WO1999025379A1 - Modified antibodies with enhanced ability to elicit an anti-idiotype response - Google Patents

Modified antibodies with enhanced ability to elicit an anti-idiotype response Download PDF

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Publication number
WO1999025379A1
WO1999025379A1 PCT/US1998/024303 US9824303W WO9925379A1 WO 1999025379 A1 WO1999025379 A1 WO 1999025379A1 US 9824303 W US9824303 W US 9824303W WO 9925379 A1 WO9925379 A1 WO 9925379A1
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antigen
receptor
virus
immunoglobulin molecule
amino acid
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PCT/US1998/024303
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French (fr)
Inventor
Ronald M. Burch
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Euro-Celtique, S.A.
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Priority to AU14598/99A priority Critical patent/AU737457C/en
Priority to KR1020007005263A priority patent/KR20010015817A/en
Priority to EP98958584A priority patent/EP1030684A4/en
Priority to CA002309990A priority patent/CA2309990A1/en
Priority to JP2000520812A priority patent/JP2002507544A/en
Priority to IL13611398A priority patent/IL136113A0/en
Priority to BR9815580-6A priority patent/BR9815580A/en
Publication of WO1999025379A1 publication Critical patent/WO1999025379A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • C07K16/4258—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig
    • C07K16/4266—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig against anti-tumor receptor Ig
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C07K16/3046—Stomach, Intestines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/18—Kallidins; Bradykinins; Related peptides
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C07K2318/10—Immunoglobulin or domain(s) thereof as scaffolds for inserted non-Ig peptide sequences, e.g. for vaccination purposes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to modified immunoglobulins, and vaccine compositions thereof, in which one or more variable region cysteine residues that form intrachain disulfide bonds have been replaced with amino acid residues that do not contain a sulfhydryl group and, therefore, do not form disulfide bonds.
  • the present invention also relates to use of the vaccine compositions of the invention to treat or prevent certain diseases and disorders, particularly cancers and infectious diseases.
  • the basic unit of immunoglobulin structure is a complex of four polypeptides — two identical low molecular weight or "light” chains and two identical high molecular weight or “heavy” chains, linked together by both noncovalent associations and by disulfide bonds.
  • Each light and heavy chain of an antibody has a variable region at its amino terminus and a constant domain at its carboxyl terminus ( Figure 1).
  • the variable regions are distinct 5 for each antibody and contain the antibody antigen binding site.
  • Each variable domain is comprised of four relatively conserved framework regions and three regions of sequence hypervariability termed complementarity determining regions or CDRs ( Figure 2). For the most part, it is the CDRs that form the antigen binding site and confer antigen specificity.
  • the constant regions are more highly conserved than the variable domains, with slight Q variations due to haplotypic differences.
  • variable region heavy chains are composed of multiple domains (CHI, CH2, CH3...CHx), the number depending upon the particular antibody class.
  • the CHI region is separated from the CH2 region by a hinge region which allows flexibility in the antibody. .
  • the variable region of each light chain aligns with the variable region of each heavy chain, and the constant region of each light chain aligns with the first constant region of each heavy chain.
  • the CH2-CHx domains of the constant region of a heavy chain form an "Fc region" which is responsible for the effector functions of the immunoglobulin molecule, such as complement binding and binding to the Fc receptors expressed by lymphocytes, granulocytes, monocyte lineage cells, killer cells, mast cells and other immune effector cells.
  • each domain is composed of a sandwich of two parallel extended protein layers of about 100 amino acids in length which are connected by a single disulfide bond (See Roitt et al., Immunology. 3rd Edition, London; Mosby, 1993, p 4.4).
  • Each of the two extended protein layers of the domain in turn, contains two "anti- parallel" adjacent strands which adopt a beta-sheet conformation. (See, e.g., Stryer, 1975, Biochemistry. WH Freeman and Co., p. 950).
  • Each of the domains has a similar three- dimensional structure based on the immunoglobulin fold.
  • Passive immunotherapy involves the administration of antibodies to a patient without eliciting a concommitant immune response.
  • a specific antibody from one animal is injected as an immunogen into a suitable second animal, the injected antibody will elicit an immune response.
  • Antibody therapy is conventionally characterized as passive since the patient is not the source of the antibodies. However, the term passive is misleading because the patient can produce anti-idiotypic secondary antibodies which in turn provoke an immune response which is cross-reactive with the original antigen.
  • Immunotherapy where the patient generates secondary antibodies is often more therapeutically effective than passive immunotherapy because the patient's own immune system continues to fight the cells bearing the particular antigen well after the initial infusion of antibody.
  • antibodies produced initially during an immune response or introduced into an organism will carry unique new epitopes to which the organism is not tolerant, and therefore will elicit production of secondary antibodies (termed “Ab2”), some of which are directed against the idiotype (i.e., the antigen binding site) of the primary antibody (termed “Abl”), i.e., the antibody that was initially produced or introduced exogenously.
  • idiotype i.e., the antigen binding site
  • Abl the primary antibody
  • termed “Ab3" tertiary antibodies
  • the secondary antibodies will have a binding site which is an analog of the original antigen, and thus will reproduce the "internal image" of the original antigen. And, the tertiary or Ab3 antibodies that recognize this antigen binding site of the Ab2 antibody will also recognize the original antigen ( Figure 4).
  • anti-idiotypic antibodies have binding sites that are similar in conformation and charge to the antigen, and can elicit the same or greater response than that of the cancer antigen itself.
  • Administration of an exogenous antibody that can elicit a strong anti-idiotypic response can thus serve as an effective vaccine, by maintaining a constant immune response.
  • anti-idiotypic vaccines have comprised murine antibodies because the anti- idiotypic response occurs as part of the typical human anti-mouse antibody (HAMA) response.
  • HAMA human anti-mouse antibody
  • a strong anti-idiotypic cascade has been observed when Abl has been structurally damaged (Madiyalakan et al., 1995, Hybridoma 14:199-203), rendering the antibody more foreign.
  • There has been direct administration to the subject of exogenously produced anti- idiotype antibodies that are raised against the idiotype of an anti-tumor antibody U.S. Patent No. 4,918,14.
  • the subject's body After administration, the subject's body will produce anti-antibodies which not only recognize these anti-idiotype antibodies, but also recognize the original tumor epitope, thereby directing complement activation and other immune system responses to a foreign entity to attack the tumor cell that expresses the tumor epitope.
  • the present invention is based upon the realization of the present inventors that an antibody in which one or more variable region cysteine residues that form one or more intrachain disulfide bonds have been replaced with amino acid residues that do not contain sulfhydryl groups, such that the particular disulfide bonds do not form, elicit a much stronger 0 anti-idiotype response than an antibody in which the variable region disulfide bonds are intact.
  • the present invention provides modified immunoglobulin molecules or antibodies (and functionally active fragments, derivatives and analogs thereof), and vaccine compositions containing these immunoglobulin molecules, wherein the variable region of the immunoglobulin is subject to decreased conformational constraints, such as, but not limited to, by breaking one or more intrachain or interchain disulfide bonds.
  • the invention provides modified immunoglobulins that comprise a variable region and are identical, except for one or more amino acid substitutions in said variable region, to a second immunoglobulin molecule, said second immunoglobulin molecule being capable of
  • immunospecifically binding i.e., specific binding of the immunoglobulin to its antigen as determined by any method known in the art for determining antibody-antigen binding, which excludes non-specific binding but not necessarily cross-reactivity with other antigens
  • said one or more amino acid substitutions being the substitution of one or more amino acid residues that do not have a sulfhydryl group at one or more positions corresponding to one or more cysteine residues that form a disulfide bond in said second immunoglobulin molecule.
  • the second immunoglobulin molecule can immunospecifically bind a cancer antigen; in other preferred embodiments, the second immunoglobulin molecule can immunospecifically bind an antigen of an infectious disease agent or a cellular receptor for an infectious disease agent.
  • the invention further provides methods of eliciting an anti-idiotype response in a subject by administering the modified immunoglobulins of the invention.
  • the modified immunoglobulins of the invention can be used to treat or prevent cancer, specifically by administering an immunoglobulin molecule of the invention, which immunoglobulin molecule was derived (i.e., by modification according to the invention to 0 replace one or more variable region cysteine residues that form an intrachain disulfide bond with an amino acid residue that does not contain a sulfhydryl group) from an immunoglobulin molecule that can immunospecifically bind a cancer antigen, the expression of which cancer antigen is associated with the particular type of cancer.
  • an immunoglobulin molecule of the invention which immunoglobulin molecule was derived (i.e., by modification according to the invention to 0 replace one or more variable region cysteine residues that form an intrachain disulfide bond with an amino acid residue that does not contain a sulfhydryl group) from an immunoglobulin molecule that can immunospecifically bind a cancer antigen, the expression of which cancer antigen is associated with the particular type of cancer.
  • the modified immunoglobulin molecules of the invention can be used to 5 treat or prevent an infectious disease by administering an immunoglobulin molecule derived from an immunoglobulin molecule that can immunospecifically bind an antigen of or a cellular receptor for the infectious disease agent causing the infectious disease.
  • the invention also provides methods of producing the modified immunoglobulin molecules of the invention and vaccine compositions containing the modified 0 immunoglobulin molecules of the invention.
  • Figure 1 A schematic diagram showing the structure of the light and heavy chain of an immunoglobulin molecule, each chain consisting of a variable region positioned at the 5 amino terminal region (H 2 N-) and a constant region positioned at a carboxyl terminal region (-COOH).
  • FIG. 1 A schematic diagram of an IgG showing the four framework regions (FR1, FR2, FR3 and FR4) and three complementarity determining regions (CDR1, CDR2 and CDR3) in the variable regions of the light and heavy chains (labeled as V L and V H ,
  • the constant region domains are indicated as C L for the light chain constant domain and CHstay CH 2 and CH 3 for the three domains of the heavy chain constant region.
  • Fab indicates the portion of the antibody fragment which includes the variable region domains of both light and heavy chains and the C L and CH, domains.
  • Fc indicates the constant region fragment containing the CH 2 and CH 3 domains.
  • FIG. 35 A schematic diagram of an antibody structure as shown in Figure 2, but drawn to emphasize that each domain (the loop structures labeled as V L , V H , C L , CH,, CH 2 , and CH 3 , respectively) is structurally defined by a disulfide bond (indicated with darkest lines) that maintains the three-dimensional structure (Roitt et al., Immunology, Second Edition, London: Gower Medical Publishing, 1989, p 5.3).
  • Figure 4 A schematic diagram showing the development of internal image bearing anti-idiotype antibodies (Ab2) and anti-anti-idiotype antibodies (Ab3) from idiotype antibodies (Abl) directed against a ligand in an anti-idiotypic cascade.
  • Figure 5 Modification of the variable region of an immunoglobulin by replacing cysteine residues in the variable regions with alanine residues to break a variable region intrachain disulfide bond.
  • CHI, CH2 and CH3 are constant regions.
  • V H is the heavy chain variable region and V L is the light chain variable region.
  • FIGs 6A-C The structure of the expression vector pMRROlO.l, which contains a human kappa light chain constant region sequence.
  • B The structure of the expression vector pGammal that contains a sequence encoding a human IgGl constant region (CHI, CH2, CH3) heavy chain and hinge region sequences.
  • C The structure of the expression vector pNEPuDGV which contains a sequence encoding the kappa constant domain of the light chain and the constant domain and hinge region of the heavy chain. For all three vectors see Bebbington et al., 1991, Methods in Enzymology 2:136-145. Figures 7 A and B.
  • A The amino acid sequence and corresponding nucleotide sequence including the leader sequence for the consensus light chain variable region ConVLl.
  • B The amino acid and corresponding nucleotide sequences for the consensus heavy chain variable region ConVHl including the leader sequence.
  • FIGS 8A-B (A) Amino acid and corresponding nucleotide sequence of 2CAVLCOL1, which is the light chain variable region sequences of an antibody derived from mAb31.1, in which alanine residues have been substituted for cysteine residues at positions 23 and 88, which residues are boxed. (B) Amino acid and corresponding nucleotide sequence of 2CAVHCOL1, which is the heavy chain variable region sequence of an antibody derived from mAb31.1 , in which alanine residues have been substituted for cysteine residues at positions 23 and 88, which residues are boxed.
  • Figures 9A-D (A) Oligonucleotide sequences for the oligonucleotides used to assemble 2CAVHCOL1, the heavy chain variable region gene specific to human colon cancer antigen. (B) Oligonucleotide sequences for the oligonucleotides used to assemble the 2CAVLCOL1 light chain variable region gene specific to human colon cancer antigen. (C) Oligonucleotide sequences for the oligonucleotides used to assemble the light chain consensus region referred to as ConVLl. (D) Oligonucleotide sequences for the oligonucleotides used to assemble the heavy chain consensus region referred to as ConVLl .
  • Figure 10 A schematic diagram of the general steps that were followed for the assembly of an engineered gene encoding the synthetic modified antibody specific to human colon cancer antigen.
  • FIGS 12A-D Results of competition binding assay of the biotin-labeled anti- colon carcinoma cell antibody to LS-174T cells in the presence of antisera from mice vaccinated with vehicle alone, control antibody that binds the colon carcinoma cell antibody but has not been modified, and peptides CDR1, CDR2, CDR3, CDR4, CDR5, and CDR6, 5 having the CDR sequences containing the bradykinin receptor binding site expressed as percent of control binding to LS-174T cells.
  • B Results of competition binding assay of the biotin-labeled anti- colon carcinoma cell antibody to LS-174T cells in the presence of antisera from mice vaccinated with vehicle alone, control antibody that binds the colon carcinoma cell antibody but has not been modified, and peptides CDR1, CDR2, CDR3, CDR4, CDR5, and CDR6, 5 having the CDR sequences containing the bradykinin receptor binding site expressed as percent of control binding to LS-174T cells.
  • B results of competition binding assay
  • C 0 Diagram showing the binding of a biotin-labeled (indicated by the "b") antibody (inverted Y) to antigen (solid triangles).
  • D Diagram showing the inhibition of binding of the biotin- labeled (indicated by the "b") antibody (inverted Y) by anti-idiotype antibodies (solid arrows) to antigen (solid triangles).
  • the present invention provides modified immunoglobulins (particularly antibodies and functionally active fragments, derivatives, and analogs thereof) that elicit a stronger - 0 immune response, particularly a stronger anti-idiotypic response, than the corresponding unmodified immunoglobulins.
  • the modified immunoglobulins of the invention are immunoglobulins that, when unmodified, immunospecifically bind an antigen, and are modified to decrease the conformational constraints on one variable region of the immunoglobulin molecule, preferably, such that at least one of the cysteines that participates in forming an intrachain disulfide bond in the variable region of the immunoglobulin has been replaced with an amino acid residue that does not have a sulfhydryl group and, therefore, does not form a disulfide bond, thereby decreasing the conformational constraints of at least one of the variable regions of the immunoglobulin (Figure 5).
  • the modified immunoglobulin molecule is derived from an immunoglobulin molecule that is capable of immunospecifically binding a cancer antigen; in other preferred embodiments, the modified immunoglobulin molecule is derived from an immunoglobulin that is capable of immunospecifically binding an antigen of an infectious disease agent or a cellular receptor for an infectious disease agent.
  • the invention also provides vaccine compositions containing the modified immunoglobulin molecules of the invention. Additionally, the invention provides methods of generating an anti-idiotype response in a subject by administration of the modified immunoglobulin molecules of the invention.
  • the invention provides methods of treating or preventing 5 cancer by administration of a modified immunoglobulin molecule of the invention which, in its unmodified state, is capable of immunospecifically binding a cancer antigen, the expression of which is associated with the particular cancer.
  • Administration of the modified immunoglobulin elicits an anti-idiotype reaction in the subject, leading to the production, by the subject, of antibodies specific for the cancer antigen.
  • the modified immunoglobulin in its unmodified state, is capable of binding an antigen of an infectious disease agent or a cellular receptor for an infectious disease agent.
  • Such immunoglobulins can be used to treat or prevent the infectious disease caused by the infectious disease agent.
  • the modified immunoglobulins, particularly antibodies, of the invention are immunoglobulins that, at least in the unmodified state, can immunospecifically bind an , relief antigen and have been modified to enhance their ability to elicit an anti-idiotype response.
  • Such immunoglobulins are modified to reduce the conformational constraints on a variable region of the immunoglobulin, e.g., by removing or reducing intrachain or interchain disulfide bonds, chemical modification, or any other method known in the art.
  • the invention provides a first immunoglobulin molecule that comprises a variable region and that is identical, except for one or more amino acid substitutions in the variable region, to a second immunoglobulin molecule, the second immunoglobulin molecule being capable of immunospecifically binding an antigen, the amino acid substitutions being the substitution of one or more amino acid residues that do not have a sulfhydryl group at one or more positions corresponding to one or more cysteine residues that form a disulfide bond in said second immunoglobulin molecule.
  • the invention also provides nucleic acids containing a nucleotide sequence encoding a modified immunoglobulin of the invention.
  • Identifying the cysteine residues that form a disulfide bond in a variable region of a particular antibody can be accomplished by any method known in the art. For example, but not by way of limitation, it is well known in the art that the cysteine residues that form 0 intrachain disulfide bonds are highly conserved among antibody classes and across species. Thus, the cysteine residues that participate in disulfide bond formation can be identified by sequence comparison with other antibody molecules in which it is known which residues form a disulfide bond.
  • Table 1 provides a list of the positions of disulfide bond forming cysteine residues
  • Table 1 (derived from Kabat et al, 1991, sequences of Proteins of Immunological Interest, 5th Ed., U.S. Department of Health and Human Services, Bethesda, Maryland).
  • Position numbers enclosed by () indicate that the protein was not sequenced to that position, but the residue is inferred by comparison to known sequences.
  • cysteine residues that form the intrachain disulfide bonds are the residues at positions 23 and 88 of the light chain variable domain and the residues at positions 22 and 92 of the heavy chain variable domain.
  • the position numbers refer to the residue corresponding to that residue in the consensus sequences as defined in Kabat, (1991, Sequences of Proteins of Immunological Interest, 5th Ed., U.S. Department of Health and Human Services, Bethesda, Maryland) or as indicated in the heavy and light chain variable region sequences depicted in Figures 7 A and B, respectively ("corresponding" means as determined by aligning the particular antibody sequence with the consensus sequence or the heavy or light chain variable region sequence depicted in Figure 7A or B).
  • the modified immunoglobulin molecule is an antibody in which the residues at positions 23 and/or 88 of the light chain are substituted with an amino acid residue that does not contain a sulfhydryl group and/or the residues at positions 22 and/or 92 of the heavy chain are substituted with an amino acid residue that does not contain a sulfhydryl group.
  • the amino acid residue that substitutes for the disulfide bond forming cysteine residue is any amino acid residue that does not contain a sulfhydryl group, e.g., alanine, arginine, asparagine, aspartate (or aspartic acid), glutamine, glutamate (or glutamic acid), glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine.
  • the cysteine residue is replaced with a glycine, serine, threonine, tyrosine, asparagine, or glutamine residue, most preferably, with an alanine residue.
  • disulfide bond forming cysteine residue may be replaced by a nonclassical amino acid or chemical amino acid analog that does not contain a sulfhydryl group (for example, but not by way of limitation, using routine protein synthesis methods).
  • Non-classical amino acids include, but are not limited, to the D-isomers of the common amino acids, cc-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, ⁇ -Abu, e-Ahx, -amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, fluoro-amino acids
  • the amino acid can be D (dextrorotary) or L (levorotary).
  • the disulfide bond forming residue is deleted.
  • the substitution of the disulfide bond forming residue is in the heavy chain variable region or is in the light chain variable region or is in both the heavy chain and light chain variable regions.
  • one of the residues that forms a particular disulfide bond is replaced (or deleted) or, alternatively, both residues that form a particular disulfide bond may be replaced (or deleted).
  • the invention provides immunoglobulin molecules that have one or more amino acid substitutions relative to the second immunoglobulin molecule of a disulfide bond forming residue in the variable region with an amino acid residue that does not contain a sulfhydryl group and that additionally have one or more other amino acid substitutions (i.e., that are not a replacement of a disulfide bond forming residue with a residue that does not contain a sulfhydryl group).
  • the invention provides a first immunoglobulin molecule comprising a variable region and which is identical, except for one or more amino acid substitutions in said variable region, to a second immunoglobulin molecule, said second immunoglobulin molecule being capable of immunospecifically binding an antigen, in which at least one of said one or more amino acid substitutions are the substitution of an amino acid residue that does not have a sulfhydryl group at one or more positions corresponding to one or more cysteine residues that form a disulfide bond in said second immunoglobulin molecule.
  • the amino acid substitutions that are not the substitution of a disulfide bond forming cysteine residue with a residue that does not have a sulfhydryl group are not stabilizing changes.
  • Stabilizing changes are defined as those amino acid changes that increase the stability of the antibody molecule.
  • Such stabilizing amino acid changes are those changes that substitute an amino acid that is not common at that particular position in the particular antibody molecule (e.g., as defined by the consensus sequences for a number of antibody molecules provided in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., U.S.
