubilization.
  • the resulting suspension is centrifuged at 24,300g for 45 minutes at 6°C and the pellet is discarded.
  • the optical density of the supernatant is determined at 280 nm and if the OD 280 is above 30, additional denaturing buffer is added to obtain an OD 2g0 of approximately 25.
  • the supernatant is slowly diluted into cold (4-7 °C) refolding buffer (50 mM Tris-HCl, 10 mM CaCl 2 , 50 mM KC1, 0.1 mM PMSF, pH 8.0) until a 1 :10 to 1:100 dilution is reached (final volume 70-120 liters).
  • the refolding buffer should be prepared at least one day prior to use, to allow sufficient time for it to cool to 4°C. The best results will be obtained when the supernatant is slowly added to the refolding buffer over a two hour period, with gentle mixing.
  • the solution is left undisturbed for at least twenty hours and then filtered through a Millipore Pellicon tangential flow apparatus at 4°C with four to six 0.45- ⁇ m microporous membranes (HVLP 000 C5).
  • the filtrate is concentrated to 1 to 2 liters using a Pellicon apparatus with four to six 10,000 NMWL cassettes (SK1PA156A4), again at 4°C.
  • the concentrated crude sFv sample is buffer exchanged at 4°C into 20 mM 2-[N-morpholino]ethanesulfonic acid (Mes), 0.3 mM CaCl 2 , pH 6.0, using the Pellicon ultrafiltration apparatus equipped with four to six 10,000 NMWL cassettes.
  • the sample is then chromatographed on a Waters Accell Plus CM ion- exchange (RCM) column (4.7 x 30.0 cm).
  • RCM Waters Accell Plus CM ion- exchange
  • the material Prior to loading on the HPLC, the material is filtered through a 0.22- ⁇ m filter and the Accell column is equilibrated with Buffer A (40 mM Mes, 1 mM CaCl 2 , pH 6.0). Following sample loading, the Accell column is eluted over a 55-minute period with a linear gradient of Buffer A and Buffer B (40 mM Mes, 100 mM CaCl 2 , pH 7.0). (See Table 1).
  • the Accell Plus CM column has a capacity of about 3 g and thus all the crude sFv sample can normally be loaded in a single run.
  • the fractions are analyzed using 4-20% Novex SDS-PAGE gels and the peak fractions are pooled.
  • sFv proteins have an extinction coefficient of about 2.0 mg ml "1 cm '1 at 280 nm and this can be used to determine protein concentration.
  • the sFv sample is eluted from the PolyCAT A column with a 50-min linear gradient of Buffer D and Buffer E (40 mM Mops, 10 mM Ca acetate, pH 8.0). See Table 2.
  • the sFv proteins will often elute between 20 and 26 min when this gradient is used. This corresponds to an eluting solvent composition of approximately 70% Buffer D and 30% Buffer E.
  • This purification procedure yields sFv proteins that are more than 95% pure as examined by SDS-PAGE and Scatchard analysis. Modifications ofthe above procedure may be dictated by the isoelectric point of the particular sFv being purified, which is often between 8.0 and 9.3.
  • the polyalkylene glycols (PAGs) employed in the practice ofthe present invention which, as indicated above, are preferably activated by reaction with a coupler, can be reacted with any of several groups that may be present attached to the chain ofthe single chain antigen binding molecules, e.g. terminal carboxyl groups, thiol groups, phenolic hydroxyl groups, or primary amino groups located at the chain terminus or along the chain. It is preferred to react activated PAGs with primary amine groups, especially those occurring along the peptide chain. It is most preferred that the activated PAGs be coupled to the e amino groups of lysine residues as well as cysteine residues in the polypeptide.
  • PAGs polyalkylene glycols
  • the reaction between the PAG and the single chain polypeptide is normally carried out in solution, preferably an aqueous buffer solution providing a pH in the range of from about 6 to about 10, preferably from about 7 to about 9, most preferably from about 7 to about 8.
  • aqueous buffer solution providing a pH in the range of from about 6 to about 10, preferably from about 7 to about 9, most preferably from about 7 to about 8.
  • the reaction between the PAG and the single chain polypeptide will normally be run under conditions that will not give rise to denaturation, e.g. mild temperatures and no more agitation than necessary.
  • the reaction will preferably be run at a temperature in the range of from about 4° C to about 25° C. More preferably, the reaction will be run at room temperature, i.e. from about 20° C to about 25° C.
  • the amount of PAG employed relative to the amount of single chain polypeptide will be dependent upon the desired nature ofthe reaction product. Where, for example, it is desired to react a PAG with each lysine residue along the polypeptide chain, an amount of PAG at least equimolar to the lysine concentration will be required. It will be advantageous to employ an excess of PAG, where possible, in order to increase the reaction rate and the likelihood of a complete reaction. Clearly, if fewer than all ofthe possible reaction sites along the polypeptide chain are to be derivatized, correspondingly less PAG will be used.
  • molar excesses of PAG's it has been determined that molar excesses on the order of 2 - 100 of the PAG can be used; molar excesses of 2 - 10 are preferred.
  • the time required for the reaction will depend upon a number of factors, such as reaction temperature, concentration of reactants, and whether full or partial reaction is desired.
  • the course of the reaction can be monitored by conventional means, such as the analysis of periodic samples by size exclusion chromatography or gel electrophoresis.
  • the reaction can conveniently be terminated when desired by the addition of a compound having a primary amine group, e.g. glycine, to scavenge the excess PAG.
  • reaction time of about 15 - 120 minutes will typically be required to fully react the PAG with the primary amine groups of the lysine residues of the single chain polypeptide at room temperature.
  • the skilled practitioner will understand that the time for conjugation, as well as the amount and type of PAG, must not be such as to inactivate the polypeptide being employed.
  • Purification ofthe PAG/single chain polypeptide reaction product can be effected by means commonly employed by those skilled in the art, such as, for example, size exclusion chromatography, ion-exchange chromatography, ultrafiltration, dialysis, and the like. Solutions of the reaction product can, if desired, be concentrated with a rotary evaporator and can be obtained in the dry state by lyophilization.
  • the resulting adduct is expected to be useful both diagnostically and therapeutically, exhibiting, as compared to the unreacted single chain polypeptide, decreased immunogenicity, increased circulating life, and increased stability while maintaining an acceptable level of activity.
  • the single chain antigen binding polypeptide can be reacted with the activated branched polyethylene glycol polymers discussed above in an aqueous reaction medium which can be buffered, depending on the pH requirements ofthe nucleophile.
  • the optimum pH for the reaction is generally between about 6.5 and about 8.0 and preferably about 7.4 for polypeptides.
  • the optimum reaction conditions for the sFv stability, reaction efficiency, etc. is within the level of ordinary skill in the art.
  • the preferred temperature range is between 4°C and 37 °C. The reaction temperature cannot exceed the temperature at which the nucleophile may denature or decompose. It is preferred that the nucleophile be reacted with an excess ofthe activated branched polymer.
  • the cojugate is recovered and purified, for example, by diafiltration, column chromatography, combinations thereof, or the like.
  • the polyalkylene oxide conjugated sFv may further be modified by conjugating a diagnostic or therapeutic agent to the polyalkylene oxide conjugated sFv.
  • a diagnostic or therapeutic agent to the polyalkylene oxide conjugated sFv.
  • the general method of preparing an antibody conjugate according to the invention is described in Shih, L.B., etal, Cancer Res. 57:4192 (1991); Shih, L.B., and D.M. Goldenberg, Cancer Immunol. Immunother. 37:197 (1990); Shih, L.B., et al, Intl. J. Cancer 46:1101 (1990); Shih, L.B., et al, Intl. J.
  • the indirect method involves reacting an antibody (or sFv), whose polyalkylene oxide has a functional group, with a carrier polymer loaded with one or plurality of bioactive molecules, such as, peptides, lipids, nucleic acids (i.e., phosphate-lysine complexes), drug, toxin, chelator, boron addend or detectable label molecule(s).
  • a carrier polymer loaded with one or plurality of bioactive molecules, such as, peptides, lipids, nucleic acids (i.e., phosphate-lysine complexes), drug, toxin, chelator, boron addend or detectable label molecule(s).
  • the polyalkylene oxide conjugated sFv may be directly conjugated with a diagnostic or therapeutic agent.
  • the general procedure is analogous to the indirect method of conjugation except that a diagnostic or therapeutic agent is directly attached to an oxidized sFv component. See Hansen et al, U.S. Patent No. 5,443,953, incorporated herein by reference.
  • the polyalkylene oxide conjugated sFv can be attached to a derivative of the particular drug, toxin, chelator, boron addend or label to be loaded, in an activated form, preferably a carboxyl-activated derivative, prepared by conventional means, e.g., using dicyclohexylcarbodumide (DCC) or a water soluble variant thereof, to form an intermediate adduct.
  • DCC dicyclohexylcarbodumide
  • drugs and toxins which have a cytotoxic effect on tumor cells or microorganisms that may infect a human and cause a lesion, in addition to the specific illustrations given above. They are to be found in compendia of drugs and toxins, such as the Merck Index and the like. Any such drug can be loaded onto a carrier or directly onto a polyalkylene oxide conjugated sFv by conventional means well known in the art, and illustrated by analogy to those described above.
  • Chelators for radiometals or magnetic resonance enhancers are also well known in the art. Typical are derivatives of ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTP A). These typically have groups on the side chain by which the chelator can be attached to a carrier or directly onto a polyalkylene oxide conjugated sFv. Such groups include, e.g., a benzylisothiocyanate, by which the DTP A or EDTA can be coupled to the reactive group of an sFv.
  • EDTA ethylenediaminetetraacetic acid
  • DTP A diethylenetriaminepentaacetic acid
  • Labels such as radioisotopes, enzymes, fluorescent compounds, electron transfer agents, and the like can also be linked to carrier or directly onto a polyalkylene oxide conjugated sFv by conventional methods well known to the art. These labels and the sFv conjugates prepared from them can be used for immunoassays and for immunohistology, much as the sFv conjugate prepared by direct attachment ofthe labels to the sFv.
  • the loading ofthe conjugates according to the present invention with a plurality of labels can increase the sensitivity of assays or histological procedures, where only low extent of binding ofthe sFv to target antigen is achieved.