  • Such other amino acid substitutions can be any amino acid substitution that does not alter the ability of the modified immunoglobulin to elicit the formation of anti-anti-idiotype antibodies, e.g., as determined, for example, as described in Section 5.5, infra.
  • such other amino acid substitutions include substitutions of functionally equivalent amino acid residues.
  • one or more amino acid residues can be substituted by another amino acid of a similar polarity which acts as a functional equivalent. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • the modified antibodies of the invention can be derived from antibodies that are capable of immunospecifically binding any antigen.
  • the modified antibodies are derived from antibodies that are capable of immunospecifically binding a cancer antigen, more preferably a tumor antigen.
  • the modified antibodies are derived from antibodies that are capable of binding polymorphic epithelial mucin antigen, human colon carcinoma-associated protein antigen, human colon carcinoma-associated carbohydrate antigen, human milk fat globule, or is an antigen of a cancer of the breast, ovary, uterus, prostate, bladder, lung, skin, colon, pancreas, gastrointestinal track, B lymphocytes or T lymphocytes or any other cancer characterized by the expression of specific antigens, e.g., those discussed in Section 5.2.1, infra.
  • the modified antibody is derived from Mab 31.1 (available from the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2201 under No. 12314), Mab 33.28 (under No. 12315) or Mab HMFG-1 (see PCT Publication Q WO90/05142 and PCT Publication WO92/04380).
  • the modified antibodies of the invention are derived from antibodies that are capable of immunospecifically binding an antigen of an infectious disease agent or a cellular receptor for an infectious disease agent.
  • the antigen of the infectious disease agent is a bacterial antigen, a viral 5 antigen, or an antigen of a parasite, or any other antigen of an infectious disease agent, such as those infectious disease agents described in Section 5.2.2, infra.
  • the immunoglobulin molecules of the invention can be of any type, class, or subclass of immunoglobulin molecules.
  • the immunoglobulin molecule is an antibody molecule, more preferably of a type selected from the group consisting of IgG, IgE, IgM, IgD and IgA, most preferably is an IgG molecule.
  • the immunoglobulin molecule is a T cell receptor, a B cell receptor, a cell-surface adhesion molecule such as the co-receptors CD4, CD8, or CD 19, or an invariant domain of an MHC molecule.
  • the modified immunoglobulin can be derived from any naturally occurring antibody, preferably a monoclonal antibody, or can be derived from a synthetic or engineered antibody.
  • the modified immunoglobulin molecules are derived from an antibody in which a binding site for a member of a binding pair or a portion of an antigen is inserted into or replaces all or a portion of one of the CDRs in the variable region, for example as described in co-pending United States Patent application Serial
  • the synthetic antibodies are antibodies that immunospecifically bind to a first member of a binding pair where at least one of the CDRs of the antibody contains a binding site for the first member of the binding pair, which binding site is derived from an amino acid sequence of the other member of the binding pair.
  • the amino acid sequence of the binding site is not found naturally within the CDR.
  • at least one of the CDRs may contain a portion of an antigen, particularly an epitope.
  • the amino acid sequence of the binding site may be identified by any method known in the art. For example, in some instances, the sequence of a member of a binding pair has already been determined to be directly involved in binding the other member of the binding pair. In this case, such a sequence can be used to construct the CDR of a synthetic antibody that specifically recognizes the other member of the binding pair.
  • the amino acid sequence for the binding site in the one member of the binding pair for the other member of the binding pair is not known, it can be determined by any method known in the art, for example, but not limited to, molecular modeling methods or empirical methods, e.g., by assaying portions (e.g., peptides) of the member for binding to the other member, or by making mutations in the member and determining which mutations prevent binding.
  • the binding pair can be any two molecules, including proteins, nucleic acids, carbohydrates, or lipids, that interact with each other, although preferably the binding partner from which the binding site is derived is a protein molecule.
  • the modified immunoglobulin contains a binding sequence for a cancer antigen, an infectious disease antigen, a cellular receptor for a pathogen, or a receptor or ligand that participates in a receptor-ligand binding pair.
  • the binding pair is a protein-protein interaction pair which is either homotypic interaction (i.e., is the interaction between two of the same proteins) or a heterotypic interaction (i.e., is the interaction between two different proteins).
  • the first member is a member of a ligand-receptor binding pair, preferably, of a receptor-ligand binding pair in which the ligand binds to the receptor and thereby elicits a physiological response, such as intracellular signaling.
  • the ligand or receptor can be a hormone, autocoid, growth factor, cytokine or neurotransmitter, or receptor for a hormone, autocoid, growth factor, cytokine, or neurotransmitter, or any receptor or ligand involved in signal transduction.
  • one member of the binding pair is ligand such as, but not limited to, cholecystokinin, galanin, IL-1, IL-2, IL-4, IL-5, IL-6, IL- 11, a chemokine, leptin, a protease, neuropeptide Y, neurokinin-1, neurokinin-2, neurokinin- 3, bombesin, gastrin, corticotropin releasing hormone, endothelin, melatonin, somatostatin, vasoactive intestinal peptide, epidermal growth factor, tumor necrosis factor, dopamine,
  • ligand such as, but not limited to, cholecystokinin, galanin, IL-1, IL-2, IL-4, IL-5, IL-6, IL- 11, a chemokine, leptin, a protease, neuropeptide Y, neurokinin-1, neurokinin-2, neurokinin- 3, bombesin, gastri
  • one member of the binding pair is a receptor, such as, but not limited to, an opioid receptor, a glucose transporter, a glutamate receptor, an orphanin receptor, erythropoietin receptor, insulin receptor, tyrosine kinase (TK)-receptor, KIT stem cell factor receptor, nerve growth factor receptor, insulin-like growth factor receptor, granulocyte-colony stimulating factor receptor,
  • a receptor such as, but not limited to, an opioid receptor, a glucose transporter, a glutamate receptor, an orphanin receptor, erythropoietin receptor, insulin receptor, tyrosine kinase (TK)-receptor, KIT stem cell factor receptor, nerve growth factor receptor, insulin-like growth factor receptor, granulocyte-colony stimulating factor receptor,
  • TK tyrosine kinase
  • one of the members of the binding pair is a ligand gated ion channel, such as but not limited to a calcium channel, a sodium channel, or a potassium channel.
  • the invention provides modified immunoglobulins that
  • the invention provides synthetic modified antibodies that immunospecifically bind a receptor and are agonists of the receptor, for example, but not by way of limitation, the endorphin, enkephalin, or nociceptin receptors.
  • the modified immunoglobulin does not bind the fibronectin receptor.
  • the binding sequence is not Arg-Gly-Asp, is not a multimer of a binding sequence, and preferably is not a multimer of the sequence Arg-Gly-Asp.
  • the modified immunoglobulin has a CDR that contains a binding site for a transcription factor.
  • the modified immunoglobulin does not bind to a specific DNA sequence, particularly does not bind to a transcription factor binding site.
  • the modified immunoglobulin has at least one CDR that contains an amino acid sequence of a binding site for a cancer antigen or a tumor antigen (e.g., as described in detail in section 5.2.1, infra.), more preferably the antigen is human colon carcinoma-associated antigen or epithelial mucin antigen.
  • at least one CDR of the modified immunoglobulin contains an amino acid sequence for a binding site for a human milk fat globule receptor.
  • the modified immunoglobulin has at least one CDR that contains an amino acid sequence of a binding site for an antigen of a tumor of the breast, ovary, uterus, prostate, bladder, lung, skin, pancreas, colon, gastrointestinal tract, B lymphocytes, or T lymphocytes.
  • At least one CDR of the modified antibody contains an amino acid sequence for a binding site for an antigen of an infectious disease agent (e.g., as described in detail in section 5.2.2, infra.), or a binding site for a cellular receptor of an infectious disease agent, preferably where the binding site is not an amino acid sequence of a Plasmodium antigen, or is not the binding site Asn-Ala-Asn-Pro or Asn-Val- Asp-Pro.
  • the modified antibody has a CDR that contains the binding site for a bacterial or viral enzyme.
  • the synthetic antibody may be built upon (i.e., the binding site sequences inserted into the CDR of) the sequence of a naturally occurring or previously existing antibody or may be synthesized from known antibody consensus sequences, such as the consensus sequences for the light and heavy chain variable regions in Figures 7 A and B, or any other antibody consensus or germline (i.e., unrecombined genomic sequences) sequences (e.g., those antibody consensus and germline sequences described in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5 th edition, NIH Publication No. 91-3242, pp 2147- 2172).
  • known antibody consensus sequences such as the consensus sequences for the light and heavy chain variable regions in Figures 7 A and B
  • any other antibody consensus or germline (i.e., unrecombined genomic sequences) sequences e.g., those antibody consensus and germline sequences described in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5 th edition,
  • Each antibody molecule has six CDR sequences, three on the light chain and three on the heavy chain, and five of these CDRs are germline CDRs (i.e., are directly derived from the germline genomic sequence of the animal, without any recombination) and one of the CDRs is a non-germline CDR (i.e., differs in sequence from the germline genomic sequence of the animal and is generated by recombination of the germline sequences).
  • Whether a CDR is a germline or non-germline sequence can be determined by sequencing the CDR and then comparing the sequence with known germline sequences, e.g., as listed in Kabat et al.
  • the CDR that contains the amino acid sequence of the binding site or antigen is a germline CDR or, alternatively, is a non-germline CDR.
  • the binding site or antigen sequence can be inserted into any of the CDRs of the antibody, and it is within the skill in the art to insert the binding site into different CDRs of the antibody and then screen the resulting modified antibodies for the ability to bind to the particular member of the binding pair, e.g.
  • a CDR of either the heavy or light chain variable region is modified to contain the amino acid sequence of the binding site or antigen.
  • the modified antibody contains a variable domain in which the first, second or third CDR of the heavy variable region or the first, second or third CDR of the light chain variable region contains the amino acid sequence of the binding site or antigen.
  • more than one CDR contains the amino acid sequence of the binding site or antigen or more than one CDR each contains a different binding site for the same molecule or contains a different binding site for a different molecule.
  • two, three, four, five or six CDRs have been engineered to contain a binding site for the first member of the binding pair.
  • one or more CDRs contain a binding site for the first member of a binding pair and one or more other CDRs contain a binding site for a molecule on the surface of an immune cell, such as, but not limited to, a T cell, B cell, NK cell, K cell, TIL cell or neutrophil.
  • a modified antibody having a binding site for a cancer antigen or an infectious disease antigen and a binding site for a molecule on the surface of an immune cell can be used to target the immune cell to a cancer cell bearing the cancer antigen or to the infectious disease agent.
  • the binding site or antigen amino acid sequence is either inserted into the CDR without replacing any of the amino acid sequence of the CDR itself or, alternatively, the binding site or antigen amino acid sequence replaces all or a portion of the amino acid sequence of the CDR.
  • the binding site amino acid sequence replaces 1, 2, 5, 8, 10, 15, or 20 amino acids of the CDR sequence.
  • the amino acid sequence of the binding site or antigen present in the CDR can be the minimal binding site necessary for the binding of the member of the binding pair or for eliciting an immune response against the antigen(which can be determined empirically by any method known in the art); alternatively, the sequence can be greater than the minimal binding site or antigen sequence necessary for the binding of the member of the binding pair or eliciting of an immune response against the antigen.
  • the binding site or antigen amino acid sequence is at least 4 amino acids in length, or is at least 6, 8, 10, 15, or 20 amino acids in length. In other embodiments the binding site amino acid sequence is no more than 10, 15, 20, or 25 amino acids in length, or is 5-10, 5-15, 5-20, 10- 15, 10-20 or 10-25 amino acids in length.
  • the total length of the CDR i.e., the combined length of the binding site sequence and the rest of the CDR sequence
  • CDRs have been observed to have a range of numbers of amino acid residues, and the observed size ranges for the CDRs (as denoted by the abbreviations indicated in figure 2) are provided in Table 2.
  • the CDR containing the binding site or antigen portion is within the size range provided for that particular CDR in Table 2, i.e., if it is the first CDR of the light chain, LI, the CDR is 10 to 17 amino acid residues; if it is the second CDR of the light chain, L2, the CDR is 7 amino acid residues; if it is the third CDR of the light chain, L3, the CDR is 7 to 11 amino acid residues; if it is the first CDR of the heavy chain, HI, the CDR is 5 to 7 amino acid residues; if it is the second CDR of the heavy chain, H2, the CDR is 9 to 12 amino acid residues; and if it is the third CDR of the heavy chain, H3, the CDR is 2 to 25 amino acid residues.
  • the CDR containing the binding site is 5-10, 5-15, 5-20, 11-15, 11-20, 11-25, or 16-25 amino acids in length. In other embodiments, the CDR containing the binding site is at least 5, 10, 15, or 20 amino acids or is no more than 10, 15, 20, 25, or 30 amino acids in length.
  • the modified antibodies can be further altered and screened to select an antibody having higher affinity or specificity.
  • Antibodies having higher affinity or specificity for the target antigen may be generated and selected by any method known in the art.
  • the nucleic acid encoding the synthetic modified antibody can be mutagenized, either randomly, i.e., by chemical or site-directed mutagenesis, or by making particular mutations at specific positions in the nucleic acid encoding the modified antibody, and then screening the antibodies exposed from the mutated nucleic acid molecules for binding affinity for the target antigen.
  • Screening can be accomplished by testing the expressed antibody molecules individually or by screening a library of the mutated sequences, e.g., by phage display techniques (see, e.g., U. S. Patent Nos. 5,223,409; 5,403,484; and 5,571,698, all by Ladner et al; PCT Publication WO 92/01047 by McCafferty et al. or any other phage display technique known in the art).
  • phage display techniques see, e.g., U. S. Patent Nos. 5,223,409; 5,403,484; and 5,571,698, all by Ladner et al; PCT Publication WO 92/01047 by McCafferty et al. or any other phage display technique known in the art).
  • the invention provides a functionally active fragment, derivative or analog of the modified immunoglobulin molecules of the invention.
  • Functionally active means that the fragment, derivative or analog is able to elicit anti-anti- idiotype antibodies (i.e., tertiary antibodies or Ab3 antibodies) that recognize the same antigen that the antibody from which the fragment, derivative or analog is derived recognized (e.g., as determined by the methods described in Section 5.5, infra).
  • anti-anti- idiotype antibodies i.e., tertiary antibodies or Ab3 antibodies
  • the antigenicity of the idiotype of the immunoglobulin molecule may be enhanced by deletion of framework and CDR sequences that are N-terminal to the particular CDR sequence that specifically recognizes the antigen.
  • synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art.
  • the invention includes modified immunoglobulin molecules that have one disulfide bond forming cysteine residue in a variable region domain replaced with an amino acid residue that does not contain a sulfhydryl group and in which a portion of that variable domain has been deleted N-terminal to the CDR sequence that recognizes the antigen.
  • modified antibodies of the invention such as, but not limited to, F(ab') 2 fragments, which contain the variable region, the light chain constant region and the CHI domain of the heavy chain can be produced by pepsin digestion of the antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • the invention also provides heavy chain and light chain dimers of the modified antibodies of the invention, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs) (e.g., as described in U.S. Patent 4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54), or any other molecule with the same specificity as the modified antibody of the invention. Techniques have been developed for the production of "chimeric antibodies"
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a constant domain from a human immunoglobulin, e.g., humanized antibodies.
  • the modified immunoglobulin of the invention is a humanized antibody, more preferably an antibody having a variable domain in which the framework regions are from a human antibody and the CDRs are from an antibody of a non- human animal, preferably a mouse (see, International Patent Application No. PCT/GB8500392 by Neuberger et al. and Celltech Limited).
  • CDR grafting is another method of humanizing antibodies. It involves reshaping murine antibodies in order to transfer full antigen specificity and binding affinity to a human framework (Winter et al. U.S. Patent No. 5,225,539).
  • CDR-grafted antibodies have been successfully constructed against various antigens, for example, antibodies against IL-2 receptor as described in Queen et al., 1989 (Proc. Natl. Acad. Sci. USA 86:10029); antibodies against cell surface receptors-CAMPATH as described in Riechmann et al. (1988, Nature, 332:323); antibodies against hepatitis B in Cole et al. (1991, Proc. Natl. Acad. Sci.
  • CDR-grafted antibodies are generated in which the CDRs of the murine monoclonal antibody are grafted into a human antibody. Following grafting, most antibodies benefit from additional amino acid changes in the framework region to maintain affinity, presumably because framework residues are necessary to maintain CDR conformation, and some framework residues have been demonstrated to be part of the antigen binding site. However, in order to preserve the framework region so as not to introduce any antigenic site, the sequence is compared with established germline sequences followed by computer modeling.
  • the invention provides fusion proteins of the modified immunoglobulins of the invention (or functionally active fragments thereof), for example in which the modified immunoglobulin is fused via a covalent bond (e.g., a peptide bond), at either the N-terminus or the C-terminus to an amino acid sequence of another protein (or portion thereof, preferably an at least 10, 20 or 50 amino acid portion of the protein) that is not the modified immunoglobulin.
  • a covalent bond e.g., a peptide bond
  • the modified immunoglobulin, or fragment thereof is covalently linked to the other protein at the N-terminus of the constant domain.
  • the invention provides fusion proteins in which the modified immunoglobulin is covalently linked to IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, ⁇ -interferon, MHC derived peptide, G-CSF, TNF, porins, NK cell antigens, or cellular endocytosis receptor.
  • the modified immunoglobulins of the invention include analogs and derivatives that are either modified, i.e, by the covalent attachment of any type of molecule as long as such covalent attachment does not prevent the modified immunoglobulin from generating an anti- idiotypic response (e.g., as determined by any of the methods described in Section 5.5, infra).
  • the derivatives and analogs of the modified immunoglobulins include those that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the analog or derivative may contain one or more non-classical amino acids, e.g., as listed above in this Section.
  • the present invention provides methods of eliciting production of anti-idiotype antibodies and anti-anti-idiotype antibodies in a subject by the administration of a therapeutic (termed herein "Therapeutic").
  • Therapeutics include the modified 0 immunoglobulins of the invention, and functionally active fragments, analogs, and derivatives thereof (e.g., as described in Section 5.1, supra), and nucleic acids encoding the modified antibodies of the invention, and functionally active fragments and derivatives thereof (e.g., as described in Section 5.1, supra).
  • the methods of the invention use a modified antibody that is derived from a human antibody; in other embodiments, the methods of the invention use a modified antibody that is derived from a chimeric or humanized antibody.
  • vaccine compositions e.g., as described in Section 5.3, infra
  • modified antibodies of the invention are administered to the subject to elicit the production of an antibody (i.e., the anti-idiotype antibody or Ab2) that specifically recognizes the idiotype of the modified antibody, the Ab2, in turn, elicits the production anti-anti-idiotype antibodies (Ab3) that specifically recognize the idiotype of Ab2, such that these Ab3 antibodies have the same or similar binding specificity as the modified antibody.
  • an antibody i.e., the anti-idiotype antibody or Ab2
  • Ab3 anti-anti-idiotype antibodies
  • the invention provides methods of administering the modified antibodies of the invention to elicit an anti-idiotype response, i.e., to generate Ab2 and Ab3 type antibodies.
  • the invention provides methods of administering the modified antibodies of the invention to one subject to generate Ab2 antibodies, isolating the Ab2 antibodies, and then administering the Ab2 antibodies to a second subject to generate Ab3 type antibodies in
  • the invention provides a method of generating an anti-idiotype response in a subject comprising administering an amount of first immunoglobulin molecule (or functionally active fragment, analog, or derivative thereof) sufficient to induce an anti- idiotype response, said first immunoglobulin comprising a variable region and being identical, except for one or more amino acid substitutions in said variable region, to a second immunoglobulin molecule, said second immunoglobulin molecule being capable of immunospecifically binding an antigen, said one or more amino acid substitutions being the substitution of an amino acid residue that does not have a sulfhydryl group at one or more positions corresponding to one or more cysteine residues that form a disulfide bond in said second immunoglobulin molecule.
  • the method further provides isolating the anti-idiotype antibody that recognizes the idiotype of said second immunoglobulin molecule, and administering to a second subject the anti-idiotype antibody.
  • the modified antibodies of the invention may be used to induce an anti-idiotype response to infectious agents and diseased or abnormal cells, such as but not limited to, bacteria, parasites, fungi, viruses, tumors and cancers.
  • the modified antibodies of the invention may be used to either treat or prevent any disease or disorder amenable to treatment or prevention by generating an anit-anti-idiotypic response to a particular antigen.
  • the modified antibodies may be used for the treatment of autoimmune disease, such as, but not limited to rheumatoid arthritis, lupus, ulcerative colitis, or psoriasis, or for the treatment of allergies.
  • the methods and vaccine compositions of the present invention may be used to elicit a humoral and/or a cell-mediated response against a modified immunoglobulin in a subject.
  • the methods and compositions of the invention elicit a humoral response in a subject.
  • the methods and compositions of the invention elicit a cell-mediated response in a subject.