  • Boron addends e.g., carboranes
  • carboranes when attached to single-chain antigen binding molecules and targeted to lesions, can be activated by thermal neutron irradiation and converted to radioactive atoms which decay by alpha emission to produce highly cytotoxic short-range effects.
  • Carboranes can be made with carboxyl functions on pendant side chains, as is well known in the art.
  • Loading of drugs on the carrier will depend upon the potency ofthe drug, the efficiency of sFv targeting and the efficacy ofthe conjugate once it reaches its target. In most cases, it is desirable to load at least 20, preferably 50, and often 100 or more molecules of a drug on a carrier.
  • the ability to partially or completely detoxify a drug as a conjugate according to the invention, while it is in circulation, can reduce systemic side effects ofthe drug and permit its use when systemic administration of the unconjugated drug would be unacceptable.
  • Administration of more molecules of the drug, but conjugated to the sFv on a carrier, according to the present invention permits therapy while mitigating systemic toxicity.
  • Toxins will often be less heavily loaded than drugs, but it will still be advantageous to load at least 5, preferably 10 and in some cases 20 or more molecules of toxin on a carrier and load at least one carrier chain on the sFv for targeted delivery.
  • the polyalkylene oxide conjugated sFv (SCA) polypeptide conjugates of the present invention are expected to have much longer circulating half lifes and reduced immunogenicity in vivo. This may solve a potential limitation relating to very rapid blood clearance of some sFv proteins. It would also reduce or eliminate concerns about repeated administration of a therapeutic sFv which may otherwise provoke an immune response in the patient.
  • the choice of the particular cysteine and/or oligo-lysine mutant combinations may allow one to achieve circulating lives over a considerable range depending on the specific polyalkylene oxide conjugated sFv variant polypeptide. This would allow sFv to be administered for the therapeutic use of choice.
  • a diagnostic or therapeutic agent is a molecule or atom which is conjugated to an antibody and useful for diagnosis or for therapy. The immunoreactivity ofthe antibody is retained. Diagnostic or therapeutic agents include drugs, toxins, chelators, boron compounds and detectable labels. See “Conjugates" section, supra, for further details.
  • the diagnostic or therapeutic agent may be, but is not limited to, at least one selected from a nucleic acid, a compound, a protein, an element, a lipid, an antibody, a saccharide, an isotope, a carbohydrate, an imaging agent, a lipoprotein, a glycoprotein, an enzyme, a detectable probe, or any combination thereof, which may be detectably labeled as for labeling antibodies, as described herein.
  • labels include, but are not limited to, enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds or metals, chemiluminescent compounds and bioluminescent compounds.
  • any other known diagnostic or therapeutic agent can be used in a method ofthe present invention.
  • a therapeutic agent used in the present invention may have a therapeutic effect on the target cell, the effect selected from, but not limited to, correcting a defective gene or protein, a drug action, a toxic effect, a growth stimulating effect, a growth inhibiting effect, a metabolic effect, a catabolic affect, an anabolic effect, an antiviral effect, an antibacterial effect, a hormonal effect, a neurohumoral effect, a cell differentiation stimulatory effect, a cell differentiation inhibitory effect, a neuromodulatory effect, an antineoplastic effect, an anti-tumor effect, an insulin stimulating or inhibiting effect, a bone marrow stimulating effect, a pluripotent stem cell stimulating effect, an immune system stimulating effect, and any other known therapeutic effects that may be provided by a therapeutic agent delivered to a cell via a delivery system according to the present invention.
  • the sFv conjugate ofthe present invention may be used for protection, suppression or treatment of infection or disease.
  • protection from infection or disease as used herein is intended “prevention,” “suppression” or “treatment.”
  • Prevention involves administration of a glycosylated sFv conjugate prior to the induction ofthe disease.
  • Suppression involves administration ofthe composition prior to the clinical appearance ofthe disease.
  • Treatment involves administration ofthe protective composition after the appearance of the disease. It will be understood that in human and veterinary medicine, it is not always possible to distinguish between “preventing” and “suppressing” since the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events. Therefore, it is common to use the term “prophylaxis” as distinct from “treatment” to encompass both “preventing” and “suppressing” as defined herein.
  • the term “protection,” as used herein, is meant to include “prophylaxis.”
  • Such additional therapeutic agents which can further comprise a therapeutic agent or composition ofthe present invention may be selected from, but are not limited to, known and new compounds and compositions including antibiotics, steroids, cytotoxic agents, vasoactive drugs, antibodies and other therapeutic modalities.
  • antibiotics used in the treatment of bacterial shock such as gentamycin, tobramycin, nafcillin, parenteral cephalosporins, etc; adrenal corticosteroids and analogs thereof, such as methyl prednisolone, mitigate the cellular injury caused by endotoxins; vasoactive drugs, such as alpha receptor blocking agent (e.g., phenoxybenzamine), beta receptor agonists (e.g., isoproterenol), and dopamine are agents suitable for treating septic shock.
  • alpha receptor blocking agent e.g., phenoxybenzamine
  • beta receptor agonists e.g., isoproterenol
  • dopamine are agents suitable for treating septic shock.
  • Polyalkylene oxide conjugated sFv ofthe invention may also be used for diagnosis of disease and to monitor therapeutic response.
  • Other uses of polyalkylene oxide conjugated sFv proteins are specific targeting of pro-drug activating enzymes to tumor cells by a bispecific molecule with specificity for tumor cells and enzyme.
  • Polyalkylene oxide conjugated sFv may be used for specific delivery of drug to an in vivo target, such as a tumor, delivery of radioactive metals for tumor radioimmunodiagnosis or radioimmunotherapy (Goldenberg, D.M., Am. J. Med.
  • Affinity purification is made possible by affixing the polyalkylene oxide conjugated sFv protein to a support, with the antigen-binding sites exposed to and in contact with the ligand molecule to be separated, and thus purified.
  • Biosensors generate a detectable signal upon binding of a specific antigen to an antigen-binding molecule, with subsequent processing of the signal.
  • Polyalkylene oxide conjugated sFv proteins when used as the antigen-binding molecule in biosensors, may change conformation upon binding, thus generating a signal that may be detected.
  • the invention is also directed to a method of detecting an antigen suspected of being in a sample by contacting the sample with the polyalkylene oxide conjugated sFv that is labeled.
  • a sample may comprise at least one compound, mixture, surface, solution, emulsion, suspension, mixture, cell culture, fermentation culture, cell, tissue, secretion and/or derivative or extract thereof.
  • Such samples can also include, e.g., animal tissues, such as blood, lymph, cerebrospinal fluid (CNS), bone marrow, gastrointestinal contents, and portions, cells or internal and external secretions of skin, heart, lung and respiratory system, liver, spleen, kidney, pancreas, gall bladder, gastrointestinal tract, smooth, skeletal or cardiac muscle, circulatory system, reproductive organs, auditory system, the autonomic and central nervous system, and extracts or cell cultures thereof.
  • animal tissues such as blood, lymph, cerebrospinal fluid (CNS), bone marrow, gastrointestinal contents, and portions, cells or internal and external secretions of skin, heart, lung and respiratory system, liver, spleen, kidney, pancreas, gall bladder, gastrointestinal tract, smooth, skeletal or cardiac muscle, circulatory system, reproductive organs, auditory system, the autonomic and central nervous system, and extracts or cell cultures thereof.
  • CNS cerebrospinal fluid
  • Such samples can be measured using methods ofthe present invention in vitro, in
  • Such samples can also include environmental samples such as earth, air or water samples, as well as industrial or commercial samples such as compounds, mixtures, surfaces, aqueous chemical solutions, emulsions, suspensions or mixtures.
  • samples that can be used in methods ofthe present invention include cell culture and fermentation media used for growth of prokaryotic or eukaryotic cells and or tissues, such as bacteria, yeast, mammalian cells, plant cells and insect cells.
  • prokaryotic or eukaryotic cells and or tissues such as bacteria, yeast, mammalian cells, plant cells and insect cells.
  • These uses include detectably-labeled forms ofthe polyalkylene oxide conjugated sFv protein.
  • Types of labels are well-known to those of ordinary skill in the art. They include radiolabeling, chemiluminescent labeling, fluorochromic labeling, and chromophoric labeling.
  • Other uses include imaging the internal structure of an animal (including a human) by administering an effective amount of a labeled form of the polyalkylene oxide conjugated sFv protein and measuring detectable radiation associated with the animal. They also include improved immunoassays, including sandwich immunoassay, competitive immunoassay, and other immunoassays wherein the labeled antibody can be replaced by the PEGylated sFv protein of this invention. See, e.g., Kohler etal, Nature 256:495 (1975); Kohler et al, Eur. J. Immunol. 6:511 (1976); Kohler et al, Eur. J. Immunol. 6:292 (1976); Hammerling et al.
  • Conjugates ofthe present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, such as by admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation are described, for example, in Remington 's Pharmaceutical Sciences, 18th ed., Osol, A., ed., Mack, Easton PA (1990). In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain a therapeutically effective amount ofthe immunoconjugate, either alone, or with a suitable amount of canier vehicle.
  • Controlled release preparations may be achieved by the use of polymers to complex or absorb the immunoconjugate of the present invention.
  • the controlled delivery may be exercised by selecting appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl pynolidone, ethylene- vinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate).
  • the rate of drug release may also be controlled by altering the concentration of such macromolecules.
  • Another possible method for controlling the duration of action comprises incorporating the therapeutic agents into particles of a polymeric substance such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, by the use of hydroxymethylcellulose or gelatin-microcapsules or poly(methylmethacrylate) microcapsules, respectively, or in a colloid drug delivery system, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules, or in macroemulsions.
  • a colloid drug delivery system for example, liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules, or in macroemulsions.
  • the immunoconjugate may be provided to a patient by means well known in the art.
  • Such means of introduction include oral means, intranasal means, subcutaneous means, intramuscular means, intravenous means, intra-arterial means, or parenteral means.
  • Intravenous, intraarterial or intrapleural administration is normally used for lung, breast, and leukemic tumors.
  • Intraperitoneal administration is advised for ovarian tumors.
  • Intrathecal administration is advised for brain tumors and leukemia.
  • Subcutaneous administration is advised for Hodgkin's disease, lymphoma and breast carcinoma.
  • Catheter perfusion is useful for metastatic lung, breast or germ cell carcinomas of the liver.