  • the methods and compositions of the invention elicit both a humoral and a cell-mediated response.
  • the subjects to which the present invention is applicable may be any mammalian or vertebrate species, which include, but are not limited to, cows, horses, sheep, pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, mice, rats, monkeys, rabbits, chimpanzees, and humans.
  • the subject is a human.
  • the compositions and methods of the invention can be used either to prevent a disease or disorder, or to treat a particular disease or disorder, where an anti-idiotypic response against a particular immunoglobulin molecule is effective to treat or prevent the disease or disorder.
  • Cancers including, but not limited to, neoplasms, tumors, metastases, or any disease or disorder characterized by uncontrolled cell growth, can be treated or prevented by administration of a modified immunoglobulin (or functionally active fragment, derivative or analog thereof) of the invention, or a nucleic acid encoding the modified immunoglobulin, or functionally active fragment, derivative or analog thereof), which modified immunoglobulin is derived from an immimoglobulin that specifically recognizes one or more antigens associated with the cancer cells of the cancer to be treated or prevented.
  • Whether a particular Therapeutic is effective to treat or prevent a certain type of cancer can be determined by any method known in the art, for example but not limited to, those methods described in Section 5.5, infra.
  • cancers associated with the following cancer antigens may be treated or prevented by administration of a modified antibody of the invention derived from an antibody that recognizes these cancer antigens: KS 1/4 pan- 0 carcinoma antigen (Perez and Walker, 1990, J. Immunol. 142:32-37; Bumal, 1988,
  • Hybridoma 7(4):407-415 ovarian carcinoma antigen (CA125) (Yu et al., 1991, Cancer Res. 51(2):48-475), prostatic acid phosphate (Tailor et al., 1990, Nucl. Acids Res. 18(1):4928), prostate specific antigen (Henttu and Vihko, 1989, Biochem. Biophys. Res. Comm. 10(2):903-910; Israeli et al., 1993, Cancer Res. 53:227-230), melanoma-associated antigen 15 p97 (Estin et al., 1989, J. Natl. Cancer Instit.
  • melanoma antigen gp75 (Vijayasardahl et al., 1990, J. Exp. Med. 171(4):1375-1380), high molecular weight melanoma antigen (HMW-MAA) (Natali et al., 1987, Cancer 59:55-3; Mittelman et al., 1990, J. Clin. Invest. 86:2136-2144)), prostate specific membrane antigen, carcinoembryonic antigen (CEA) (Foon et al., 1994, Proc. Am. Soc. Clin. Oncol.
  • ganglioside GM2 Livingston et al., 1994, J. Clin. Oncol. 12:1036-1044
  • ganglioside GM3 Hoon et al., 1993, Cancer Res. 53:5244-5250
  • tumor-specific transplantation type of - 0 cell-surface antigen TSTA
  • virally-induced tumor antigens including T-antigen DNA tumor viruses and envelope antigens of RNA tumor viruses
  • oncofetal antigen-alpha- fetoprotein such as CEA of colon
  • bladder tumor oncofetal antigen Hellstrom et al., 1985, Cancer. Res.
  • differentiation antigen such as human lung carcinoma antigen L6, L20 (Hellstrom et al., 1986, Cancer Res. 46:3917-3923), antigens of fibrosarcoma, human leukemia T cell antigen-Gp37 (Bhattacharya-Chatterjee et al., 1988, J. oflmmun. 141:1398-1403), neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR (Epidermal growth factor receptor), HER2 antigen (pi 85TM ⁇ ), polymorphic epithelial mucin (PEM) (Hilkens et al., 1992, Trends in Bio. Chem. Sci.
  • PEM polymorphic epithelial mucin
  • malignant human lymphocyte antigen-APO-1 (Bernhard et al., 1989, Science 245:301-304), differentiation antigen (Feizi, 1985, Nature 314:53-57) such as I antigen found in fetal erthrocytes and primary endoderm, I(Ma) found in gastric adencarcinomas, Ml 8 and M39 found in breast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl, VIM-D5,and D,56-22 found in colorectal cancer, TRA-1-85 (blood group H), C14 found in colonic adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten, Le y found in 0 embryonal carcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells , E, series (blood group B) found in pancreatic cancer, FC10.2 found in embryonal carcinoma
  • the antigen is a T cell receptor derived peptide from a cutaneous T cell lymphoma (see Edelson, 1998, The Cancer Journal 4:62).
  • the subject being treated with the modified 0 antibody of this invention may, optionally, be treated with other cancer treatments such as surgery, radiation therapy or chemotherapy.
  • the Therapeutic of the invention used to treat or prevent cancer may be administered in conjunction with one or a combination of chemotherapeutic agents including, but not limited to, methotrexate, taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, 5 nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, an etoposide, a campathecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel,
  • Malignancies and related disorders that can be treated or prevented by administration of the invention include but are not limited to those listed in Table 3 (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia):
  • malignancy or dysproliferative changes are treated or prevented in the 25 ovary, bladder, breast, colon, lung, skin, pancreas, or uterus.
  • sarcoma, melanoma, or leukemia is treated or prevented.
  • the Therapeutics of the invention can also be administered to treat premalignant 30 conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders listed in Table 3.
  • Such prophylactic or therapeutic use is indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see 35 Robbins and Angell, 197, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp.
  • Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function.
  • endometrial hyperplasia often precedes endometrial cancer.
  • Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells.
  • Atypical metaplasia involves a somewhat disorderly metaplastic epithelium.
  • Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells.
  • Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.
  • the presence of one or more characteristics of a transformed phenotype, or of a malignant phenotype, displayed in vivo or displayed in vitro by a cell sample from a patient can indicate the desirability of prophylactic/therapeutic administration of the vaccine composition.
  • characteristics of a transformed phenotype include morphology changes, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, protease release, increased sugar transport, decreased serum requirement, expression of fetal antigens, disappearance of the 250,000 dalton cell surface protein, etc. (see also id., at pp. 84-90 for characteristics associated with a transformed or malignant phenotype).
  • leukoplakia a benign-appearing hyperplastic or dysplastic lesion of the epithelium, or Bowen's disease, a carcinoma in situ, are pre-neoplastic lesions indicative of the desirability of prophylactic intervention.
  • fibrocystic disease cystic hyperplasia, mammary dysplasia, particularly adenosis (benign epithelial hyperplasia) is indicative of the desirability of prophylactic intervention.
  • a patient which exhibits one or more of the following predisposing factors for malignancy is treated by administration of an effective amount of the Therapeutic of the invention: a chromosomal translocation associated with a malignancy (e.g., the Philadelphia chromosome for chrome myelogenous leukemia, t(14;18) for follicular lymphoma, etc.), familial polyposis or Gardner's syndrome (possible forerunners of colon cancer), benign monoclonal gammopathy (a possible forerunner of multiple myeloma), and a first degree kinship with persons having a cancer or precancerous disease showing a Mendelian (genetic) inheritance pattern (e.g., familial polyposis of the colon, Gardner's syndrome, hereditary exostosis, polyendocrine adenomatosis, medullary thyroid carcinoma with amyloid production and pheochromocytoma, Peutz-Jeghers syndrome, neurofibromatos
  • Therapeutic of the invention is administered to a 0 human patient to prevent progression to ovary, breast, colon, lung, pancreatic, skin, prostate, gastrointestinal, B lymphocyte, T lymphocyte or uterine cancer, melanoma or sarcoma.
  • the invention also provides methods of treating or preventing infectious diseases by 5 administration of a Therapeutic of the invention, in particular a modified immunoglobulin molecule (or functionally active fragment, derivative or analog thereof, or a nucleic acid encoding the modified immunoglobulin, or functionally active fragment, analog or derivative thereof) that is derived from an immunoglobulin molecule that can immunospecifically bind an antigen of the agent causing the infectious disease or a cellular 0 receptor for the infectious disease agent.
  • the infectious agents include, but are not limited to viruses, bacteria, fungi, protozoa, and parasites.
  • infectious diseases are treated or prevented by administration of a modified immunoglobulin of the invention (or functionally active fragment, derivative or analog thereof, or nucleic acid encoding the same) that is derived
  • influenza virus hemagglutinin Genbank accession no. JO2132; Air, 1981, Proc. Natl. Acad. Sci. USA 78:739-743; Newton et al., 1983, Virology 128:495-501
  • human respiratory syncytial virus G glycoprotein Genbank accession no. Z33429; Garcia et al., 1994, J. Virol.; Collins et al, 1984, Proc. Natl. Acad. Sci. USA
  • antigen of equine influenza virus or equine herpesvirus e.g., equine influenza virus type A/ Alaska 91 neuraminidase, equine influenza virus type A/Miami 63 neuraminidase, equine influenza virus type A/Kentucky 81 neuraminidase, equine herpesvirus type 1 glycoprotein B, equine herpesvirus type 1 glycoprotein D
  • antigen of bovine respiratory syncytial virus or bovine parainfluenza virus e.g., bovine respiratory syncytial virus attachment protein (BRSV G), bovine respiratory syncytial virus fusion protein (BRSV F), bovine respiratory syncytial virus nucleocapsid protein (BRSV ⁇ ), bovine parainfluenza virus type 3 fusion protein, and the bovine parainfluenza
  • infectious diseases are treated or prevented by administration of a modified immunoglobulin (or functionally active fragment, derivative, or analog thereof, or nucleic acid encoding the same) that recognizes a cellular receptor for aninfectiusdisease agent, for example but not by way of limitation, such cellular receptors, along with their corresponding pathogens are listed in Table 4.
  • a modified immunoglobulin or functionally active fragment, derivative, or analog thereof, or nucleic acid encoding the same
  • Viral diseases that can be treated or prevented by the methods of the present invention include, but are not limited to, those caused by hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsachie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), any picornaviridae, enteroviruses, caliciviridae, any of the Norwalk group of viruses, togaviruses (such as Dengue virus), al
  • Bacterial diseases that can be treated or prevented by the methods of the present invention are caused by bacteria including, but not limited to, gram negative and gram positive bacteria, mycobacteria rickettsia, mycoplasma, Neisseria spp. (e.g., Neisseria mennigitidis and Neisseria gonorrhoeae), legionella, Vibrio cholerae, Streptococci, such as
  • Streptococcus pneumoniae corynebacteria diphtheriae, clostridium tetani, bordetella pertussis, Haemophilus spp. (e.g., influenzae), Chlamydia spp., Enterotoxigenic Escherichia coli, Shigella spp. etc., and bacterial diseases such as Syphilis, Lyme's disease, etc.
  • Protozoal diseases that can be treated or prevented by the methods of the present invention are caused by protozoa including, but not limited to, plasmodia, eimeria, leishmania, kokzidioa, trypanosoma, fungi, such as Candida, etc.
  • the Therapeutic of the invention is administered in conjunction with an appropriate antibiotic, anti-fungal, anti-viral or any other drug useful in treating or preventing the infectious disease.
  • GENE THERAPY Gene therapy refers to treatment or prevention of a disease performed by the administration of a nucleic acid to a subject who has a disease associated with the expression of the antigen which is recognized by the immunoglobulin molecule from which the modified immunoglobulin molecule was derived.
  • the disease or disorder may be a cancer associated with the expression of a particular cancer or tumor agent or an infectious disease associated with the expression of a particular antigen of an infectious disease agent or for which the infectious disease agent binds a particular cellular receptor.
  • the therapeutic nucleic acid encodes a sequence that 0 produces intracellularly (without a leader sequence) or intercellularly (with a leader sequence), a modified immunoglobulin.
  • the therapeutic nucleic acid comprises an expression vector that expresses the modified immunoglobulin molecule.
  • Delivery of the nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector or a delivery
  • the nucleic acid is directly administered in vivo, where it is expressed to produce the antibodies. This can be accomplished by any of numerous methods
  • nucleic acid expression vector e.g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in biopolymers (e.g., poly- ⁇ -l->4-N-acetylglucosamine polysaccharide; see U.S. Patent No.
  • biopolymers e.g., poly- ⁇ -l->4-N-acetylglucosamine polysaccharide; see U.S. Patent No.
  • nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
  • nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180 dated April 16,
  • nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
  • single chain antibodies such as neutralizing antibodies, which bind to intracellular epitopes can also be administered.
  • Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population by utilizing, for example, techniques such as those described in Marasco et al. (Marasco et al., 1993, Proc. Natl. Acad. Sci. USA 90:7889-
  • Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion
  • Adeno-associated virus has also been proposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300).
  • the form and amount of therapeutic nucleic acid envisioned for use depends on the type of disease and the severity of its desired effect, patient state, etc., and can be determined by one skilled in the art.
  • the invention also provides vaccine formulations containing Therapeutics of the invention, which vaccine formulations are suitable for administration to elicit a protective immune (humoral and/or cell mediated) response against certain antigens, e.g., for the treatment and prevention of diseases.
  • a protective immune humoral and/or cell mediated
  • Suitable preparations of such vaccines include injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection, may also be prepared.
  • the preparation may also be emulsified, or the polypeptides encapsulated in liposomes.
  • the active immunogenic ingredients are often mixed with 0 excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, buffered saline, dextrose, glycerol, ethanol, sterile isotonic aqueous buffer or the like and combinations thereof.
  • the vaccine preparation may also include minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance 5 the effectiveness of the vaccine.
  • adjuvants which may be effective, include, but are not limited to: aluminim hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl- nor-muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L- alanine-2-( 1 '-2'-dipalmitoyl-sn-glycero-3 -hydroxyphosphoryloxy)-ethylamine.
  • the effectiveness of an adjuvant may be determined by measuring the induction of anti-idiotype antibodies directed against the injected immunoglobulin formulated with the particular adjuvant.
  • composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • Oral formulation can include standard carriers 5 such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active
  • composition is administered by injection, an ampoule of sterile diluent can be provided so that the ingredients may be mixed prior to administration.
  • the lyophilized modified immunoglobulin of the invention is provided in a first container; a second container comprises diluent consisting of an aqueous solution of 50% glycerin, 0.25% phenol, and an antiseptic (e.g., 0.005% brilliant
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the vaccine formulations of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the vaccine formulations of the invention.
  • Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • Composition comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • the subject to which the vaccine is administered is preferably a mammal, most preferably a human, but can also be a non-human animal including but not limited to cows, horses, sheep, pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, mice and rats.
  • cows horses, sheep, pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, mice and rats.
  • Many methods may be used to introduce the vaccine formulations of the invention; these include but are not limited to oral, intracerebral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal routes, and via scarification (scratching through the top layers of skin, e.g., using a bifurcated needle) or any other standard routes of immunization.
  • scarification is employed.
  • the precise dose of the modified immunoglobulin molecule to be employed in the formulation will also depend on the route of administration, and the nature of the patient, and should be decided according to the judgment of the practitioner and each patient's circumstances according to standard clinical techniques.
  • An effective immunizing amount is that amount sufficient to produce an immune response to the modified immunoglobulin molecule in the host (i.e., an anti-idiotype reaction) to which the vaccine preparation is administered. Effective doses may also be extrapolated from dose-response curves derived from animal model test systems.
  • the modified immunoglobulins of the invention can be produced by any method known in the art for the synthesis of immunoglobulins, in particular, by chemical synthesis or by recombinant expression, and is preferably produced by recombinant expression techniques.
  • Recombinant expression of the modified immunoglobulin of the invention, or fragment, derivative or analog thereof, requires construction of a nucleic acid that encodes the modified immunoglobulin.
  • a nucleic acid encoding the modified immunoglobulin may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the modified immunoglobulin, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR, e.g., as exemplified in Section 6, infra.
  • the nucleic acid encoding the modified immunoglobulin may be generated from a nucleic acid encoding the immunoglobulin from which the modified immunoglobulin was derived. If a clone containing the nucleic acid encoding the particular immunoglobulin is not available, but the sequence of the immunoglobulin molecule is known, a nucleic acid encoding the immunoglobulin may be obtained from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by hybridization using an oligonucleotide probe specific for the particular gene sequence.
  • a suitable source e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin
  • immunoglobulins specific for a particular antigen may be generated by any method known in the art, for example, by immunizing an animal, such as a rabbit, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or, as 5 described by Kozbon et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in
  • a clone encoding at least the Fab portion of the immunoglobulin can be obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246:1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (see, Q e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).
  • nucleic acid encoding at least the variable domain of the immunoglobulin molecule may be introduced into any available cloning vector, and may be introduced into a vector containing the nucleotide sequence encoding the constant region of the immunoglobulin molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; U.S. Patent No. 5,122,464; and Bebbington, 1991, Methods in Enzymology 2:136-145).
  • Vectors containing the complete light or heavy chain for co-expression with the nucleic acid to allow the expression of a complete antibody molecule are also available, see Id.
  • the nucleic acid encoding the immimoglobulin can be modified to introduce the nucleotide substitutions or deletion necessary to substitute (or delete) the one or more variable region cysteine residues participating in an intrachain disulfide bond with an amino acid residue that does not contain a sulfhydyl group, along with any other desired amino acid substitutions, deletions or insertions.
  • modifications can be carried out by any method known in the art for the introduction of specific mutations or deletions in a nucleotide sequence, for example, but not limited to, chemical muagenesis, in vitro site directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551), PCR based methods, etc.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a constnat region derived from a human immunoglobulin, e.g., humanized antibodies.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., 1988, Science 242:1038-1041).
  • Antibody fragments which recognize specific epitopes may be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • the vector for the production of the immunoglobulin molecule may be produced by recombinant D ⁇ A technology using techniques well known in the art.
  • the modified immunoglobulin molecule can then be recombinantly expressed and isolated by any method known in the art, for example, using the method described in Section 6, supra, (see also Bebbington, 1991, Methods in Enzymology 2:136-145).
  • COS cells or any other appropriate cultured cells, can be transiently or non-transiently transfected with the expression vector encoding the modified immunoglobulin, cultured for an appropriate period of time to permit immunoglobulin expression, and then the supernatan can be harvested from the COS cells, which supernatant contains the secreted, expressed modified immunoglobulin.
  • the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce the immunoglobulin of the invention.
  • the host cells used to express the recombinant antibody of the invention may be any suitable host cells used to express the recombinant antibody of the invention.
  • bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells, especially for the expression of whole recombinant immunoglobulin molecules.
  • mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalo virus is an effective expression system for immunoglobulins (Foecking et al., 198, Gene 45:101; Cockett et al.,
  • host-expression vector systems may be utilized to express the modified immunoglobulin molecules of the invention.
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the
  • appropriate nucleotide coding sequences express the immunoglobulin molecule of the invention in situ.
  • suitable nucleotide coding sequences include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing immunoglobulin coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing
  • immunoglobulin coding sequences insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the immunoglobulin coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing immunoglobulin coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adeno virus late promoter; the vaccinia virus 7.5K promoter).
  • recombinant virus expression vectors e.g., baculovirus
  • a number of expression vectors may be advantageously selected depending upon the use intended for the immunoglobulin molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an immunoglobulin molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983 , EMBO J.
  • pG ⁇ X vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S- transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pG ⁇ X vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • the virus grows in Spodoptera frugiperda cells.
  • the immunoglobulin coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • an AcNPV promoter for example the polyhedrin promoter
  • a number of viral-based expression systems may be utilized.
  • the immunoglobulin coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination.
  • Insertion in a non-essential region of the viral genome will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81 :355-359).
  • Specific initiation signals may also be required for efficient translation of inserted immunoglobulin coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • the efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:51-544).
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have charac- teristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38.
  • cell lines which stably express the immunoglobulin molecule may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the immunoglobulin molecule.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the immunoglobulin molecule.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11 :223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk " , hgprt " or aprt " cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance
  • any fusion protein may be readily purified by utilizing an antibody
  • Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897).
  • the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of
  • the tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni 2+, nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.
  • the expression levels of the immunoglobulin molecule can be increased by vector
  • 35 amplified region is associated with the immunoglobulin gene, production of the immunoglobulin will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).
  • the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, N ⁇ twre 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA
  • the coding sequences for the heavy and light chains may comprise cD ⁇ A or genomic D ⁇ A.
  • the modified immunoglobulin molecule of the invention may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange,
  • modified antibodies of the invention can be screened or assayed in a variety of ways for efficacy in treating or preventing a particular disease .
  • the immunopotency of a vaccine formulation containing the modified antibody of the invention can be determined by monitoring the anti-idiotypic response of test animals following immunization with the vaccine. Generation of a humoral response may be taken
  • Test animals may include mice, rabbits, chimpanzees and eventually human subjects.
  • a vaccine made in this invention can be made to infect chimpanzees experimentally. However, since chimpanzees are a protected species, the antibody response to a vaccine of the invention can
  • the immune response of the test subjects can be analyzed by various approaches such as the reactivity of the resultant immune serum to antibodies, as assayed by known techniques, e.g., enzyme linked immunosorbent assay (ELISA), immunoblots, radioimmunoprecipitations, etc.; or protection from infection and/or attenuation of disease symptoms in immunized hosts.
  • ELISA enzyme linked immunosorbent assay
  • the vaccine composition of the invention may be tested in rabbits for the ability to induce an anti-idiotypic response to the modified immunoglobulin molecule.
  • rabbits for example, male specific-pathogen-free (SPF) young adult New Zealand White rabbits may be used.
  • SPF specific-pathogen-free
  • the test group of rabbits each receives an effective amount of the vaccine.
  • a control group of rabbits receives an injection in 1 mM Tris-HCl pH 9.0 of the vaccine containing a naturally occurring antibody.
  • Blood samples may be drawn from the rabbits every one or two weeks, and serum analyzed for anti- idiotypic antibodies to the modified immunoglobulin molecule and anti-anti-idiotypic antibodies specific for the antigen against which the modified antibody was directed using, e.g., a radioimmunoassay (Abbott Laboratories). The presence of anti-idiotypic antibodies may be assayed using an ELISA. Because rabbits may give a variable response due to their outbred nature, it may also be useful to test the vaccines in mice.
  • a modified antibody of the invention may be tested by first administering the modified antibody to a test subject, either animal or human, and then isolating the anti- anti-idiotypic antibodies (i.e., the Ab3 antibodies) generated as part of the anti-idiotype response to the injected modified antibody.
  • a test subject either animal or human
  • isolating the anti- anti-idiotypic antibodies i.e., the Ab3 antibodies
  • the isolated Ab3 may then be tested for the ability to bind the particular antigen (e.g., a tumor antigen, antigen of an infectious disease agent by any immunoassays known in the art, for example, but not limited to, radioimmunoassays, ELISA, "sandwich” immunoassay, gel diffusion precipitin reactions, immunodiffusion assays, western blots, precipitation reactions, agglytination assays, complement fixation assays, immunofluorescence assays, protein A assays, immunoelectrophoresis assays, etc.
  • immunoassays known in the art, for example, but not limited to, radioimmunoassays, ELISA, "sandwich” immunoassay, gel diffusion precipitin reactions, immunodiffusion assays, western blots, precipitation reactions, agglytination assays, complement fixation assays, immunofluorescence assays, protein A assays, immunoelec
  • the efficacy of the isolated Ab3 for treating cancer, a tumor, or other neoplastic disease is screened by culturing cancer or tumor cells from a patient, contacting the cells with the Ab3 antibody to be tested, and comparing the proliferation or survival of the contacted cells with the proliferation or survival of cells not so contacted with the Ab3 antibody, wherein a lower level of proliferation or survival of the contacted cells indicates that the Ab3 antibody (which was elicited by immunization with the modified antibody of the invention) is effective to treat the cancer in the patient.
  • cell proliferation can be assayed by measuring 3 H-thymidine incorporation, by direct cell count, by detecting changes in transcriptional activity of known genes such as proto-oncogenes (e.g.,fos, myc) or cell cycle markers; cell viability can be assessed by trypan blue staining, differentiation can be assessed visually based on changes in morphology, etc.
  • proto-oncogenes e.g.,fos, myc
  • cell cycle markers e.g., cell cycle markers
  • cell viability can be assessed by trypan blue staining
  • differentiation can be assessed visually based on changes in morphology, etc.
  • the level of the antigen against which the modified antibody is directed is measured at suitable time intervals before, during, or after therapy. Any change or absence of change in the amount of the antigen can be identified and correlated with the effect of the treatment on the subject.
  • the serum levels of an antigen bears a direct relationship with severity of a cancer, such as breast cancer, and poor prognosis. Generally, a decrease in the level of antigen is associated with efficacious treatment.
  • the efficacy of the modified antibody can be monitored by measuring the level of the antigen of the infectious disease agent at suitable times before, during and after therapy, where a decrease in the levels of the antigen indicates that the modified antibody is efficacious.
  • the approach that can be taken is to determine the levels of antigen at different time points and to compare these values with a baseline level.
  • the baseline level can be either the level of the marker present in normal, disease free individuals; and/or the levels present prior to treatment, or during remission of disease, or during periods of stability. These levels can then be correlated with the disease course or treatment outcome.
  • the levels of antigen can be determined by any method well known in the art. For example, a certain antigen can be quantitated by known immunodiagnostic methods such as western blotting immunoprecipitation using any antibody against a certain antigen.
  • the strength of the immune response in vivo to the modified immunogluobulin may be determined by any method known in the art, for example, but not limited to, delayed hypersensitivity skin tests and assays of the activity of cytolytic T-lymphocytes in vitro. Delayed hypersensitivity skin tests are of great value in the testing of the overall immunocompetence and cellular immunity to an antigen. Proper technique of skin testing requires that the antigens be stored sterile at 4°C, protected from light and reconstituted shortly before use. A 25- or 27-gauge need ensures intradermal, rather than subcutaneous, administration of antigen. Twenty-four and 48 hours after intradermal administration of the antigen, the largest dimensions of both erythema and induration are measured with a ruler. Hypoactivity to any given antigen or group of antigens is confirmed by testing with higher concentrations of antigen or, in ambiguous circumstances, by a repeat test with an intermediate test.
  • T-lymphocytes isolated from the immunized subject are restimulated with cells bearing the antigen against which the modified antibody was directed in 3 ml RPMI medium containing 10% fetal calf serum.
  • 33% secondary mixed lymphocyte culture supernatant or IL-2 is included in the culture medium as a source of T cell growth factors.
  • the isolated T cells are cultured with or without the cells bearing the antigen. After six days, the cultures are tested for cytotoxity in a 4 hour 51 Cr- release assay. The spontaneous 51 Cr-release of the targets should reach a level less than 20% if immunization was effective (Heike et al., J. Immunotherapy 15:15-174).
  • the modified immunoglobulins may be tested for efficacy by monitoring the subject for improvement or recovery from the particular disease or condition associated with the antigen against which the modified antibody is directed.
  • the progress of the particular tumor or cancer may be followed by any diagnostic or screening method known for monitoring cancer or a tumor.
  • the cancer or tumor progress may be monitored by assaying the levels of the particular cancer or tumor antigen (or another antigen associated with the particular cancer or tumor) either in the serum of the subject or by injecting a labeled antibody specific for the antigen.
  • CT computed tomographic
  • sonograms sonograms
  • other imaging techniques such as computed tomographic (CT) scan or sonograms, or any other imaging method, may be used to monitor the progression of the cancer or tumor.
  • Biopsies may also be performed. Before carrying out such trials in humans, the tests for efficacy of the modified immunoglobulins can be performed in animal models of the particular cancer or tumor.
  • the efficacy of the modified antibody can be assayed by administering the modified antibody to a subject (either a human subject or an animal model for the disease) and then monitoring either the levels of the particular infectious disease agent or symptoms of the particular infectious disease.
  • the levels of the infectious disease agent may be determined by any method known in the art for assaying the levels of an infectious disease agent, e.g., the viral titer, in the case of a virus, or bacterial levels (for example, by culturing of a sample from the patient), etc.
  • the levels of the infectious disease agent may also be determined by measuring the levels of the antigen against which the modified immunoglobulin was directed or another antigen of the infectious disease agent. A decrease in the levels of the infectious disease agent or an amelioration of the symptoms of the infectious disease indicates that the modified antibody is effective.
  • This example describes the construction of a modified antibody derived from the monoclonal antibody MAb31.1 (hybridoma secreting Mab31.1 is available from the American Type Tissue Collection as accession No. HB12314). Mab31.1 recognizes an antigen expressed by human colon carcinomas.
  • the modified antibody of the invention based on Mab31.1, was engineered to have variable region cysteine residues of both the heavy and light chain variable regions substituted with alanine. Therefore, the resulting modified antibody, was missing intrachain disulfide bonds in either the heavy and light chain variable regions.
  • the strategy for construction of the modified antibody was to construct two engineered genes that encoded the heavy and light chain variable regions wherein specific cysteine residues, known to be important in intra-chain disulfide bonding , were altered to alanine. Alanine residues were substituted for the cysteine residues at positions 22 and 92 of the heavy chain variable region of the antibody derived from Mab31.1 or at positions 23 and 88 of the Mab31.1 light chain variable region of the antibody derived from Mab31.1.
  • groups of olionucleotides were assembled (as discussed below) and inserted into an appropriate vector providing constant regions.
  • variable region genes encoding CDRs lacking intrachain disulfide bonds the following strategy was performed.
  • oligonucleotides were annealed to create cohesive double stranded DNA fragments (as diagramed in Figure 10, Step 1). Specifically, oligonucleotides of about 80 bases in length corresponding to the sequences of interest with 20 base overlapping regions were synthesized using automated techniques of GenoSys Biotech Inc. The specific sequences of each of these oligonucleotides. The specific sequences of these oligonucleotides are presented in Figures 9 A and 9B.
  • Figure 9A list the group often oligos used in engineering a heavy chain variable region gene called 2CAVHCOL1. 2CAVHCOL1 lacked 2 cysteine residues as compared to the consensus heavy chain variable gene.
  • Figure 9B lists the group of 12 oligos used in the engineering of the light chain variable region gene called 2CAVLCOL1.
  • 2CAVLCOL1 lacked two cysteine residues as compared to the consensus light chain variable region gene.
  • groups of 10 or 12 oligos were combined as described below and as presented in Figure 10, where the identities of oligos 1 to 10 indicated in Figure 10 are provided in Table 5.
  • each oligonucleotide was 5' phosphorylated as follows: 25 ⁇ l of each oligo was incubated for 1 hour in the presence of T4 polynucleotide kinase and 50mM ATP at 37 °C. The reactions were stopped by heating for 5 minutes at
  • oligo 1 + oligo 10, oligo 2 + oligo 9, oligo 3 + oligo 8, oligo 4 + oligo 7, oligo 5 + oligo 6 complementary oligonucleotides (oligo 1 + oligo 10, oligo 2 + oligo 9, oligo 3 + oligo 8, oligo 4 + oligo 7, oligo 5 + oligo 6), as shown in Figure 10, were then mixed in sterile microcentrifuge tubes and annealed by heating the tube in a water bath at 65 °C for 5 minutes followed by cooling at room temperature for 30 minutes. Annealing resulted in short double strand DNA
  • cohesive double stand DNA fragments were ligated into longer strands ( Figure 10, Steps 2-4), until the engineered variable region gene was assembled.
  • cohesive double strand DNA fragments were ligated in the presence of T4 DNA ligase and lOmM ATP for 2 hours in a water bath maintained at 16°C. Annealed
  • oligo 1/10 was mixed with annealed oligo 2/9, and annealed oligo 3/8 was mixed with annealed oligo 4/7.
  • the resulting oligos were labeled oligo 1/10/2/9 and oligo 3/8/4/7.
  • oligo 3/8/4/7 was ligated to oligo 5/6.
  • the resulting oligo 3/8/4/7/5/6 was then ligated to oligo 1/10/2/9 resulted in a full length variable region gene.
  • modified variable region genes were then purified by gel electrophoresis. To remove unligated excess of oligos and other incomplete DNA fragments, ligated product was run on 1% low melting agarose gel at constant 110 V for 2 hours. The major band containing full length DNA product was cut out and placed in a
  • the gel slice was digested with f3-Agrase I at 40°C for 3 hours.
  • the DNA was recovered by precipitation with 0.3 M NaOAc and isopropanol at — 20 °C for 1 hour followed by centrifugation at 12,000 rpm for 15 minutes.
  • the purified DNA pellet was resuspended in 50 ⁇ l of TE buffer, pH 8.0.
  • the engineered variable region gene was then amplified by PCR. Specifically, 100 ng of the engineered variable region gene was mixed with 25mM dNTPs, 200 ng of primers and 5 U of high fidelity thermostable Pfu DNA polymerase in buffer. Resulting PCR product was analyzed on 1% agarose gel.
  • pUC19 is a 2686 base pair, a high copy number E. coli plasmid vector containing a 54 base pair polylinker cloning site in lacZ and an Amp selection marker.
  • lO ⁇ g of pUC19 was linearized with Hinc II (50 U) for 3 hours at 37°C resulting in a vector with blunt end sequence 5' GTC.
  • linear vector DNA was dephosphorylated with 25 U of calf intestine alkaline phosphatase (CIP) for 1 hour at 37 °C.
  • the bacterial vector containing the engineered variable region gene was then used to transform bacterial cells. Specifically, freshly prepared competent DH5- ⁇ cells, 50 ⁇ l, were mixed with 1 ⁇ g of pUC19 containing the engineered variable region gene and transferred to an electroporation cuvette (0.2 cm gap; Bio-Rad). Each cuvette was pulsed at 2.5 kV/200 ohm/25 ⁇ F in an electroporator (Bio-Rad Gene Pulser). Immediately thereafter, 1 ml of SOC media was added to each cuvette and cells were allowed to recover for 1 hour at 37°C in centrifuge tubes.
  • a single transformant colony was picked and grown overnight in a 3 ml LB/Amp sterile glass tube with constant shaking at 37°C.
  • the plasmid DNA was isolated using Easy Prep columns (Pharmacia Biotech.) and suspended in 100 ⁇ l of TE buffer, pH 7.5.
  • 25 ⁇ l of plasmid DNA from each colony was digested with a restriction endonuclease for 1 hour at 37 °C, and was analyzed on a 1% agarose gel.
  • plasmid DNA containing gene insert was resistant to enzyme cleavage due to loss of restriction site ( 5'..GTCGAC. 3') and migrated as closed circular (CC) DNA, while those plasmids without insert were cleaved and migrated as linear (L) double strand DNA fragment on gel.
  • Oligo 1 Oligo 2 Oligo 3 Oligo 4 Oligo 5 Oligo Oligo 7 Oligo 8 Oligo 9 Oligo 10
  • a complete antibody light chain has both a variable region and a constant region.
  • a Q complete antibody heavy chain contains a variable region, a constant region, and a hinge region.
  • a modified variable region genes 2CAVHCOL1 or 2CAVLCOL1 were inserted into vectors containing appropriate constant regions.
  • Engineered variable region genes lacking cysteine residues in the light chain were inserted into the pMRROlO.l vector Figure 6 A.
  • the pMRROlO.l vector contained a human kappa light chain constant region. f Insertion of the engineered light chain variable region into this vector gave a complete light chain sequence.
  • the engineered variable region gene lacking cysteine residues in the heavy chain were inserted into the pGAMMAl vector Figure 6B.
  • the pGAMMAl vector contained human and IgGl constant region and hinge region sequences. Insertion of the engineered heavy chain variable region gene into this vector gave a complete heavy Q chain sequence.
  • COS cells an African green monkey kidney cell line, CV-1, transformed with an origin-defective SV40 virus
  • COS cells an African green monkey kidney cell line, CV-1, transformed with an origin-defective SV40 virus
  • the antibody expression vector was transferred to C0S7 cells (obtained from the American Type Culture Collection).
  • the transfected cells were grown in Dulbecco's modified Eagle's Medium and transfected with the expression vectors using calcium precipitation (Sullivan et al., FEBS Lett. 285:120-123, 1991). The transfected cells were cultured for 72 hours after which supernatants were collected.
  • the modified antibody was expressed and isolated as indicated in Section 6.4, supra. The binding capacity and specificity were then assayed using LS-174T cells WiDR cells (a human colon cancer cell line) and an antigen derived from these cells.
  • FIG. 11 provides the results of the dot blot analysis. The figure demonstrated that the labeled antibody bound to the LS-174T cells. Additionally, the unlabeled antibody competed with biotinylated antibody binding, indicating specificity of binding of the antibody derived from Mab31.1 to tumor cell antigens.
  • LS-174T cells are human colon carcinoma cells which express antigen recognized by the Mab31.1 antibody. Peptides containing the sequence of one of the CDRs of the Mab31.1 antibody were generated. These peptides, the modified antibody and the control antibody derived from Mab31.1 were administered to mice in order to generate antisera against the CDR regions of Mab31.1 and the antibodies. Blood samples from mice were drawn two weeks and four weeks following injection. Antisera from the immuized mice were used in binding competition assays presented in Figures 12A and B. Antisera and biotinylated antibodies were assayed for their ability to bind LS- 174T cells.
  • antisera raised to the CDR3 and CDR4 peptides dramatically competed for control antibody (antibody derived from Mab31.1) binding to LS-174T cells. Additionally, antisera raised against CDR1 and CDR2 also significantly competed for control antibody binding to LS-174T cells. Additionally, antisera from nice injected with the 2CAVHCOL1 and 2CAVLCOL1 antibodies (i.e., the modified antibodies having the cysteine to alanine change in the variable region) competed for binding with the biotinylated antibody derived from Mab31.1 better than antiserum from mice injected with the antibody derived from Mab31.1 ( Figure 12B). This result indicates that administration of the antibodies having the cysteine to alanine change in the variable region elicit an anti-idiotype antibodies that recognize the colon carcinoma cell antigen better than antibodies with variable region intra-chain disulfide bonds.
  • EXAMPLE 7 PRODUCTION OF A SYNTHETIC MODIFIED ANTIBODY CONTAINING HMFG-1 SEQUENCE
  • HMFG-1 was an antibody to known to bind polymorphic epithelial mucin (PEM) (Stewart et al., 1990, J Clin Oncol 8:1941-50; Kosmas et al., 1994, Cancer 73:3000-3010).
  • PEM polymorphic epithelial mucin
  • the antigenic determinant of PEM with the sequence ProAspThrArgPro was inserted into the variable chain region by methods of the invention. This short sequence is a highly immunogenic region of human polymorphic epithelial mucin (Gendler et al.,
  • HMFG-1 (Table 6) were replaced with ProAspThrArgPro using the oligonucleotide method described in section 6 supra, also in Figure 10.
  • Antiidiotype synthetic HMFG-1 antibodies were produced which immunospecifically bound to HMFG-1, using the known sequences for the variable regions of the light and heavy chains of the HMFG-1.
  • PCR Polymerase chain reaction
  • the engineered variable regions gene constructed to contain nucleotide sequence encoding HMFG-1 is shown in Figure 13.
  • the engineered variable region gene was inserted into appropriate vectors for antibody production, such as the pNEPuDGV vector, as described in Section 6, supra.
  • Other methods for constructing engineered genes may be used, including but not limited to those methods described by Jayaraman et al., 1989, Nucleic Acids Res. 17:4403; Sandhu et al., 1992, BioTechniques 12:14; Barnett and Erfie, 1990, H. Nucleic Acids Res 18:3094; Ciccarelli et al., 1991, Nucleic Acids Res 19:6007; Michaels et al., 1992, BioTechniques 12:45, incorporated by reference herein.
  • VL CDR2 Sequences residues 50 51 52 53 54 55 56

Abstract

The invention relates to modified immunoglobulin molecules in which one or more variable region residues that form intrachain disulfide bonds are substituted with amino acid residues that do not contain sulfydryl groups, such that the intrachain disulfide bond does not form. Such immunoglobulin molecules have an enhanced ability to elicit an anti-idiotype response. The invention further provides for the methods of prevention and treatment of cancer and/or infectious diseases using the modified immunoglobulins of the invention.

Description

MODIFIED ANTIBODIES WITH ENHANCED ABILITY TO ELICIT AN ANTI-IDIOTYPE RESPONSE
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of Provisional application Serial No. 60/065,716, filed November 14, 1997, and Provisional application Serial No. 60/081,403, filed April 10, 1998, both of which are incorporated by reference herein in their entireties.
1. FIELD OF THE INVENTION
The present invention relates to modified immunoglobulins, and vaccine compositions thereof, in which one or more variable region cysteine residues that form intrachain disulfide bonds have been replaced with amino acid residues that do not contain a sulfhydryl group and, therefore, do not form disulfide bonds. The present invention also relates to use of the vaccine compositions of the invention to treat or prevent certain diseases and disorders, particularly cancers and infectious diseases.
2. BACKGROUND OF THE INVENTION 2.1. IMMUNOGLOBULIN STRUCTURE The basic unit of immunoglobulin structure is a complex of four polypeptides — two identical low molecular weight or "light" chains and two identical high molecular weight or "heavy" chains, linked together by both noncovalent associations and by disulfide bonds. Each light and heavy chain of an antibody has a variable region at its amino terminus and a constant domain at its carboxyl terminus (Figure 1). The variable regions are distinct 5 for each antibody and contain the antibody antigen binding site. Each variable domain is comprised of four relatively conserved framework regions and three regions of sequence hypervariability termed complementarity determining regions or CDRs (Figure 2). For the most part, it is the CDRs that form the antigen binding site and confer antigen specificity. The constant regions are more highly conserved than the variable domains, with slight Q variations due to haplotypic differences.
Based on their amino acid sequences, light chains are classified as either kappa or lambda. The constant region heavy chains are composed of multiple domains (CHI, CH2, CH3...CHx), the number depending upon the particular antibody class. The CHI region is separated from the CH2 region by a hinge region which allows flexibility in the antibody. . The variable region of each light chain aligns with the variable region of each heavy chain, and the constant region of each light chain aligns with the first constant region of each heavy chain. The CH2-CHx domains of the constant region of a heavy chain form an "Fc region" which is responsible for the effector functions of the immunoglobulin molecule, such as complement binding and binding to the Fc receptors expressed by lymphocytes, granulocytes, monocyte lineage cells, killer cells, mast cells and other immune effector cells.