  • Intralesional administration is useful for lung and breast lesions.
  • compositions according to the invention may be administered parenterally in combination with conventional injectable liquid earners such as sterile pyrogen-free water, sterile peroxide-free ethyl oleate, dehydrated alcohol, or propylene glycol.
  • conventional injectable liquid earners such as sterile pyrogen-free water, sterile peroxide-free ethyl oleate, dehydrated alcohol, or propylene glycol.
  • Conventional pharmaceutical adjuvants for injection solution such as stabilizing agent, solubilizing agents and buffers, such as ethanol, complex forming agents such as ethylene diamine tetraacetic acid, tartrate and citrate buffers, and high-molecular weight polymers such as polyethylene oxide for viscosity regulation may be added.
  • Such compositions may be injected intramuscularly, intraperitoneally, or intravenously.
  • carriers and diluents include albumin and/or other plasma protein components such as low density lipoproteins, high density lipoproteins and the lipids with which these serum proteins are associated.
  • These lipids include phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine and neutral lipids such as triglycerides.
  • Lipid carriers also include, without limitation, tocopherol.
  • At least one polyalkylene oxide conjugated sFv linked to a therapeutic agent according to the invention may be administered by any means that achieve their intended purpose, for example, to treat various pathologies, such as cell inflammatory, allergy, tissue damage or other related pathologies.
  • a typical regimen for preventing, suppressing, or treating various pathologies comprises administration of an effective amount of an sFv conjugate, administered over a period of one or several days, up to and including between one week and about 24 months.
  • the dosage ofthe present invention administered in vivo or in vitro will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature ofthe effect desired.
  • the ranges of effective doses provided below are not intended to limit the invention and represent prefened dose ranges.
  • the most preferred dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. See, e.g., Berkow et al, eds., Merck Manual, 16th edition, Merck and Co., Rahway, N.J.
  • Effective amounts of a diagnostic/pharmaceutical compound or composition ofthe present invention are from about 0.001 ⁇ g to about 100 mg/kg body weight, administered at intervals of 4-72 hours, for a period of 2 hours to 5 years, or any range or value therein, such as 0.01-1.0, 1.0- 10, 10-50 and 50-100 mg/kg, at intervals of 1-4, 6-12, 12-24 and 24-72 hours, for a period of 0.5, 1.0-2.0, 2.0-4.0 and 4.0-7.0 days, or 1, 1-2, 2-4, 4-52 or more weeks, or 1, 2, 3-10, 10-20, 20-60 or more years, or any range or value therein.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients which are known in the art.
  • Pharmaceutical compositions such as tablets and capsules can also be prepared according to routine methods. See, e.g., Berker, supra, Goodman, supra, Avery, supra and Ebadi, supra, which are entirely incorporated herein by reference, including all references cited therein.
  • compositions comprising at least one type of sFv conjugate of the invention, or, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 types of sFv conjugates, ofthe present invention may be contained in an amount effective to achieve its intended purpose.
  • a pharmaceutical composition may contain suitable pharmaceutically acceptable earners, such as excipients, earners and/or auxiliaries which facilitate processing ofthe active compounds into preparations which can be used pharmaceutically.
  • compositions may also include suitable solutions for administration intravenously, subcutaneously, dermally, orally, mucosally or rectally, and contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active component (i.e., the sFv) together with the excipient.
  • Pharmaceutical compositions for oral administration include tablets and capsules.
  • Compositions which can be administered rectally include suppositories. See, e.g., Berker, supra, Goodman, supra, Avery, supra and Ebadi, supra. Additional lipid and lipoprotein drug delivery systems that may be included herein are described more fully in Annals NY. Acad. Sci. 507:775-88, 98-103, and 252-271, which disclosure is hereby incorporated by reference.
  • compositions may also be formulated into orally administrable compositions containing one or more physiologically compatible earners or excipients, and may be solid or liquid in form.
  • These compositions may, if desired, contain conventional ingredients such as binding agents, for example, syrups, acacia, gelatin, sorbitol, tragacanth, or polyvinylpynolidone; fillers, such as lactose, mannitol, starch, calcium phosphate, sorbitol, cyclodextran, or methylcellulose; lubricants such as magnesium stearate, high molecular weight polymers such as polyethylene glycols, high molecular weight fatty acids such as stearic acid or silica; disintegrants such as starch; acceptable wetting agents as, for example, sodium lauryl sulfate.
  • binding agents for example, syrups, acacia, gelatin, sorbitol, tragacanth, or polyvinylpynolidone
  • the oral compositions may assume any convenient form, such as tablets, capsules, lozenges, aqueous or oily suspensions, emulsions, or dry products suitable for reconstitution with water or other liquid medium prior to use.
  • the liquid oral forms may, of course, contain flavors, sweeteners, preservatives such as methyl or propyl p-hydroxybenzoates; suspending agents such as sorbitol, glucose or other sugar syrup, methyl, hydroxymethyl, or carboxymethyl celluloses or gelatin; emulsifying agents such as lecithin or sorbitan monooleate or thickening agents.
  • Non-aqueous compositions may also be formulated which comprise edible oils as, for example, fish-liver or vegetable oils. These liquid compositions may conveniently be encapsulated in, for example, gelatin capsules in a unit dosage amount.