As seen in Figure 3, the light and heavy chains of an IgG molecule form the variable region domain and the constant region domain. Each domain is composed of a sandwich of two parallel extended protein layers of about 100 amino acids in length which are connected by a single disulfide bond (See Roitt et al., Immunology. 3rd Edition, London; Mosby, 1993, p 4.4). Each of the two extended protein layers of the domain, in turn, contains two "anti- parallel" adjacent strands which adopt a beta-sheet conformation. (See, e.g., Stryer, 1975, Biochemistry. WH Freeman and Co., p. 950). Each of the domains has a similar three- dimensional structure based on the immunoglobulin fold.
2.2. IMMUNOTHERAPY AND ANTI-IDIOTYPE ANTIBODIES
In modern medicine, immunotherapy or vaccination has virtually eradicated diseases such as polio, tetanus, tuberculosis, chicken pox, measles, hepatitis, etc. The approach using vaccinations has exploited the ability of the immune system to prevent infectious diseases. Use of immunotherapy has also been explored for cancer therapy. The era of tumor immunology began with experiments by Prehn and Main, who showed that antigens on the methylcholanthrene (MCA)-induced sarcomas were tumor specific in that transplantation assays could not detect these antigens in normal tissue of the mice (Prehn et al., 1957, J. Natl. Cancer Inst. 18:79-778). This notion was confirmed by further experiments demonstrating that tumor specific resistance against MCA-induced tumors could be elicited in the autochthonous host, that is, the mouse in which the tumor originated (Klein et al., 1990, Cancer Res. 20:151-1572).
There are many reasons why immunotherapy is desired for use in cancer patients. First, if cancer patients are immunosuppressed in surgery, with anesthesia and subsequent chemotherapy, it may worsen the immunosuppression, then with appropriate immunotherapy in the preoperative period, this immunosuppression may be prevented or reversed. This could lead to fewer infectious complications and accelerated wound healing. Second, tumor bulk is minimal following surgery and immunotherapy is most likely to be effective in this situation. A third reason is the possibility that tumor cells are shed into the circulation at surgery and effective immunotherapy applied at this time can eliminate these cells. There are two types of immunotherapy, the "active immunotherapy" and the "passive immunotherapy". In "active immunotherapy", an antigen is administered in the form of a vaccine, to a patient, so as to elicit a protective immune response. "Passive immunotherapy" involves the administration of antibodies to a patient without eliciting a concommitant immune response. When a specific antibody from one animal is injected as an immunogen into a suitable second animal, the injected antibody will elicit an immune response. Antibody therapy is conventionally characterized as passive since the patient is not the source of the antibodies. However, the term passive is misleading because the patient can produce anti-idiotypic secondary antibodies which in turn provoke an immune response which is cross-reactive with the original antigen. Immunotherapy where the patient generates secondary antibodies is often more therapeutically effective than passive immunotherapy because the patient's own immune system continues to fight the cells bearing the particular antigen well after the initial infusion of antibody.
In an anti-idiotype response, antibodies produced initially during an immune response or introduced into an organism will carry unique new epitopes to which the organism is not tolerant, and therefore will elicit production of secondary antibodies (termed "Ab2"), some of which are directed against the idiotype (i.e., the antigen binding site) of the primary antibody (termed "Abl"), i.e., the antibody that was initially produced or introduced exogenously. These secondary antibodies or Ab2 likewise will have an idiotype, which will induce production of tertiary antibodies (termed "Ab3"), some of which will recognize the antigen binding site of Ab2, and so forth. This is known as the "network" theory. Some of the secondary antibodies will have a binding site which is an analog of the original antigen, and thus will reproduce the "internal image" of the original antigen. And, the tertiary or Ab3 antibodies that recognize this antigen binding site of the Ab2 antibody will also recognize the original antigen (Figure 4).
Therefore, anti-idiotypic antibodies have binding sites that are similar in conformation and charge to the antigen, and can elicit the same or greater response than that of the cancer antigen itself. Administration of an exogenous antibody that can elicit a strong anti-idiotypic response can thus serve as an effective vaccine, by maintaining a constant immune response.
To date, anti-idiotypic vaccines have comprised murine antibodies because the anti- idiotypic response occurs as part of the typical human anti-mouse antibody (HAMA) response. A strong anti-idiotypic cascade has been observed when Abl has been structurally damaged (Madiyalakan et al., 1995, Hybridoma 14:199-203), rendering the antibody more foreign. There has been direct administration to the subject of exogenously produced anti- idiotype antibodies that are raised against the idiotype of an anti-tumor antibody (U.S. Patent No. 4,918,14). After administration, the subject's body will produce anti-antibodies which not only recognize these anti-idiotype antibodies, but also recognize the original tumor epitope, thereby directing complement activation and other immune system responses to a foreign entity to attack the tumor cell that expresses the tumor epitope.
However, while anti-idiotypic vaccines are desirable targets and several have been identified, the ability to deliver antibodies that reproducibly cause the generation of such an anti-idiotypic response is not currently possible. (Foon et al., 1995, J. Clin. Invest. 9:334- 342; Madiyalakan et al., 1995, Hybridoma 14:199-203). One of the reasons for the failure to 0 generate an anti-idiotypic response is that, Abl, while exogenous, is still very similar to "self, as all antibodies have very similar structures, and anti-idiotypic responses to self molecules tend to be very limited. Thus, there is a need in the art for methods of reliably generating an anti-idiotype response to a specific antibody.
5 3. SUMMARY OF THE INVENTION
The present invention is based upon the realization of the present inventors that an antibody in which one or more variable region cysteine residues that form one or more intrachain disulfide bonds have been replaced with amino acid residues that do not contain sulfhydryl groups, such that the particular disulfide bonds do not form, elicit a much stronger 0 anti-idiotype response than an antibody in which the variable region disulfide bonds are intact.
Accordingly, the present invention provides modified immunoglobulin molecules or antibodies (and functionally active fragments, derivatives and analogs thereof), and vaccine compositions containing these immunoglobulin molecules, wherein the variable region of the immunoglobulin is subject to decreased conformational constraints, such as, but not limited to, by breaking one or more intrachain or interchain disulfide bonds. Specifically, the invention provides modified immunoglobulins that comprise a variable region and are identical, except for one or more amino acid substitutions in said variable region, to a second immunoglobulin molecule, said second immunoglobulin molecule being capable of
,-. immunospecifically binding (i.e., specific binding of the immunoglobulin to its antigen as determined by any method known in the art for determining antibody-antigen binding, which excludes non-specific binding but not necessarily cross-reactivity with other antigens) an antigen, said one or more amino acid substitutions being the substitution of one or more amino acid residues that do not have a sulfhydryl group at one or more positions corresponding to one or more cysteine residues that form a disulfide bond in said second immunoglobulin molecule. In preferred embodiments, the second immunoglobulin molecule can immunospecifically bind a cancer antigen; in other preferred embodiments, the second immunoglobulin molecule can immunospecifically bind an antigen of an infectious disease agent or a cellular receptor for an infectious disease agent. The invention further provides methods of eliciting an anti-idiotype response in a subject by administering the modified immunoglobulins of the invention. In particular embodiments, the modified immunoglobulins of the invention can be used to treat or prevent cancer, specifically by administering an immunoglobulin molecule of the invention, which immunoglobulin molecule was derived (i.e., by modification according to the invention to 0 replace one or more variable region cysteine residues that form an intrachain disulfide bond with an amino acid residue that does not contain a sulfhydryl group) from an immunoglobulin molecule that can immunospecifically bind a cancer antigen, the expression of which cancer antigen is associated with the particular type of cancer. Additionally, in other embodiments, the modified immunoglobulin molecules of the invention can be used to 5 treat or prevent an infectious disease by administering an immunoglobulin molecule derived from an immunoglobulin molecule that can immunospecifically bind an antigen of or a cellular receptor for the infectious disease agent causing the infectious disease.
The invention also provides methods of producing the modified immunoglobulin molecules of the invention and vaccine compositions containing the modified 0 immunoglobulin molecules of the invention.
4. BRIEF DESCRIPTION OF THE FIGURES
Figure 1. A schematic diagram showing the structure of the light and heavy chain of an immunoglobulin molecule, each chain consisting of a variable region positioned at the 5 amino terminal region (H2N-) and a constant region positioned at a carboxyl terminal region (-COOH).
Figure 2. A schematic diagram of an IgG showing the four framework regions (FR1, FR2, FR3 and FR4) and three complementarity determining regions (CDR1, CDR2 and CDR3) in the variable regions of the light and heavy chains (labeled as VL and VH,
- „ respectively). The constant region domains are indicated as CL for the light chain constant domain and CH„ CH2 and CH3 for the three domains of the heavy chain constant region. Fab indicates the portion of the antibody fragment which includes the variable region domains of both light and heavy chains and the CL and CH, domains. Fc indicates the constant region fragment containing the CH2 and CH3 domains.
35 Figure 3. A schematic diagram of an antibody structure as shown in Figure 2, but drawn to emphasize that each domain (the loop structures labeled as VL, VH, CL, CH,, CH2, and CH3, respectively) is structurally defined by a disulfide bond (indicated with darkest lines) that maintains the three-dimensional structure (Roitt et al., Immunology, Second Edition, London: Gower Medical Publishing, 1989, p 5.3).
Figure 4. A schematic diagram showing the development of internal image bearing anti-idiotype antibodies (Ab2) and anti-anti-idiotype antibodies (Ab3) from idiotype antibodies (Abl) directed against a ligand in an anti-idiotypic cascade.
Figure 5. Modification of the variable region of an immunoglobulin by replacing cysteine residues in the variable regions with alanine residues to break a variable region intrachain disulfide bond. CHI, CH2 and CH3 are constant regions. VH is the heavy chain variable region and VL is the light chain variable region.
Figures 6A-C. (A). The structure of the expression vector pMRROlO.l, which contains a human kappa light chain constant region sequence. (B). The structure of the expression vector pGammal that contains a sequence encoding a human IgGl constant region (CHI, CH2, CH3) heavy chain and hinge region sequences. (C) The structure of the expression vector pNEPuDGV which contains a sequence encoding the kappa constant domain of the light chain and the constant domain and hinge region of the heavy chain. For all three vectors see Bebbington et al., 1991, Methods in Enzymology 2:136-145. Figures 7 A and B. (A) The amino acid sequence and corresponding nucleotide sequence including the leader sequence for the consensus light chain variable region ConVLl. (B) The amino acid and corresponding nucleotide sequences for the consensus heavy chain variable region ConVHl including the leader sequence.
Figures 8A-B. (A) Amino acid and corresponding nucleotide sequence of 2CAVLCOL1, which is the light chain variable region sequences of an antibody derived from mAb31.1, in which alanine residues have been substituted for cysteine residues at positions 23 and 88, which residues are boxed. (B) Amino acid and corresponding nucleotide sequence of 2CAVHCOL1, which is the heavy chain variable region sequence of an antibody derived from mAb31.1 , in which alanine residues have been substituted for cysteine residues at positions 23 and 88, which residues are boxed.
Figures 9A-D. (A) Oligonucleotide sequences for the oligonucleotides used to assemble 2CAVHCOL1, the heavy chain variable region gene specific to human colon cancer antigen. (B) Oligonucleotide sequences for the oligonucleotides used to assemble the 2CAVLCOL1 light chain variable region gene specific to human colon cancer antigen. (C) Oligonucleotide sequences for the oligonucleotides used to assemble the light chain consensus region referred to as ConVLl. (D) Oligonucleotide sequences for the oligonucleotides used to assemble the heavy chain consensus region referred to as ConVLl .
Figure 10. A schematic diagram of the general steps that were followed for the assembly of an engineered gene encoding the synthetic modified antibody specific to human colon cancer antigen.
Figure 11. Dot blot showing the result of an assay for the competition of binding of the antibody derived from mAB31.1, but not having the cysteine to alanine changes with the same antibody which is biotin labeled to an antigen preparation derived from LS-174 T- cells. The concentration of the unlabeled antibody is indicated as nM unlabeled antibody. 0 The "blk" lane has no antigen.
Figures 12A-D. (A)Results of competition binding assay of the biotin-labeled anti- colon carcinoma cell antibody to LS-174T cells in the presence of antisera from mice vaccinated with vehicle alone, control antibody that binds the colon carcinoma cell antibody but has not been modified, and peptides CDR1, CDR2, CDR3, CDR4, CDR5, and CDR6, 5 having the CDR sequences containing the bradykinin receptor binding site expressed as percent of control binding to LS-174T cells. (B). Results of competition binding assays of the biotin-labeled anti-colon carcinoma cell antibody to LS-174T cells in the presence of antisera from mice vaccinated with vehicle alone, control antibody that binds the colon carcinoma cell antibody, but has not been modified, 2CAVHCOL1, and 2CAVLCOL1. (C) 0 Diagram showing the binding of a biotin-labeled (indicated by the "b") antibody (inverted Y) to antigen (solid triangles). (D) Diagram showing the inhibition of binding of the biotin- labeled (indicated by the "b") antibody (inverted Y) by anti-idiotype antibodies (solid arrows) to antigen (solid triangles).
Figure 13. Nucleotide sequence for the light chain variable region having a CDR
25 containing a binding sequence for HMFG1.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention provides modified immunoglobulins (particularly antibodies and functionally active fragments, derivatives, and analogs thereof) that elicit a stronger -0 immune response, particularly a stronger anti-idiotypic response, than the corresponding unmodified immunoglobulins. In particular, the modified immunoglobulins of the invention are immunoglobulins that, when unmodified, immunospecifically bind an antigen, and are modified to decrease the conformational constraints on one variable region of the immunoglobulin molecule, preferably, such that at least one of the cysteines that participates in forming an intrachain disulfide bond in the variable region of the immunoglobulin has been replaced with an amino acid residue that does not have a sulfhydryl group and, therefore, does not form a disulfide bond, thereby decreasing the conformational constraints of at least one of the variable regions of the immunoglobulin (Figure 5). In preferred embodiments of the invention, the modified immunoglobulin molecule is derived from an immunoglobulin molecule that is capable of immunospecifically binding a cancer antigen; in other preferred embodiments, the modified immunoglobulin molecule is derived from an immunoglobulin that is capable of immunospecifically binding an antigen of an infectious disease agent or a cellular receptor for an infectious disease agent. 0 The invention also provides vaccine compositions containing the modified immunoglobulin molecules of the invention. Additionally, the invention provides methods of generating an anti-idiotype response in a subject by administration of the modified immunoglobulin molecules of the invention.
In specific embodiments, the invention provides methods of treating or preventing 5 cancer by administration of a modified immunoglobulin molecule of the invention which, in its unmodified state, is capable of immunospecifically binding a cancer antigen, the expression of which is associated with the particular cancer. Administration of the modified immunoglobulin elicits an anti-idiotype reaction in the subject, leading to the production, by the subject, of antibodies specific for the cancer antigen. In another specific embodiment, 0 the modified immunoglobulin, in its unmodified state, is capable of binding an antigen of an infectious disease agent or a cellular receptor for an infectious disease agent. Such immunoglobulins can be used to treat or prevent the infectious disease caused by the infectious disease agent.
For clarity of disclosure, and not by way of limitation, the detailed description of the
25 invention is divided into the subsections which follow.
5.1. MODIFIED ANTIBODIES
The modified immunoglobulins, particularly antibodies, of the invention are immunoglobulins that, at least in the unmodified state, can immunospecifically bind an ,„ antigen and have been modified to enhance their ability to elicit an anti-idiotype response. Such immunoglobulins are modified to reduce the conformational constraints on a variable region of the immunoglobulin, e.g., by removing or reducing intrachain or interchain disulfide bonds, chemical modification, or any other method known in the art. Specifically, the invention provides a first immunoglobulin molecule that comprises a variable region and that is identical, except for one or more amino acid substitutions in the variable region, to a second immunoglobulin molecule, the second immunoglobulin molecule being capable of immunospecifically binding an antigen, the amino acid substitutions being the substitution of one or more amino acid residues that do not have a sulfhydryl group at one or more positions corresponding to one or more cysteine residues that form a disulfide bond in said second immunoglobulin molecule. The invention also provides nucleic acids containing a nucleotide sequence encoding a modified immunoglobulin of the invention.
Identifying the cysteine residues that form a disulfide bond in a variable region of a particular antibody can be accomplished by any method known in the art. For example, but not by way of limitation, it is well known in the art that the cysteine residues that form 0 intrachain disulfide bonds are highly conserved among antibody classes and across species. Thus, the cysteine residues that participate in disulfide bond formation can be identified by sequence comparison with other antibody molecules in which it is known which residues form a disulfide bond.
Table 1 provides a list of the positions of disulfide bond forming cysteine residues
15 for a number of antibody molecules.
Table 1 (derived from Kabat et al, 1991, sequences of Proteins of Immunological Interest, 5th Ed., U.S. Department of Health and Human Services, Bethesda, Maryland).
Disulfide bond-forming 0 Variable domain cysteines
Species Subgroup (positions)
Human kappa light I 23,88 Human kappa light II 23,88 Human kappa light III 23,88 Human kappa light IV 23,88
ΔJ Human lambda light I 23,88 Human lambda light II 23,88 Human lambda light mi 23,88 Human lambda light IV 23,88 Human lambda light V 23,88 Human lambda light VI 23,88 Mouse kappa light I 23,88
30 Mouse kappa light II 23,88 Mouse kappa light III 23,88 Mouse kappa light IV 23,88 Mouse kappa light V 23,88 Mouse kappa light VI 23,88 Mouse kappa light VII 23,88
-._- Mouse kappa light Miscellaneous 23,88 Disulfide bond-forming
Variable domain cysteines
Species Subgroup (positions)
Mouse lambda light 23,88 Chimpanzee lambda light 23,88
Rat kappa light 23,88
Rat lambda light 23,88
Rabbit kappa light 23,88
Rabbit lambda light 23,88
Dog kappa light 23,88
Pig kappa light 23 (88) pig lambda light 23,88
Guinea pig lambda light 23 (88)
Sheep lambda light 23,88
Chicken lambda light 23,88
Turkey lambda light 23 (88)
Ratfish lambda light 23 (88)
Shark kappa light 23,88 Human heavy I 22,92
Human heavy II 22,92
Human heavy III 22,92
Mouse heavy 1 (A) 22,92
Mouse heavy 1 (B) 22,92
Mouse heavy 11 (A) 22,92
Mouse heavy 11 (B) 22,92 Mouse heavy 11 (C) 22,92
Mouse heavy III (A) 22,92
Mouse heavy 111(B) 22,92
Mouse heavy HI (C) 22,92
Mouse heavy III (D) 22,92
Mouse heavy V (A) 22,92
Mouse heavy V (B) 22,92 Mouse heavy Miscellaneous 22,92
Rat heavy 22,92
Rabbit heavy 22,92
Guinea pig heavy 22,92
Cat heavy 22 (92)
Dog heavy 22,92
Pig heavy 22 (92) Mink heavy 22 (92)
Sea lion heavy 22 (92)
Seal heavy 22 (92)
Chicken heavy 22,92
Duck heavy 22 (92)
Goose heavy 22 (92)
Pigeon heavy 22 (92) Turkey heavy 22 (92) Disulfide bond-forming
Variable domain cysteines
Species Subgroup (positions)
Caiman heavy 22, 92
Xenopus frog heavy 22,92
Elops heavy 22,92
Goldfish heavy 22,92
Ratfish heavy 22 (92)
Shark heavy 22,92
Position numbers enclosed by () indicate that the protein was not sequenced to that position, but the residue is inferred by comparison to known sequences.
Notably, for all of the antibody molecules listed in Table 1, the cysteine residues that form the intrachain disulfide bonds are the residues at positions 23 and 88 of the light chain variable domain and the residues at positions 22 and 92 of the heavy chain variable domain. The position numbers refer to the residue corresponding to that residue in the consensus sequences as defined in Kabat, (1991, Sequences of Proteins of Immunological Interest, 5th Ed., U.S. Department of Health and Human Services, Bethesda, Maryland) or as indicated in the heavy and light chain variable region sequences depicted in Figures 7 A and B, respectively ("corresponding" means as determined by aligning the particular antibody sequence with the consensus sequence or the heavy or light chain variable region sequence depicted in Figure 7A or B).
Accordingly, in one embodiment of the invention, the modified immunoglobulin molecule is an antibody in which the residues at positions 23 and/or 88 of the light chain are substituted with an amino acid residue that does not contain a sulfhydryl group and/or the residues at positions 22 and/or 92 of the heavy chain are substituted with an amino acid residue that does not contain a sulfhydryl group.
In the modified immunoglobulin of the invention, the amino acid residue that substitutes for the disulfide bond forming cysteine residue is any amino acid residue that does not contain a sulfhydryl group, e.g., alanine, arginine, asparagine, aspartate (or aspartic acid), glutamine, glutamate (or glutamic acid), glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine. In a preferred embodiment, the cysteine residue is replaced with a glycine, serine, threonine, tyrosine, asparagine, or glutamine residue, most preferably, with an alanine residue.