  • compositions according to the present invention may also be administered, if appropriate, either topically as an aerosol or, formulated with conventional bases as a cream or ointment.
  • compositions of the present invention can also be administered by incorporating the active ingredient into colloidal earners, such as liposomes.
  • colloidal earners such as liposomes.
  • Liposome technology is well known in the art, having been described by Allison et al, Nature 252:252-254 (1974), and Dancy et al, J. Immunol. 720:1109-1113 (1978).
  • the frozen cell paste from three 10-liter fermentations (600-900 g) was thawed overnight at 4°C and gently resuspended at 4°C in 50 mM Tris-HCl, 1.0 mM EDTA, 100 mM KC1, 0.1 mM PMSF, pH 8.0 (lysis buffer), using 10 liters of lysis buffer for every kilogram of wet cell paste.
  • the chilled mixture was passed three times through a Manton-Gaulin cell homogenizer to totally lyse the cells.
  • the cell homogenizer raised the temperature of the cell lysate to 25 ⁇ 5°C
  • the cell lysate was cooled to 5 + 2°C with a Lauda/Brinkman chilling coil after each pass. Complete lysis was verified by visual inspection under a microscope. The cell lysate was centrifuged at 24,300g for 30 minutes at 6°C using a
  • Sorvall RC-5B centrifuge The pellet containing the insoluble single-chain antigen-binding protein was retained, and the supernatant was discarded. The pellet was washed by gently scraping it from the centrifuge bottles and resuspending it in 5 liters of lysis buffer/kg of wet cell paste. The resulting 3.0- to 4.5-liter suspension was again centrifuged at 24,300g for 30 minutes at 6°C, and the supernatant was discarded. This washing ofthe pellet removes soluble E. coli proteins and can be repeated as many as five times. At any time during this washing procedure the material can be stored as a frozen pellet at -20°C. A substantial time saving in the washing steps can be accomplished by utilizing a Pellicon tangential flow apparatus equipped with 0.22- ⁇ m microporous filters, in place of centrifugation.
  • the washed pellet was solubilized at 4°C in freshly prepared 6 M guanidine hydrochloride, 50 mM Tris-HCl, 10 mM CaCl 2 , 50 mM KC1, pH 8.0 (dissociating buffer), using 9 ml/g of pellet. If necessary, a few quick pulses from a Heat Systems Ultrasonics tissue homogenizer can be used to complete the solubilization. The resulting suspension was centrifuged at 24,3 OOg for 45 minutes at 6°C and the pellet was discarded. The optical density of the supernatant was determined at 280 nm and if the OD 280 was above 30, additional dissociating buffer was added to obtain an OD 280 of approximately 25.
  • GuHCl extract is slowly added to the refolding buffer over a two hour period, with gentle mixing.
  • the solution was filtered through a 0.2 ⁇ m Millipore Millipak 200. This filtration step may be optionally preceded by a centrifugation step.
  • the filtrate was concentrated to 1 to 2 liters using an Amicon spiral cartridge with 10,000 MWCO cartridge, again at 4°C.
  • the concentrated crude antigen-binding protein sample was dialyzed against Buffer G (60 mM MOPS, 0.5 mM Ca acetate, pH 6.0 - 6.4) until the conductivity was lowered to that of Buffer G.
  • Buffer G 60 mM MOPS, 0.5 mM Ca acetate, pH 6.0 - 6.4
  • the sample was then loaded on a 21.5 x 250-mm polyaspartic acid PolyCAT A column, manufactured by Poly LC of Columbia, Maryland. If more than 60 mg of protein is loaded on this column, the resolution begins to deteriorate; thus, the concentrated crude sample often must be divided into several PolyCAT A runs.
  • Most antigen-binding proteins have an extinction coefficient of about 2.0 ml mg ⁇ cm "1 at 280 nm and this can be used to determine protein concentration.
  • the antigen-binding protein sample was eluted from the PolyCAT A column with a 50-min linear gradient from Buffer G to Buffer H (60 mM MOPS, 20 mM Ca Acetate, pH 7.5 - 8.0). Most of the single-chain proteins elute between 20 and 26 minutes when this gradient is used. This conesponds to an eluting solvent composition of approximately 70% Buffer G and 30% Buffer H. Most ofthe bivalent antigen-binding proteins elute later than 45 minutes, which conespond to over 90% Buffer H.
  • a sample of CC 49/212 single chain antigen binding molecule (MW 27000) dissolved in KPO 4 /NaCl buffer (pH 7.2) was obtained as described in Example 2.
  • the protein was found to be pure using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and size exclusion chromatography. The concentration of the protein was 0.79 mg/ml. It was further concentrated to at least 2 mg/ml using an Amicon concentrator with a 10,000 dalton nominal size cut-off, i.e., anything greater than 10K is retained.
  • the modification reaction i.e., the coupling of the SC-PEG to the CC 49/212, was canied out in 50 mM KPO 4 , 150 mM NaCl buffer, which was the storage buffer the protein was supplied in.
  • the pH was raised from 7.2 to 7.5.
  • SC-PEG (MW 5,000) was added in a 50x molar excess to protein.