Additionally, the disulfide bond forming cysteine residue may be replaced by a nonclassical amino acid or chemical amino acid analog that does not contain a sulfhydryl group (for example, but not by way of limitation, using routine protein synthesis methods). Non-classical amino acids include, but are not limited, to the D-isomers of the common amino acids, cc-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, e-Ahx, -amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary). In an alternative embodiment, the disulfide bond forming residue is deleted. In specific embodiments, the substitution of the disulfide bond forming residue is in the heavy chain variable region or is in the light chain variable region or is in both the heavy chain and light chain variable regions. In other specific embodiments, one of the residues that forms a particular disulfide bond is replaced (or deleted) or, alternatively, both residues that form a particular disulfide bond may be replaced (or deleted). In other embodiments, the invention provides immunoglobulin molecules that have one or more amino acid substitutions relative to the second immunoglobulin molecule of a disulfide bond forming residue in the variable region with an amino acid residue that does not contain a sulfhydryl group and that additionally have one or more other amino acid substitutions (i.e., that are not a replacement of a disulfide bond forming residue with a residue that does not contain a sulfhydryl group).
In particular, the invention provides a first immunoglobulin molecule comprising a variable region and which is identical, except for one or more amino acid substitutions in said variable region, to a second immunoglobulin molecule, said second immunoglobulin molecule being capable of immunospecifically binding an antigen, in which at least one of said one or more amino acid substitutions are the substitution of an amino acid residue that does not have a sulfhydryl group at one or more positions corresponding to one or more cysteine residues that form a disulfide bond in said second immunoglobulin molecule.
In a preferred embodiment, the amino acid substitutions that are not the substitution of a disulfide bond forming cysteine residue with a residue that does not have a sulfhydryl group, are not stabilizing changes. Stabilizing changes are defined as those amino acid changes that increase the stability of the antibody molecule. Such stabilizing amino acid changes are those changes that substitute an amino acid that is not common at that particular position in the particular antibody molecule (e.g., as defined by the consensus sequences for a number of antibody molecules provided in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., U.S. Department of Health and Human Services, Bethesda, Maryland) with a residue that is common at that particular position, e.g., is the amino acid at that position in the consensus sequence for that antibody molecule (see PCT Publication WO 96/02574, dated February 1, 1996 by Steipe et al.).
Such other amino acid substitutions can be any amino acid substitution that does not alter the ability of the modified immunoglobulin to elicit the formation of anti-anti-idiotype antibodies, e.g., as determined, for example, as described in Section 5.5, infra. For example, such other amino acid substitutions include substitutions of functionally equivalent amino acid residues. For example, one or more amino acid residues can be substituted by another amino acid of a similar polarity which acts as a functional equivalent. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
The modified antibodies of the invention can be derived from antibodies that are capable of immunospecifically binding any antigen. In a preferred embodiment, the modified antibodies are derived from antibodies that are capable of immunospecifically binding a cancer antigen, more preferably a tumor antigen. In specific embodiments, the modified antibodies are derived from antibodies that are capable of binding polymorphic epithelial mucin antigen, human colon carcinoma-associated protein antigen, human colon carcinoma-associated carbohydrate antigen, human milk fat globule, or is an antigen of a cancer of the breast, ovary, uterus, prostate, bladder, lung, skin, colon, pancreas, gastrointestinal track, B lymphocytes or T lymphocytes or any other cancer characterized by the expression of specific antigens, e.g., those discussed in Section 5.2.1, infra. In preferred embodiments, the modified antibody is derived from Mab 31.1 (available from the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2201 under No. 12314), Mab 33.28 (under No. 12315) or Mab HMFG-1 (see PCT Publication Q WO90/05142 and PCT Publication WO92/04380).
In another specific embodiment, the modified antibodies of the invention are derived from antibodies that are capable of immunospecifically binding an antigen of an infectious disease agent or a cellular receptor for an infectious disease agent. In preferred embodiments, the antigen of the infectious disease agent is a bacterial antigen, a viral 5 antigen, or an antigen of a parasite, or any other antigen of an infectious disease agent, such as those infectious disease agents described in Section 5.2.2, infra.
The immunoglobulin molecules of the invention can be of any type, class, or subclass of immunoglobulin molecules. In a preferred embodiment, the immunoglobulin molecule is an antibody molecule, more preferably of a type selected from the group consisting of IgG, IgE, IgM, IgD and IgA, most preferably is an IgG molecule. Alternatively, the immunoglobulin molecule is a T cell receptor, a B cell receptor, a cell-surface adhesion molecule such as the co-receptors CD4, CD8, or CD 19, or an invariant domain of an MHC molecule. The modified immunoglobulin can be derived from any naturally occurring antibody, preferably a monoclonal antibody, or can be derived from a synthetic or engineered antibody. In one aspect of the invention, the modified immunoglobulin molecules are derived from an antibody in which a binding site for a member of a binding pair or a portion of an antigen is inserted into or replaces all or a portion of one of the CDRs in the variable region, for example as described in co-pending United States Patent application Serial
No. , entitled "Immunoglobulin Molecules Having A Synthetic Variable Region And
Modified Specificity", by Burch, filed November 13, 1998 (attorney docket no. 6750-016), which is incorporated by reference herein in its entirety.
In particular, the synthetic antibodies are antibodies that immunospecifically bind to a first member of a binding pair where at least one of the CDRs of the antibody contains a binding site for the first member of the binding pair, which binding site is derived from an amino acid sequence of the other member of the binding pair. In one aspect of the invention, the amino acid sequence of the binding site is not found naturally within the CDR. Additionally, at least one of the CDRs may contain a portion of an antigen, particularly an epitope.
The amino acid sequence of the binding site may be identified by any method known in the art. For example, in some instances, the sequence of a member of a binding pair has already been determined to be directly involved in binding the other member of the binding pair. In this case, such a sequence can be used to construct the CDR of a synthetic antibody that specifically recognizes the other member of the binding pair. If the amino acid sequence for the binding site in the one member of the binding pair for the other member of the binding pair is not known, it can be determined by any method known in the art, for example, but not limited to, molecular modeling methods or empirical methods, e.g., by assaying portions (e.g., peptides) of the member for binding to the other member, or by making mutations in the member and determining which mutations prevent binding. The binding pair can be any two molecules, including proteins, nucleic acids, carbohydrates, or lipids, that interact with each other, although preferably the binding partner from which the binding site is derived is a protein molecule. In preferred embodiments, the modified immunoglobulin contains a binding sequence for a cancer antigen, an infectious disease antigen, a cellular receptor for a pathogen, or a receptor or ligand that participates in a receptor-ligand binding pair.
In specific embodiments, the binding pair is a protein-protein interaction pair which is either homotypic interaction (i.e., is the interaction between two of the same proteins) or a heterotypic interaction (i.e., is the interaction between two different proteins).
10 In a specific embodiment, the first member is a member of a ligand-receptor binding pair, preferably, of a receptor-ligand binding pair in which the ligand binds to the receptor and thereby elicits a physiological response, such as intracellular signaling. By way of example, and not by way of limitation, the ligand or receptor can be a hormone, autocoid, growth factor, cytokine or neurotransmitter, or receptor for a hormone, autocoid, growth factor, cytokine, or neurotransmitter, or any receptor or ligand involved in signal transduction. (For reviews of signal transduction pathways, see, e.g., Campbell, 1997, J. Pediat. L31:S42-S44; Hamilton, 1997, J. Leukoc. Biol. 62:145-155; Soede-Bobok & Touw, 1997, J. Mol. Med. 75:470-477; Heldin, 1995, Cell 80:213-223; Kishimoto et al., 1994, Cell I l -lsTl; Miyajima et al., 1992, Annu. Rev. Immunol. 10:295-331; and Cantley et al.,
20 1991, Cell 64:281-302.). In specific embodiments, one member of the binding pair is ligand such as, but not limited to, cholecystokinin, galanin, IL-1, IL-2, IL-4, IL-5, IL-6, IL- 11, a chemokine, leptin, a protease, neuropeptide Y, neurokinin-1, neurokinin-2, neurokinin- 3, bombesin, gastrin, corticotropin releasing hormone, endothelin, melatonin, somatostatin, vasoactive intestinal peptide, epidermal growth factor, tumor necrosis factor, dopamine,
25 endothelin, or a receptor for any of these ligands. In other embodiments, one member of the binding pair is a receptor, such as, but not limited to, an opioid receptor, a glucose transporter, a glutamate receptor, an orphanin receptor, erythropoietin receptor, insulin receptor, tyrosine kinase (TK)-receptor, KIT stem cell factor receptor, nerve growth factor receptor, insulin-like growth factor receptor, granulocyte-colony stimulating factor receptor,
30 somatotropin receptor, glial-derived neurotrophic factor receptor or gp39 receptor, G-protein receptor class or B2-adrenergic receptor, or a ligand that binds any of these receptors. In another embodiment, one of the members of the binding pair is a ligand gated ion channel, such as but not limited to a calcium channel, a sodium channel, or a potassium channel. In certain embodiments, the invention provides modified immunoglobulins that
3 immunospecifically bind a receptor and are antagonists the ligand that binds that receptor, for example, but not by way of limitation, are antagonists of endorphin, enkephalin or nociceptin. In other embodiments, the invention provides synthetic modified antibodies that immunospecifically bind a receptor and are agonists of the receptor, for example, but not by way of limitation, the endorphin, enkephalin, or nociceptin receptors. In a preferred embodiment, the modified immunoglobulin does not bind the fibronectin receptor. In another preferred embodiment, the binding sequence is not Arg-Gly-Asp, is not a multimer of a binding sequence, and preferably is not a multimer of the sequence Arg-Gly-Asp. In other specific embodiments, the modified immunoglobulin has a CDR that contains a binding site for a transcription factor. In a preferred aspect, the modified immunoglobulin does not bind to a specific DNA sequence, particularly does not bind to a transcription factor binding site.
In preferred embodiments, the modified immunoglobulin has at least one CDR that contains an amino acid sequence of a binding site for a cancer antigen or a tumor antigen (e.g., as described in detail in section 5.2.1, infra.), more preferably the antigen is human colon carcinoma-associated antigen or epithelial mucin antigen. In other embodiments, at least one CDR of the modified immunoglobulin contains an amino acid sequence for a binding site for a human milk fat globule receptor. In other embodiments, the modified immunoglobulin has at least one CDR that contains an amino acid sequence of a binding site for an antigen of a tumor of the breast, ovary, uterus, prostate, bladder, lung, skin, pancreas, colon, gastrointestinal tract, B lymphocytes, or T lymphocytes.
In other preferred embodiments of the invention, at least one CDR of the modified antibody contains an amino acid sequence for a binding site for an antigen of an infectious disease agent (e.g., as described in detail in section 5.2.2, infra.), or a binding site for a cellular receptor of an infectious disease agent, preferably where the binding site is not an amino acid sequence of a Plasmodium antigen, or is not the binding site Asn-Ala-Asn-Pro or Asn-Val- Asp-Pro. In additional embodiments, the modified antibody has a CDR that contains the binding site for a bacterial or viral enzyme.
The synthetic antibody may be built upon (i.e., the binding site sequences inserted into the CDR of) the sequence of a naturally occurring or previously existing antibody or may be synthesized from known antibody consensus sequences, such as the consensus sequences for the light and heavy chain variable regions in Figures 7 A and B, or any other antibody consensus or germline (i.e., unrecombined genomic sequences) sequences (e.g., those antibody consensus and germline sequences described in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication No. 91-3242, pp 2147- 2172).
Each antibody molecule has six CDR sequences, three on the light chain and three on the heavy chain, and five of these CDRs are germline CDRs (i.e., are directly derived from the germline genomic sequence of the animal, without any recombination) and one of the CDRs is a non-germline CDR (i.e., differs in sequence from the germline genomic sequence of the animal and is generated by recombination of the germline sequences). Whether a CDR is a germline or non-germline sequence can be determined by sequencing the CDR and then comparing the sequence with known germline sequences, e.g., as listed in Kabat et al. (1991, Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication No. 91-3242, pp 2147-2172). Significant variation from the known germline sequences indicates that the CDR is a non-germline CDR. Accordingly, the CDR that contains the amino acid sequence of the binding site or antigen is a germline CDR or, alternatively, is a non-germline CDR. The binding site or antigen sequence can be inserted into any of the CDRs of the antibody, and it is within the skill in the art to insert the binding site into different CDRs of the antibody and then screen the resulting modified antibodies for the ability to bind to the particular member of the binding pair, e.g. as discussed in Section 5.5, infra, or to elicit an immune response against the antigenic site, e.g., as described in Section 5.5, infra. Thus, one can determine which CDR optimally contains the binding site or antigen. In specific embodiments, a CDR of either the heavy or light chain variable region is modified to contain the amino acid sequence of the binding site or antigen. In another specific embodiment, the modified antibody contains a variable domain in which the first, second or third CDR of the heavy variable region or the first, second or third CDR of the light chain variable region contains the amino acid sequence of the binding site or antigen. In another embodiment of the invention, more than one CDR contains the amino acid sequence of the binding site or antigen or more than one CDR each contains a different binding site for the same molecule or contains a different binding site for a different molecule. In particular, embodiments, two, three, four, five or six CDRs have been engineered to contain a binding site for the first member of the binding pair. In a preferred embodiment, one or more CDRs contain a binding site for the first member of a binding pair and one or more other CDRs contain a binding site for a molecule on the surface of an immune cell, such as, but not limited to, a T cell, B cell, NK cell, K cell, TIL cell or neutrophil. For example, a modified antibody having a binding site for a cancer antigen or an infectious disease antigen and a binding site for a molecule on the surface of an immune cell can be used to target the immune cell to a cancer cell bearing the cancer antigen or to the infectious disease agent. In specific embodiments of the invention, the binding site or antigen amino acid sequence is either inserted into the CDR without replacing any of the amino acid sequence of the CDR itself or, alternatively, the binding site or antigen amino acid sequence replaces all or a portion of the amino acid sequence of the CDR. In specific embodiments, the binding site amino acid sequence replaces 1, 2, 5, 8, 10, 15, or 20 amino acids of the CDR sequence.
The amino acid sequence of the binding site or antigen present in the CDR can be the minimal binding site necessary for the binding of the member of the binding pair or for eliciting an immune response against the antigen( which can be determined empirically by any method known in the art); alternatively, the sequence can be greater than the minimal binding site or antigen sequence necessary for the binding of the member of the binding pair or eliciting of an immune response against the antigen. In particular embodiments, the binding site or antigen amino acid sequence is at least 4 amino acids in length, or is at least 6, 8, 10, 15, or 20 amino acids in length. In other embodiments the binding site amino acid sequence is no more than 10, 15, 20, or 25 amino acids in length, or is 5-10, 5-15, 5-20, 10- 15, 10-20 or 10-25 amino acids in length.
In addition, the total length of the CDR (i.e., the combined length of the binding site sequence and the rest of the CDR sequence) should be of an appropriate number of amino acids to allow binding of the antibody to the antigen. CDRs have been observed to have a range of numbers of amino acid residues, and the observed size ranges for the CDRs (as denoted by the abbreviations indicated in figure 2) are provided in Table 2.
Table 2
CDR Number of residues
LI 10-17
L2 7
L3 7-11 HI 5-7
H2 9-12
H3 2-25
(compiled from data in Kabat and Wu, 1971, Ann. NY Acad. Sci.
190:382-93) While many CDR H3 regions are of 5-9 residue in length, certain CDR H3 regions have been observed that are much longer. In particular, a number of antiviral antibodies have heavy chain CDR H3 regions of 17-24 residues in length.
Accordingly, in specific embodiments of the invention, the CDR containing the binding site or antigen portion is within the size range provided for that particular CDR in Table 2, i.e., if it is the first CDR of the light chain, LI, the CDR is 10 to 17 amino acid residues; if it is the second CDR of the light chain, L2, the CDR is 7 amino acid residues; if it is the third CDR of the light chain, L3, the CDR is 7 to 11 amino acid residues; if it is the first CDR of the heavy chain, HI, the CDR is 5 to 7 amino acid residues; if it is the second CDR of the heavy chain, H2, the CDR is 9 to 12 amino acid residues; and if it is the third CDR of the heavy chain, H3, the CDR is 2 to 25 amino acid residues. In other specific embodiments, the CDR containing the binding site is 5-10, 5-15, 5-20, 11-15, 11-20, 11-25, or 16-25 amino acids in length. In other embodiments, the CDR containing the binding site is at least 5, 10, 15, or 20 amino acids or is no more than 10, 15, 20, 25, or 30 amino acids in length.
After constructing antibodies containing modified CDRs, the modified antibodies can be further altered and screened to select an antibody having higher affinity or specificity. Antibodies having higher affinity or specificity for the target antigen may be generated and selected by any method known in the art. For example, but not by way of limitation, the nucleic acid encoding the synthetic modified antibody can be mutagenized, either randomly, i.e., by chemical or site-directed mutagenesis, or by making particular mutations at specific positions in the nucleic acid encoding the modified antibody, and then screening the antibodies exposed from the mutated nucleic acid molecules for binding affinity for the target antigen. Screening can be accomplished by testing the expressed antibody molecules individually or by screening a library of the mutated sequences, e.g., by phage display techniques (see, e.g., U. S. Patent Nos. 5,223,409; 5,403,484; and 5,571,698, all by Ladner et al; PCT Publication WO 92/01047 by McCafferty et al. or any other phage display technique known in the art).
In specific embodiments, the invention provides a functionally active fragment, derivative or analog of the modified immunoglobulin molecules of the invention.
Functionally active means that the fragment, derivative or analog is able to elicit anti-anti- idiotype antibodies (i.e., tertiary antibodies or Ab3 antibodies) that recognize the same antigen that the antibody from which the fragment, derivative or analog is derived recognized (e.g., as determined by the methods described in Section 5.5, infra). Specifically, in a preferred embodiment, the antigenicity of the idiotype of the immunoglobulin molecule may be enhanced by deletion of framework and CDR sequences that are N-terminal to the particular CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art. Accordingly, in a preferred embodiment, the invention includes modified immunoglobulin molecules that have one disulfide bond forming cysteine residue in a variable region domain replaced with an amino acid residue that does not contain a sulfhydryl group and in which a portion of that variable domain has been deleted N-terminal to the CDR sequence that recognizes the antigen. Other embodiments of the invention include fragments of the modified antibodies of the invention such as, but not limited to, F(ab')2 fragments, which contain the variable region, the light chain constant region and the CHI domain of the heavy chain can be produced by pepsin digestion of the antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. The invention also provides heavy chain and light chain dimers of the modified antibodies of the invention, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs) (e.g., as described in U.S. Patent 4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54), or any other molecule with the same specificity as the modified antibody of the invention. Techniques have been developed for the production of "chimeric antibodies"
(Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Νeuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a constant domain from a human immunoglobulin, e.g., humanized antibodies.
In a preferred embodiment, the modified immunoglobulin of the invention is a humanized antibody, more preferably an antibody having a variable domain in which the framework regions are from a human antibody and the CDRs are from an antibody of a non- human animal, preferably a mouse (see, International Patent Application No. PCT/GB8500392 by Neuberger et al. and Celltech Limited).
CDR grafting is another method of humanizing antibodies. It involves reshaping murine antibodies in order to transfer full antigen specificity and binding affinity to a human framework (Winter et al. U.S. Patent No. 5,225,539). CDR-grafted antibodies have been successfully constructed against various antigens, for example, antibodies against IL-2 receptor as described in Queen et al., 1989 (Proc. Natl. Acad. Sci. USA 86:10029); antibodies against cell surface receptors-CAMPATH as described in Riechmann et al. (1988, Nature, 332:323); antibodies against hepatitis B in Cole et al. (1991, Proc. Natl. Acad. Sci. USA 88:2869); as well as against viral antigens-respiratory syncitial virus in Tempest et al. (1991, Bio-Technology 9:267). CDR-grafted antibodies are generated in which the CDRs of the murine monoclonal antibody are grafted into a human antibody. Following grafting, most antibodies benefit from additional amino acid changes in the framework region to maintain affinity, presumably because framework residues are necessary to maintain CDR conformation, and some framework residues have been demonstrated to be part of the antigen binding site. However, in order to preserve the framework region so as not to introduce any antigenic site, the sequence is compared with established germline sequences followed by computer modeling.
In other embodiments, the invention provides fusion proteins of the modified immunoglobulins of the invention (or functionally active fragments thereof), for example in which the modified immunoglobulin is fused via a covalent bond (e.g., a peptide bond), at either the N-terminus or the C-terminus to an amino acid sequence of another protein (or portion thereof, preferably an at least 10, 20 or 50 amino acid portion of the protein) that is not the modified immunoglobulin. Preferably the modified immunoglobulin, or fragment thereof, is covalently linked to the other protein at the N-terminus of the constant domain. In preferred embodiments, the invention provides fusion proteins in which the modified immunoglobulin is covalently linked to IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, γ-interferon, MHC derived peptide, G-CSF, TNF, porins, NK cell antigens, or cellular endocytosis receptor. The modified immunoglobulins of the invention include analogs and derivatives that are either modified, i.e, by the covalent attachment of any type of molecule as long as such covalent attachment does not prevent the modified immunoglobulin from generating an anti- idiotypic response (e.g., as determined by any of the methods described in Section 5.5, infra). For example, but not by way of limitation, the derivatives and analogs of the modified immunoglobulins include those that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the analog or derivative may contain one or more non-classical amino acids, e.g., as listed above in this Section.
Methods of producing the modified immunoglobulins, and fragments, analogs, and derivatives thereof, are described in Section 5.4, infra.
5.2. THERAPEUTIC UTILITY
The present invention provides methods of eliciting production of anti-idiotype antibodies and anti-anti-idiotype antibodies in a subject by the administration of a therapeutic (termed herein "Therapeutic"). Such Therapeutics include the modified 0 immunoglobulins of the invention, and functionally active fragments, analogs, and derivatives thereof (e.g., as described in Section 5.1, supra), and nucleic acids encoding the modified antibodies of the invention, and functionally active fragments and derivatives thereof (e.g., as described in Section 5.1, supra).
Generally, administration of products of a species origin or species reactivity that is 5 the same species as that of the subject is preferred. Thus, in a preferred embodiment, the methods of the invention use a modified antibody that is derived from a human antibody; in other embodiments, the methods of the invention use a modified antibody that is derived from a chimeric or humanized antibody.
Specifically, vaccine compositions (e.g., as described in Section 5.3, infra) 0 containing the modified antibodies of the invention are administered to the subject to elicit the production of an antibody (i.e., the anti-idiotype antibody or Ab2) that specifically recognizes the idiotype of the modified antibody, the Ab2, in turn, elicits the production anti-anti-idiotype antibodies (Ab3) that specifically recognize the idiotype of Ab2, such that these Ab3 antibodies have the same or similar binding specificity as the modified antibody.
25 The invention provides methods of administering the modified antibodies of the invention to elicit an anti-idiotype response, i.e., to generate Ab2 and Ab3 type antibodies. Alternatively, the invention provides methods of administering the modified antibodies of the invention to one subject to generate Ab2 antibodies, isolating the Ab2 antibodies, and then administering the Ab2 antibodies to a second subject to generate Ab3 type antibodies in
^π that second subject.
Accordingly, the invention provides a method of generating an anti-idiotype response in a subject comprising administering an amount of first immunoglobulin molecule (or functionally active fragment, analog, or derivative thereof) sufficient to induce an anti- idiotype response, said first immunoglobulin comprising a variable region and being identical, except for one or more amino acid substitutions in said variable region, to a second immunoglobulin molecule, said second immunoglobulin molecule being capable of immunospecifically binding an antigen, said one or more amino acid substitutions being the substitution of an amino acid residue that does not have a sulfhydryl group at one or more positions corresponding to one or more cysteine residues that form a disulfide bond in said second immunoglobulin molecule. In another embodiment, the method further provides isolating the anti-idiotype antibody that recognizes the idiotype of said second immunoglobulin molecule, and administering to a second subject the anti-idiotype antibody. In particular embodiments discussed in more detail in the subsections that follow, the modified antibodies of the invention may be used to induce an anti-idiotype response to infectious agents and diseased or abnormal cells, such as but not limited to, bacteria, parasites, fungi, viruses, tumors and cancers. The modified antibodies of the invention may be used to either treat or prevent any disease or disorder amenable to treatment or prevention by generating an anit-anti-idiotypic response to a particular antigen.
In other embodiments, the modified antibodies may be used for the treatment of autoimmune disease, such as, but not limited to rheumatoid arthritis, lupus, ulcerative colitis, or psoriasis, or for the treatment of allergies. The methods and vaccine compositions of the present invention may be used to elicit a humoral and/or a cell-mediated response against a modified immunoglobulin in a subject. In one specific embodiment, the methods and compositions of the invention elicit a humoral response in a subject. In another specific embodiment, the methods and compositions of the invention elicit a cell-mediated response in a subject. In a preferred embodiment, the methods and compositions of the invention elicit both a humoral and a cell-mediated response.
The subjects to which the present invention is applicable may be any mammalian or vertebrate species, which include, but are not limited to, cows, horses, sheep, pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, mice, rats, monkeys, rabbits, chimpanzees, and humans. In a preferred embodiment, the subject is a human. The compositions and methods of the invention can be used either to prevent a disease or disorder, or to treat a particular disease or disorder, where an anti-idiotypic response against a particular immunoglobulin molecule is effective to treat or prevent the disease or disorder.
5.2.1. TREATMENT AND PREVENTION OF CANCERS
Cancers, including, but not limited to, neoplasms, tumors, metastases, or any disease or disorder characterized by uncontrolled cell growth, can be treated or prevented by administration of a modified immunoglobulin (or functionally active fragment, derivative or analog thereof) of the invention, or a nucleic acid encoding the modified immunoglobulin, or functionally active fragment, derivative or analog thereof), which modified immunoglobulin is derived from an immimoglobulin that specifically recognizes one or more antigens associated with the cancer cells of the cancer to be treated or prevented. Whether a particular Therapeutic is effective to treat or prevent a certain type of cancer can be determined by any method known in the art, for example but not limited to, those methods described in Section 5.5, infra.
For example, but not by way of limitation, cancers associated with the following cancer antigens may be treated or prevented by administration of a modified antibody of the invention derived from an antibody that recognizes these cancer antigens: KS 1/4 pan- 0 carcinoma antigen (Perez and Walker, 1990, J. Immunol. 142:32-37; Bumal, 1988,
Hybridoma 7(4):407-415), ovarian carcinoma antigen (CA125) (Yu et al., 1991, Cancer Res. 51(2):48-475), prostatic acid phosphate (Tailor et al., 1990, Nucl. Acids Res. 18(1):4928), prostate specific antigen (Henttu and Vihko, 1989, Biochem. Biophys. Res. Comm. 10(2):903-910; Israeli et al., 1993, Cancer Res. 53:227-230), melanoma-associated antigen 15 p97 (Estin et al., 1989, J. Natl. Cancer Instit. 81(6):445-44), melanoma antigen gp75 (Vijayasardahl et al., 1990, J. Exp. Med. 171(4):1375-1380), high molecular weight melanoma antigen (HMW-MAA) (Natali et al., 1987, Cancer 59:55-3; Mittelman et al., 1990, J. Clin. Invest. 86:2136-2144)), prostate specific membrane antigen, carcinoembryonic antigen (CEA) (Foon et al., 1994, Proc. Am. Soc. Clin. Oncol. 13:294), polymorphic 20 epithelial mucin antigen, human milk fat globule antigen, Colorectal tumor-associated antigens such as: CEA, TAG-72 (Yokata et al., 1992, Cancer Res. 52:3402-3408), CO 17- 1 A (Ragnhammar et al., 1993, Int. J. Cancer 53:751-758); GICA 19-9 (Herlyn et al., 1982, J. Clin. Immunol. 2:135), CTA-1 and LEA, Burkitt's lymphoma antigen-38.13, CD19 (Ghetie et al., 1994, Blood 83:1329-1336), human B-lymphoma antigen-CD20 (Reff et al., 25 1994, Blood 83:435-445), CD33 (Sgouros et al., 1993, J. Nucl. Med. 34:422-430), melanoma specific antigens such as ganglioside GD2 (Saleh et al., 1993, J.Immunol., 151, 3390-3398), ganglioside GD3 (Shitara et al., 1993, Cancer Immunol. Immunother. 36:373- 380), ganglioside GM2 (Livingston et al., 1994, J. Clin. Oncol. 12:1036-1044), ganglioside GM3 (Hoon et al., 1993, Cancer Res. 53:5244-5250), tumor-specific transplantation type of -0 cell-surface antigen (TSTA) such as virally-induced tumor antigens including T-antigen DNA tumor viruses and envelope antigens of RNA tumor viruses, oncofetal antigen-alpha- fetoprotein such as CEA of colon, bladder tumor oncofetal antigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188), differentiation antigen such as human lung carcinoma antigen L6, L20 (Hellstrom et al., 1986, Cancer Res. 46:3917-3923), antigens of fibrosarcoma, human leukemia T cell antigen-Gp37 (Bhattacharya-Chatterjee et al., 1988, J. oflmmun. 141:1398-1403), neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR (Epidermal growth factor receptor), HER2 antigen (pi 85™^), polymorphic epithelial mucin (PEM) (Hilkens et al., 1992, Trends in Bio. Chem. Sci. 17:359), malignant human lymphocyte antigen-APO-1 (Bernhard et al., 1989, Science 245:301-304), differentiation antigen (Feizi, 1985, Nature 314:53-57) such as I antigen found in fetal erthrocytes and primary endoderm, I(Ma) found in gastric adencarcinomas, Ml 8 and M39 found in breast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl, VIM-D5,and D,56-22 found in colorectal cancer, TRA-1-85 (blood group H), C14 found in colonic adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten, Ley found in 0 embryonal carcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells , E, series (blood group B) found in pancreatic cancer, FC10.2 found in embryonal carcinoma cells, gastric adenocarcinoma, CO-514 (blood group Lea) found in adenocarcinoma, NS-10 found in adenocarcinomas, CO-43 (blood group Leb), G49, EGF receptor, (blood group ALeb/Ley) found in colonic adenocarcinoma, 19.9 found in colon cancer, gastric cancer 5 mucins, T5A7 found in myeloid cells, R24 found in melanoma, 4.2, GD3, Dl.l, OFA-1, G^,
OFA-2, GD2, Ml:22:25:8 found in embryonal carcinoma cells and SSEA-3, SSEA-4 found in 4-8-cell stage embryos. In another embodiment, the antigen is a T cell receptor derived peptide from a cutaneous T cell lymphoma (see Edelson, 1998, The Cancer Journal 4:62).
In other embodiments of the invention, the subject being treated with the modified 0 antibody of this invention may, optionally, be treated with other cancer treatments such as surgery, radiation therapy or chemotherapy. In particular, the Therapeutic of the invention used to treat or prevent cancer may be administered in conjunction with one or a combination of chemotherapeutic agents including, but not limited to, methotrexate, taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, 5 nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, an etoposide, a campathecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, etc.
5.2.1.1. MALIGNANCIES
-ft Malignancies and related disorders that can be treated or prevented by administration of the invention include but are not limited to those listed in Table 3 (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia):
35 TABLE 3 MALIGNANCIES AND RELATED DISORDERS
Leukemia acute leukemia acute lymphocytic leukemia acute myelocytic leukemia myeloblastic promyelocytic myelomonocytic monocytic , Q erythroleukemia chronic leukemia chronic myelocytic (granulocytic) leukemia chronic lymphocytic leukemia Polycythemia vera Lymphoma
Hodgkin's disease non-Hodgkin's disease 15 Multiple myeloma
Waldenstrόm's macroglobulinemia Heavy chain disease Solid tumors sarcomas and carcinomas fibrosarcoma myxosarcoma liposarcoma 20 chondrosarcoma osteogenic sarcoma chordoma angiosarcoma endotheliosarcoma lymphangiosarcoma lymphangioendotheliosarcoma 25 synovioma mesothelioma Ewing's tumor leiomyosarcoma rhabdomyosarcoma colon carcinoma pancreatic cancer breast cancer 30 ovarian cancer prostate cancer squamous cell carcinoma basal cell carcinoma adenocarcinoma sweat gland carcinoma sebaceous gland carcinoma papillary carcinoma ~ papillary adenocarcinomas cystadenocarcinoma medullary carcinoma bronchogenic carcinoma renal cell carcinoma hepatoma bile duct carcinoma choriocarcinoma seminoma embryonal carcinoma Wilms' tumor cervical cancer uterine cancer testicular tumor
, lung carcinoma small cell lung carcinoma bladder carcinoma epithelial carcinoma glioma astrocytoma medulloblastoma craniopharyngioma
15 ependymoma pinealoma hemangioblastoma acoustic neuroma oligodendroglioma meningioma melanoma neuroblastoma
^0 retinoblastoma
In specific embodiments, malignancy or dysproliferative changes (such as metaplasias and dysplasias), or hyperproliferative disorders, are treated or prevented in the 25 ovary, bladder, breast, colon, lung, skin, pancreas, or uterus. In other specific embodiments, sarcoma, melanoma, or leukemia is treated or prevented.
5.2.1.2. PREMALIGNANT CONDITIONS
The Therapeutics of the invention can also be administered to treat premalignant 30 conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders listed in Table 3. Such prophylactic or therapeutic use is indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see 35 Robbins and Angell, 197, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 8-79.) Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. As but one example, endometrial hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.
Alternatively or in addition to the presence of abnormal cell growth characterized as hyperplasia, metaplasia, or dysplasia, the presence of one or more characteristics of a transformed phenotype, or of a malignant phenotype, displayed in vivo or displayed in vitro by a cell sample from a patient, can indicate the desirability of prophylactic/therapeutic administration of the vaccine composition. As mentioned supra, such characteristics of a transformed phenotype include morphology changes, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, protease release, increased sugar transport, decreased serum requirement, expression of fetal antigens, disappearance of the 250,000 dalton cell surface protein, etc. (see also id., at pp. 84-90 for characteristics associated with a transformed or malignant phenotype).
In a specific embodiment, leukoplakia, a benign-appearing hyperplastic or dysplastic lesion of the epithelium, or Bowen's disease, a carcinoma in situ, are pre-neoplastic lesions indicative of the desirability of prophylactic intervention.
In another embodiment, fibrocystic disease (cystic hyperplasia, mammary dysplasia, particularly adenosis (benign epithelial hyperplasia) is indicative of the desirability of prophylactic intervention.
In other embodiments, a patient which exhibits one or more of the following predisposing factors for malignancy is treated by administration of an effective amount of the Therapeutic of the invention: a chromosomal translocation associated with a malignancy (e.g., the Philadelphia chromosome for chrome myelogenous leukemia, t(14;18) for follicular lymphoma, etc.), familial polyposis or Gardner's syndrome (possible forerunners of colon cancer), benign monoclonal gammopathy (a possible forerunner of multiple myeloma), and a first degree kinship with persons having a cancer or precancerous disease showing a Mendelian (genetic) inheritance pattern (e.g., familial polyposis of the colon, Gardner's syndrome, hereditary exostosis, polyendocrine adenomatosis, medullary thyroid carcinoma with amyloid production and pheochromocytoma, Peutz-Jeghers syndrome, neurofibromatosis of Von Recklinghausen, retinoblastoma, carotid body tumor, cutaneous melanocarcinoma, intraocular melanocarcinoma, xeroderma pigmentosum, ataxia telangiectasia, Chediak-Higashi syndrome, albinism, Fanconi's aplastic anemia, and Bloom's syndrome; see Robbins and Angell, 197, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 112-113) etc.)
In another specific embodiment, Therapeutic of the invention is administered to a 0 human patient to prevent progression to ovary, breast, colon, lung, pancreatic, skin, prostate, gastrointestinal, B lymphocyte, T lymphocyte or uterine cancer, melanoma or sarcoma.
5.2.2. TREATMENT OF INFECTIOUS DISEASES
The invention also provides methods of treating or preventing infectious diseases by 5 administration of a Therapeutic of the invention, in particular a modified immunoglobulin molecule (or functionally active fragment, derivative or analog thereof, or a nucleic acid encoding the modified immunoglobulin, or functionally active fragment, analog or derivative thereof) that is derived from an immunoglobulin molecule that can immunospecifically bind an antigen of the agent causing the infectious disease or a cellular 0 receptor for the infectious disease agent. As discussed in detail below, the infectious agents include, but are not limited to viruses, bacteria, fungi, protozoa, and parasites.
In specific embodiments, infectious diseases are treated or prevented by administration of a modified immunoglobulin of the invention (or functionally active fragment, derivative or analog thereof, or nucleic acid encoding the same) that is derived
25 from an immunoglobulin that specifically recognizes one of the following antigens of an infectious disease agent: influenza virus hemagglutinin (Genbank accession no. JO2132; Air, 1981, Proc. Natl. Acad. Sci. USA 78:739-743; Newton et al., 1983, Virology 128:495-501), human respiratory syncytial virus G glycoprotein (Genbank accession no. Z33429; Garcia et al., 1994, J. Virol.; Collins et al, 1984, Proc. Natl. Acad. Sci. USA
-n 81:783), core protein, matrix protein or other protein of Dengue virus (Genbank accession no. M19197; Hahn et al., 1988, Virology 12:17-180), measles virus hemagglutinin (Genbank accession no. M81899; Rota et al., 1992, Virology 188:135-142), herpes simplex virus type 2 glycoprotein gB (Genbank accession no. M14923; Bzik et al., 198, Virology 155:322-333), poliovirus I VP1 (Emini et al., 1983, Nature 304:99), envelope glycoproteins of HIV I, such as gpl20(Putney et al., 198, Science 234:1392-1395), hepatitis B surface antigen (Itoh et al., 198, Nature 308:19; Neurath et al., 198, Vaccine 4:34), diptheria toxin (Audibert et al., 1981, Nαtwre 289:543), streptococcus 24M epitope (Beachey, 1985, Adv. Exp. Med. Biol. 185:193), gonococcal pilin (Rothbard and Schoolnik, 1985, Adv. Exp. Med. Biol. 185:247), pseudorabies virus g50 (gpD), pseudorabies virus II (gpB), pseudorabies virus gill (gpC), pseudorabies virus glycoprotein H, pseudorabies virus glycoprotein E, transmissible gastroenteritis glycoprotein 195, transmissible gastroenteritis matrix protein, swine rotavirus glycoprotein 38, swine parvovirus capsid protein, Serpulina hydodysenteriae protective antigen, bovine viral diarrhea glycoprotein 55, newcastle disease virus hemagglutinin- neuraminidase, swine flu hemagglutinin, swine flu neuraminidase, foot and mouth disease virus, hog colera virus, swine influenza virus, african swine fever virus, Mycoplasma hypopneumoniae, infectious bovine rhinotracheitis virus (e.g., infectious bovine rhinotracheitis virus glycoprotein E or glycoprotein G), infectious laryngotracheitis virus (e.g., infectious laryngotracheitis virus glycoprotein G or glycoprotein I), a glycoprotein of La Crosse virus (Gonzales-Scarano et al., 1982, Virology 120:42), neonatal calf diarrhea virus (Matsuno and Inouye, 1983, Infection and Immunity 39:155), Venezuelan equine encephalomyelitis virus (Mathews and Roehrig, 1982, J. Immunol. 129:273), punta toro virus (Dalrymple et al., 1981, in Replication of Negative Strand Viruses, Bishop and Compans (eds.), Elsevier, ΝY, p. 17), murine leukemia virus (Steeves et al., 1974, J. Virol. 14:187), mouse mammary tumor virus (Massey and Schochetman, 1981, Virology 115:20), hepatitis B virus core protein and or hepatitis B virus surface antigen (see, e.g., U.K. Patent Publication No. GB 2034323 A published June 4, 1980; Ganem and Varmus, 1987, Ann. Rev. Biochem. 5:51-93; Tiollais et al., 1985, Nαtwre 317:489-495), antigen of equine influenza virus or equine herpesvirus (e.g., equine influenza virus type A/ Alaska 91 neuraminidase, equine influenza virus type A/Miami 63 neuraminidase, equine influenza virus type A/Kentucky 81 neuraminidase, equine herpesvirus type 1 glycoprotein B, equine herpesvirus type 1 glycoprotein D, antigen of bovine respiratory syncytial virus or bovine parainfluenza virus (e.g., bovine respiratory syncytial virus attachment protein (BRSV G), bovine respiratory syncytial virus fusion protein (BRSV F), bovine respiratory syncytial virus nucleocapsid protein (BRSV Ν), bovine parainfluenza virus type 3 fusion protein, and the bovine parainfluenza virus type 3 hemagglutinin neuraminidase), bovine viral diarrhea virus glycoprotein 48 or glycoprotein 53. In other specific embodiments, infectious diseases are treated or prevented by administration of a modified immunoglobulin (or functionally active fragment, derivative, or analog thereof, or nucleic acid encoding the same) that recognizes a cellular receptor for aninfectiusdisease agent, for example but not by way of limitation, such cellular receptors, along with their corresponding pathogens are listed in Table 4.
Table 4
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Viral diseases that can be treated or prevented by the methods of the present invention include, but are not limited to, those caused by hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsachie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), any picornaviridae, enteroviruses, caliciviridae, any of the Norwalk group of viruses, togaviruses (such as Dengue virus), alphaviruses, flaviviruses, coronaviruses, rabies virus, Marburg viruses, ebola viruses, parainfluenza virus, orthomyxoviruses, bunyaviruses, arenaviruses, reoviruses, rotaviruses, orbiviruses, human T cell leukemia virus type I, human T cell leukemia virus type II, simian immunodeficiency virus, lentiviruses, polyomaviruses, parvoviruses, Epstein-Barr virus, human herpesvirus-, cercopithecine herpes virus 1 (B virus), poxviruses, and encephalitis.