  • the coupling reaction was terminated by the addition of a 5 Ox molar excess of glycine and the extent and progress of the coupling reaction was checked as a function of time using both size exclusion chromatography using a DuPont Zorbax 250 column and SDS-PAGE.
  • the samples were checked for degree of modification by size exclusion chromatography. After concentrating the samples, the residual amine concentration on the protein was determined by titration with trinitrobenzene sulfonate and the percentage of amine groups that had reacted with the SC-PEG (the "% modification") was calculated from the results.
  • the CC49 monoclonal antibody was developed by Dr. Jeffrey Schlom's group, Laboratory of Tumor Immunology and Biology, National Cancer Institute. It binds specifically to the pan-carcinoma tumor antigen TAG-72. See Muraro, R. et al, Cancer Research 48: 4588-4596 (1988).
  • Figure 1 is a graphical representation of three competition ELISA's in which unlabeled PEG modified CC49/212 single-chain Fv (closed squares), CC49/212 single-chain Fv (open squares), CC49 IgG (open circles), and MOPC- 21 IgG (+) competed against a CC49 IgG radiolabeled with 125 I for binding to the TAG-72 antigen on a human breast melanoma extract.
  • MOPC-21 is a control antibody that does not bind to TAG-72 antigen.
  • Methoxypolyethylene glycol (m-PEG) was obtained from Union Carbide. The solvents were obtained from Aldrich Chemical of Milwaukee, Wisconsin. The methoxypoly(ethylene glycol)-N-succinimidyl carbonate (SC-PEG) was prepared as described in U.S. Patent No. 5,122,614, using m-PEG having a molecular weight of about 5,000. Each of the products prepared in Examples 5-10 was confirmed structurally by carbon- 13 NMR.
  • the branched polymer, U-PEG-OH was prepared by adding 100 mg ( 1.1 mmol) of 1 ,3-diamino-2-propanol to a solution of 10.0 g (2 mmol) of SC-PEG in 50 mL of methylene chloride. The mixture was stined for 18 hours at room temperature then filtered. Excess solvent was removed by distillation in vacuo. The residue was recrystallized from 2-propanol to yield 7.1 g of product (70% yield).
  • Example 5 The compound of Example 5 was activated with p-nitrophenyl chloroformate.
  • 5.0g (0.5 mmol) of U-PEG-OH was azeotropically dried by refluxing in 75 mL of toluene for 2 hours, resulting in the removal of 25 mL of solvent water.
  • the reaction mixture was cooled to 30 ° C, followed by the addition of 120 mg (0.6 mmol) of p-nitrophenyl chloroformate and 50 mg (0.6 mmol) of pyridine.
  • the resulting mixture was stirred for two hours at 45 °C, followed by stining overnight at room temperature.
  • reaction mixture was then filtered through CELITETM , followed by removal ofthe solvent from the filtrate by distillation in vacuo. The residue was recrystallized from 2-propanol to yield 4.2 g (81% yield) ofthe product.
  • the U-PNP-PEG of Example 6 was reacted with N-hydroxysuccinimide to form the succinimidyl carbonate ester of U-PEG.
  • a solution containing 5.0 g (0.5 mmol) of the U-PNP-PEG, 0.6 g (5 mmol) of N-hydroxysuccinimide and 0.13 g (1 mmol) of diisopropylethylamine in 40 ml of methylene chloride was refluxed for 18 hours. The solvent was then removed by distillation in vacuo, and the residue was recrystallized from 2-propanol to yield 4.2 g ofthe succinimidyl carbonate ester (82% yield).
  • Example 8 Preparation of XU-PEG-OH
  • This branched polymer was prepared by reacting the U-PNP-PEG of Example 6 with 2-(2-aminoethoxy) ethanol (i.e., the amino alcohol was reacted with the p-nitrophenyl carbonate).
  • the recrystallized product yield was 86%.
  • Example 8 The compound of Example 8 was functionalized with p-nitrophenyl carbonate as in Example 6. The recrystallized product yield was 83%.
  • succinimidyl carbonate derivative of compound prepared in Example 8 was prepared according to the process described in Example 7, by reacting N-hydroxysuccinimide with the p-nitrophenyl carbonate derivative of Example 9.
  • the recovered product yield was 84%.
  • a sample containing CC49/218 was desalted on a PD-10 column in a buffer consisting of 0.1 M sodium phosphate, pH 8.0. An equimolar amount of SC-PEG or XUS-PEG was added and the reactions were incubated at 4°C, overnight. The reactions were quenched with an excess of glycine.
  • the modified CC49/218 conjugates were GPC purified and then concentrated in a centricon- 10.
  • the assay was performed as in Example 4 above using SC-PEG modified CC49/218 and XUS-PEG modified CC49/218 along with the appropriate controls. The results are shown in Figure 4 and in Table 4 below.
  • the affinity ofthe SC-PEG modified CC49-SCA was within about 8 to 10 fold of the native CC49-SCA and the affinity the XUS-PEG modified CC49-SCA was within about 4 to 5 fold ofthe native CC49-SCA.
  • Samples #049304 and #049303 were PEG modified, whiles samples #04901 and #049302 were unmodified CC49/218 isolated from the reaction mixtures.
  • CC49-SCA dimers and trimers of CC49-SCA have been made.
  • CC49-SCA was modified with bifunctional PEG as follows: 2 ml of CC49- SCA in phosphate buffered saline (25 mM sodium phosphate, pH 7.3, 0.15 M NaCl) at a concentration of 1.5 mg/ml was modified as follows.
  • Bifunctional PEG polyethylene glycol with reactive SC at both terminal ends
  • 1.887 mg (powder) was dissolved in 0.1 ml of MOPS (3-[N-mo holino]propane)-sulfonic acid) buffered at pH 7.3. This PEG solution was added to CC49-SCA solution within 10 seconds of dissolution.
  • the multimers were assayed for binding affinity using the following assay which was modified method described in B Friquet et al. J. of Immunology Methods, 77:305-319(1985). Briefly, various amounts of a given modified single chain antibody were mixed with various amounts of the antigen mucin in PBS (phosphate buffered saline). The binding reaction was allowed to reach equilibration for at least 24 hours at 4°C. At the end of the incubation, the unbound CC49-SCA fractions were assayed by ELISA, while the bound fractions were washed away.
  • the total amount of free CC49-SCA was determined by the ELISA using the CC49-SCA sample pre-incubated in the absence ofthe antigen mucin.
  • the bound antibody was determined by subtraction ofthe free (unbound) amount from the total amount as determined by ELISA. Since the total amount ofthe CC49-SCA was known, it was also used as its own standard curve. Note that each type of CC49-SCA had its own reference control. In essence, the protocol was measuring the unbound amount of a particular version of PEG- CC49-SC A as a result ofthe binding to the antigen. Although PEG may affect the detection reagent in ELISA, this was well contained in the standard references. Therefore, the amount measured was not due to the difference of various PEG on the various versions of PEG-CC49-SCA.
  • PEG increases. As more PEG is attached to the protein, the circulation time increases. However, attachment of a few strands of high molecular weight PEG gives a better increase in circulation half-life than multiple stands of lower molecular weight PEG.
  • the linker is an SC-bond, Flan-bond, hydrazine bond, or TPC, there was no significant change in the circulation half-life. Therefore, the chemical bonds of the linkers, if not releasable, do not affect the circulation half-life. In addition, the PEG remains attached to the protein during the observable time.
  • the PK modeling was following a one-compartment, i.v.-bolus model.
  • the circulatory half-life is the time for the drug concentration in the serum to reduce to one half after equilibration is reached (about 2 min.)
  • AUC area under the curve, is the integral ofthe drug blood level over time from zero to last measurement, and is a measure of quantity of drug absorbed and in the body.
  • Mean residence time is the average amount of time a drug remains in the compartment.
  • the Flan-bond PEG was prepared as taught in U.S. Patent 5,405,877.
  • a competition binding assay of biotinylated CC49-SCA with various PEGylated CC49-SCA proteins was performed using an ELISA ofthe biotinylated CC49-SCA as detected by horseradish-peroxidase conjugated with streptoavidin
  • biotinylated CC49-SCA and PEG-CC49-SCA sample were mixed at various ratios for competition of binding to the antigen mucin on a surface.
  • the amount of biotinylated-CC49-SCA bound to antigen was then measured by SAV-HRP, which would not detect the PEG-modified CC49- SCA.
  • SAV-HRP SAV-HRP
  • PEG-CC49-SCA is a reflection ofthe relative affinity ofthe two forms of CC49- SCA for the antigen.
  • SC2 is PEG SC2000-CC49-SCA
  • GC is glyco-CC49-SCA
  • Bio.CC49-SCA is the biotinylated CC49-SCA
  • CC12 is the PEG-SC12,000-CC49-SCA
  • F5 is PEG-Flan-5000-CC49-SC A
  • HZ is CC49-SC A highly PEGylated on the carboxyl groups with MW 5000 hydrazine-PEG
  • PG is PEG-glyco-CC49-SCA and is highly PEGylated on the carbohydrate with MW 5000 hydrazine PEG
  • C20 is PEG-SC20000-CC49-SC A.
  • ADDRESSEE STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
  • TELECOMMUNICATION INFORMATION (A) TELEPHONE: 202-371-2600 (B) TELEFAX: 202-371-2540
  • GGT AAT CAA AAG AAC TAC TTG GCC TGG TAC CAG CAG AAA CCA GGG CAG 144 Gly Asn Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45
  • TCT CCT AAA CTG CTG ATT TAC TGG GCA TCC GCT AGG GAA TCT GGG GTC 192 Ser Pro Lys Leu Leu lie Tyr Trp Ala Ser Ala Arg Glu Ser Gly Val 50 55 60
  • ATC AGC TGT GTG AAG ACT GAA GAC CTG GCA GTT TAT TAC TGT CAG CAG 288 lie Ser Cys Val Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Gin Gin 85 90 95
  • Glu Trp lie Gly Tyr Phe Ser Pro Gly Asn Asp Asp Phe Lys Tyr Asn 180 185 190
  • GGT AAT CAA AAG AAC TAC TTG GCC TGG TAC CAG CAG AAA CCA GGG CAG 144 Gly Asn Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin 280 285 290
  • TCT CCT AAA CTG CTG ATT TAC TGG GCA TCC

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