Bacterial diseases that can be treated or prevented by the methods of the present invention are caused by bacteria including, but not limited to, gram negative and gram positive bacteria, mycobacteria rickettsia, mycoplasma, Neisseria spp. (e.g., Neisseria mennigitidis and Neisseria gonorrhoeae), legionella, Vibrio cholerae, Streptococci, such as
Streptococcus pneumoniae, corynebacteria diphtheriae, clostridium tetani, bordetella pertussis, Haemophilus spp. (e.g., influenzae), Chlamydia spp., Enterotoxigenic Escherichia coli, Shigella spp. etc., and bacterial diseases such as Syphilis, Lyme's disease, etc.
Protozoal diseases that can be treated or prevented by the methods of the present invention are caused by protozoa including, but not limited to, plasmodia, eimeria, leishmania, kokzidioa, trypanosoma, fungi, such as Candida, etc. In specific embodiments of the invention, the Therapeutic of the invention is administered in conjunction with an appropriate antibiotic, anti-fungal, anti-viral or any other drug useful in treating or preventing the infectious disease. 5.3. GENE THERAPY Gene therapy refers to treatment or prevention of a disease performed by the administration of a nucleic acid to a subject who has a disease associated with the expression of the antigen which is recognized by the immunoglobulin molecule from which the modified immunoglobulin molecule was derived. For example, the disease or disorder may be a cancer associated with the expression of a particular cancer or tumor agent or an infectious disease associated with the expression of a particular antigen of an infectious disease agent or for which the infectious disease agent binds a particular cellular receptor. In this embodiment of the invention, the therapeutic nucleic acid encodes a sequence that 0 produces intracellularly (without a leader sequence) or intercellularly (with a leader sequence), a modified immunoglobulin.
For general reviews of the methods of gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and 5 Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, 0 John Wiley & Sons, NY).
In one aspect, the therapeutic nucleic acid comprises an expression vector that expresses the modified immunoglobulin molecule.
Delivery of the nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector or a delivery
25 complex, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
In a specific embodiment, the nucleic acid is directly administered in vivo, where it is expressed to produce the antibodies. This can be accomplished by any of numerous methods
,0 known in the art, e.g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in biopolymers (e.g., poly-β-l->4-N-acetylglucosamine polysaccharide; see U.S. Patent No. 5,635,493), encapsulation in liposomes, microparticles, or microcapsules, or by administering it in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), etc. In another embodiment, a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180 dated April 16,
10 1992 (Wu et al.); WO 92/22635 dated December 23, 1992 (Wilson et al.); WO92/20316 dated November 26, 1992 (Findeis et al.); WO93/14188 dated July 22, 1993 (Young). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
15 Alternatively, single chain antibodies, such as neutralizing antibodies, which bind to intracellular epitopes can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population by utilizing, for example, techniques such as those described in Marasco et al. (Marasco et al., 1993, Proc. Natl. Acad. Sci. USA 90:7889-
20 7893). Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion
25 in Genetics and Development 3:499-503 present a review of adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., 1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; and Mastrangeli et al., 1993, J. Clin. Invest. 91:225-
_~ 234. Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300).
The form and amount of therapeutic nucleic acid envisioned for use depends on the type of disease and the severity of its desired effect, patient state, etc., and can be determined by one skilled in the art.
35 5.3. VACCINE FORMULATIONS AND ADMINISTRATION
The invention also provides vaccine formulations containing Therapeutics of the invention, which vaccine formulations are suitable for administration to elicit a protective immune (humoral and/or cell mediated) response against certain antigens, e.g., for the treatment and prevention of diseases.
Suitable preparations of such vaccines include injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection, may also be prepared. The preparation may also be emulsified, or the polypeptides encapsulated in liposomes. The active immunogenic ingredients are often mixed with 0 excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, buffered saline, dextrose, glycerol, ethanol, sterile isotonic aqueous buffer or the like and combinations thereof. In addition, if desired, the vaccine preparation may also include minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance 5 the effectiveness of the vaccine.
Examples of adjuvants which may be effective, include, but are not limited to: aluminim hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl- nor-muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L- alanine-2-( 1 '-2'-dipalmitoyl-sn-glycero-3 -hydroxyphosphoryloxy)-ethylamine. 0 The effectiveness of an adjuvant may be determined by measuring the induction of anti-idiotype antibodies directed against the injected immunoglobulin formulated with the particular adjuvant.
The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. Oral formulation can include standard carriers 5 such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active
-« agent. Where the composition is administered by injection, an ampoule of sterile diluent can be provided so that the ingredients may be mixed prior to administration.
In a specific embodiment, the lyophilized modified immunoglobulin of the invention is provided in a first container; a second container comprises diluent consisting of an aqueous solution of 50% glycerin, 0.25% phenol, and an antiseptic (e.g., 0.005% brilliant
35 green). The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the vaccine formulations of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Composition comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
The subject to which the vaccine is administered is preferably a mammal, most preferably a human, but can also be a non-human animal including but not limited to cows, horses, sheep, pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, mice and rats.
Many methods may be used to introduce the vaccine formulations of the invention; these include but are not limited to oral, intracerebral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal routes, and via scarification (scratching through the top layers of skin, e.g., using a bifurcated needle) or any other standard routes of immunization. In a specific embodiment, scarification is employed.
The precise dose of the modified immunoglobulin molecule to be employed in the formulation will also depend on the route of administration, and the nature of the patient, and should be decided according to the judgment of the practitioner and each patient's circumstances according to standard clinical techniques. An effective immunizing amount is that amount sufficient to produce an immune response to the modified immunoglobulin molecule in the host (i.e., an anti-idiotype reaction) to which the vaccine preparation is administered. Effective doses may also be extrapolated from dose-response curves derived from animal model test systems.
5.4. METHOD OF PRODUCING THE MODIFIED IMMUNOGLOBULINS
The modified immunoglobulins of the invention can be produced by any method known in the art for the synthesis of immunoglobulins, in particular, by chemical synthesis or by recombinant expression, and is preferably produced by recombinant expression techniques. Recombinant expression of the modified immunoglobulin of the invention, or fragment, derivative or analog thereof, requires construction of a nucleic acid that encodes the modified immunoglobulin. If the nucleotide sequence of the modified immunoglobulin is known, a nucleic acid encoding the modified immunoglobulin may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the modified immunoglobulin, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR, e.g., as exemplified in Section 6, infra. Alternatively, the nucleic acid encoding the modified immunoglobulin may be generated from a nucleic acid encoding the immunoglobulin from which the modified immunoglobulin was derived. If a clone containing the nucleic acid encoding the particular immunoglobulin is not available, but the sequence of the immunoglobulin molecule is known, a nucleic acid encoding the immunoglobulin may be obtained from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by hybridization using an oligonucleotide probe specific for the particular gene sequence.
If an immunoglobulin molecule that specifically recognizes a particular antigen is not available (or a source for a cDNA library for cloning a nucleic acid encoding such an immunoglobulin is not available), immunoglobulins specific for a particular antigen may be generated by any method known in the art, for example, by immunizing an animal, such as a rabbit, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or, as 5 described by Kozbon et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, a clone encoding at least the Fab portion of the immunoglobulin can be obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246:1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (see, Q e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).
Once a nucleic acid encoding at least the variable domain of the immunoglobulin molecule is obtained, it may be introduced into any available cloning vector, and may be introduced into a vector containing the nucleotide sequence encoding the constant region of the immunoglobulin molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; U.S. Patent No. 5,122,464; and Bebbington, 1991, Methods in Enzymology 2:136-145). Vectors containing the complete light or heavy chain for co-expression with the nucleic acid to allow the expression of a complete antibody molecule are also available, see Id. Then, the nucleic acid encoding the immimoglobulin can be modified to introduce the nucleotide substitutions or deletion necessary to substitute (or delete) the one or more variable region cysteine residues participating in an intrachain disulfide bond with an amino acid residue that does not contain a sulfhydyl group, along with any other desired amino acid substitutions, deletions or insertions. Such modifications can be carried out by any method known in the art for the introduction of specific mutations or deletions in a nucleotide sequence, for example, but not limited to, chemical muagenesis, in vitro site directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551), PCR based methods, etc.
In addition, techniques developed for the production of chimeric antibodies (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81 :851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can also be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a constnat region derived from a human immunoglobulin, e.g., humanized antibodies.
Alternatively, techniques described for the production of single chain antibodies (U.S. Patent 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., 1988, Science 242:1038-1041).
Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
Once a nucleic acid encoding the modified immunoglobulin molecule of the invention has been obtained, the vector for the production of the immunoglobulin molecule may be produced by recombinant DΝA technology using techniques well known in the art. The modified immunoglobulin molecule can then be recombinantly expressed and isolated by any method known in the art, for example, using the method described in Section 6, supra, (see also Bebbington, 1991, Methods in Enzymology 2:136-145). Briefly, COS cells, or any other appropriate cultured cells, can be transiently or non-transiently transfected with the expression vector encoding the modified immunoglobulin, cultured for an appropriate period of time to permit immunoglobulin expression, and then the supernatan can be harvested from the COS cells, which supernatant contains the secreted, expressed modified immunoglobulin.
Methods which are well known to those skilled in the art can be used to construct expression vectors containing the immimoglobulin molecule coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and Ausubel et al. (eds., 1998, Current Protocols in Molecular Biology,
15 John Wiley & Sons, NY).
The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce the immunoglobulin of the invention.
The host cells used to express the recombinant antibody of the invention may be
20 either bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells, especially for the expression of whole recombinant immunoglobulin molecules. In particular, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalo virus is an effective expression system for immunoglobulins (Foecking et al., 198, Gene 45:101; Cockett et al.,
25 1990, Bio/Technology 8:2).
A variety of host-expression vector systems may be utilized to express the modified immunoglobulin molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the
30 appropriate nucleotide coding sequences, express the immunoglobulin molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing immunoglobulin coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing
35 immunoglobulin coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the immunoglobulin coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing immunoglobulin coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adeno virus late promoter; the vaccinia virus 7.5K promoter).
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the immunoglobulin molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an immunoglobulin molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983 , EMBO J. 2 : 1791 ), in which the immunoglobulin coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGΕX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S- transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGΕX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety. In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The immunoglobulin coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the immunoglobulin coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region Εl or Ε3) will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81 :355-359). Specific initiation signals may also be required for efficient translation of inserted immunoglobulin coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:51-544).
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have charac- teristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the immunoglobulin molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the immunoglobulin molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the immunoglobulin molecule. A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11 :223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk", hgprt" or aprt" cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance
10 to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.),
15 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1.
Alternatively, any fusion protein may be readily purified by utilizing an antibody
20 specific for the fusion protein being expressed. For example, a system described by
Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of
25 six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni2+,nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.
The expression levels of the immunoglobulin molecule can be increased by vector
-„ amplification (for a review, see Bebbington and Hentschel, the Use of Vectors Based on Gene Amplification for the Expression of Cloned Genes in Mammalian Cells in DNA Cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing immimoglobulin is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the
35 amplified region is associated with the immunoglobulin gene, production of the immunoglobulin will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).
The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nαtwre 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA
10 77:2197). The coding sequences for the heavy and light chains may comprise cDΝA or genomic DΝA.
Once the modified immunoglobulin molecule of the invention has been recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange,
15 affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
5.5. DEMONSTRATION OF THERAPEUTIC UTILITY
20 The modified antibodies of the invention can be screened or assayed in a variety of ways for efficacy in treating or preventing a particular disease .
First, the immunopotency of a vaccine formulation containing the modified antibody of the invention can be determined by monitoring the anti-idiotypic response of test animals following immunization with the vaccine. Generation of a humoral response may be taken
25 as an indication of a generalized immune response, other components of which, particularly cell-mediated immunity, may be important for protection against a disease. Test animals may include mice, rabbits, chimpanzees and eventually human subjects. A vaccine made in this invention can be made to infect chimpanzees experimentally. However, since chimpanzees are a protected species, the antibody response to a vaccine of the invention can
-n first be studied in a number of smaller, less expensive animals, with the goal of finding one or two best candidate immunoglobulin molecules or best combinations of immunoglobulin molecules to use in chimpanzee efficacy studies.
The immune response of the test subjects can be analyzed by various approaches such as the reactivity of the resultant immune serum to antibodies, as assayed by known techniques, e.g., enzyme linked immunosorbent assay (ELISA), immunoblots, radioimmunoprecipitations, etc.; or protection from infection and/or attenuation of disease symptoms in immunized hosts.
As one example of suitable animal testing, the vaccine composition of the invention may be tested in rabbits for the ability to induce an anti-idiotypic response to the modified immunoglobulin molecule. For example, male specific-pathogen-free (SPF) young adult New Zealand White rabbits may be used. The test group of rabbits each receives an effective amount of the vaccine. A control group of rabbits receives an injection in 1 mM Tris-HCl pH 9.0 of the vaccine containing a naturally occurring antibody. Blood samples may be drawn from the rabbits every one or two weeks, and serum analyzed for anti- idiotypic antibodies to the modified immunoglobulin molecule and anti-anti-idiotypic antibodies specific for the antigen against which the modified antibody was directed using, e.g., a radioimmunoassay (Abbott Laboratories). The presence of anti-idiotypic antibodies may be assayed using an ELISA. Because rabbits may give a variable response due to their outbred nature, it may also be useful to test the vaccines in mice. In addition, a modified antibody of the invention may be tested by first administering the modified antibody to a test subject, either animal or human, and then isolating the anti- anti-idiotypic antibodies (i.e., the Ab3 antibodies) generated as part of the anti-idiotype response to the injected modified antibody. The isolated Ab3 may then be tested for the ability to bind the particular antigen (e.g., a tumor antigen, antigen of an infectious disease agent by any immunoassays known in the art, for example, but not limited to, radioimmunoassays, ELISA, "sandwich" immunoassay, gel diffusion precipitin reactions, immunodiffusion assays, western blots, precipitation reactions, agglytination assays, complement fixation assays, immunofluorescence assays, protein A assays, immunoelectrophoresis assays, etc. In one aspect where the modified antibody is directed against a cancer or tumor antigen, the efficacy of the isolated Ab3 for treating cancer, a tumor, or other neoplastic disease is screened by culturing cancer or tumor cells from a patient, contacting the cells with the Ab3 antibody to be tested, and comparing the proliferation or survival of the contacted cells with the proliferation or survival of cells not so contacted with the Ab3 antibody, wherein a lower level of proliferation or survival of the contacted cells indicates that the Ab3 antibody (which was elicited by immunization with the modified antibody of the invention) is effective to treat the cancer in the patient. Many assays standard in the art can be used to assess such survival and/or growth; for example, cell proliferation can be assayed by measuring 3H-thymidine incorporation, by direct cell count, by detecting changes in transcriptional activity of known genes such as proto-oncogenes (e.g.,fos, myc) or cell cycle markers; cell viability can be assessed by trypan blue staining, differentiation can be assessed visually based on changes in morphology, etc. If the modified antibody is directed against an antigen of an infectious disease agent, the isolated Ab3 may be tested for activity in any in vitro test for activity against the particular pathogen. Additionally, the modified antibodies of the invention may also be tested directly in vivo. To monitor the effect of a Therapeutic of the invention, the level of the antigen against which the modified antibody is directed is measured at suitable time intervals before, during, or after therapy. Any change or absence of change in the amount of the antigen can be identified and correlated with the effect of the treatment on the subject. In particular, in the case of cancer therapeutics, the serum levels of an antigen bears a direct relationship with severity of a cancer, such as breast cancer, and poor prognosis. Generally, a decrease in the level of antigen is associated with efficacious treatment.
When the modified antibody is directed against an antigen of an infectious disease agent, the efficacy of the modified antibody can be monitored by measuring the level of the antigen of the infectious disease agent at suitable times before, during and after therapy, where a decrease in the levels of the antigen indicates that the modified antibody is efficacious.
In a preferred aspect, the approach that can be taken is to determine the levels of antigen at different time points and to compare these values with a baseline level. The baseline level can be either the level of the marker present in normal, disease free individuals; and/or the levels present prior to treatment, or during remission of disease, or during periods of stability. These levels can then be correlated with the disease course or treatment outcome.
The levels of antigen can be determined by any method well known in the art. For example, a certain antigen can be quantitated by known immunodiagnostic methods such as western blotting immunoprecipitation using any antibody against a certain antigen.
The strength of the immune response in vivo to the modified immunogluobulin may be determined by any method known in the art, for example, but not limited to, delayed hypersensitivity skin tests and assays of the activity of cytolytic T-lymphocytes in vitro. Delayed hypersensitivity skin tests are of great value in the testing of the overall immunocompetence and cellular immunity to an antigen. Proper technique of skin testing requires that the antigens be stored sterile at 4°C, protected from light and reconstituted shortly before use. A 25- or 27-gauge need ensures intradermal, rather than subcutaneous, administration of antigen. Twenty-four and 48 hours after intradermal administration of the antigen, the largest dimensions of both erythema and induration are measured with a ruler. Hypoactivity to any given antigen or group of antigens is confirmed by testing with higher concentrations of antigen or, in ambiguous circumstances, by a repeat test with an intermediate test.
To test the activity of cytolytic T-lymphocytes, T-lymphocytes isolated from the immunized subject, e.g., by the Ficoll-Hypaque centrifugation gradient technique, are restimulated with cells bearing the antigen against which the modified antibody was directed in 3 ml RPMI medium containing 10% fetal calf serum. In some experiments, 33% secondary mixed lymphocyte culture supernatant or IL-2 is included in the culture medium as a source of T cell growth factors. In order to measure the primary response of cytolytic T- lymphocytes after immunization, the isolated T cells are cultured with or without the cells bearing the antigen. After six days, the cultures are tested for cytotoxity in a 4 hour 51Cr- release assay. The spontaneous 51Cr-release of the targets should reach a level less than 20% if immunization was effective (Heike et al., J. Immunotherapy 15:15-174).
In other aspects, the modified immunoglobulins may be tested for efficacy by monitoring the subject for improvement or recovery from the particular disease or condition associated with the antigen against which the modified antibody is directed. When the modified antibody is directed against a tumor or a cancer antigen, the progress of the particular tumor or cancer may be followed by any diagnostic or screening method known for monitoring cancer or a tumor. For example, but not by way of limitation, the cancer or tumor progress may be monitored by assaying the levels of the particular cancer or tumor antigen (or another antigen associated with the particular cancer or tumor) either in the serum of the subject or by injecting a labeled antibody specific for the antigen. Additionally, other imaging techniques, such as computed tomographic (CT) scan or sonograms, or any other imaging method, may be used to monitor the progression of the cancer or tumor. Biopsies may also be performed. Before carrying out such trials in humans, the tests for efficacy of the modified immunoglobulins can be performed in animal models of the particular cancer or tumor.
In the case of infectious diseases, the efficacy of the modified antibody can be assayed by administering the modified antibody to a subject (either a human subject or an animal model for the disease) and then monitoring either the levels of the particular infectious disease agent or symptoms of the particular infectious disease. The levels of the infectious disease agent may be determined by any method known in the art for assaying the levels of an infectious disease agent, e.g., the viral titer, in the case of a virus, or bacterial levels (for example, by culturing of a sample from the patient), etc. The levels of the infectious disease agent may also be determined by measuring the levels of the antigen against which the modified immunoglobulin was directed or another antigen of the infectious disease agent. A decrease in the levels of the infectious disease agent or an amelioration of the symptoms of the infectious disease indicates that the modified antibody is effective.
6. EXAMPLE: ANTI-IDIOTYPIC VACCINE INDUCER FOR COLON
CANCER
This example describes the construction of a modified antibody derived from the monoclonal antibody MAb31.1 (hybridoma secreting Mab31.1 is available from the American Type Tissue Collection as accession No. HB12314). Mab31.1 recognizes an antigen expressed by human colon carcinomas. The modified antibody of the invention, based on Mab31.1, was engineered to have variable region cysteine residues of both the heavy and light chain variable regions substituted with alanine. Therefore, the resulting modified antibody, was missing intrachain disulfide bonds in either the heavy and light chain variable regions.
6.1. CONSTRUCTION OF A MODIFIED ANTIBODY
The strategy for construction of the modified antibody was to construct two engineered genes that encoded the heavy and light chain variable regions wherein specific cysteine residues, known to be important in intra-chain disulfide bonding , were altered to alanine. Alanine residues were substituted for the cysteine residues at positions 22 and 92 of the heavy chain variable region of the antibody derived from Mab31.1 or at positions 23 and 88 of the Mab31.1 light chain variable region of the antibody derived from Mab31.1. In order to construct these engineered genes, groups of olionucleotides were assembled (as discussed below) and inserted into an appropriate vector providing constant regions. In order to construct variable region genes encoding CDRs lacking intrachain disulfide bonds, the following strategy was performed.
First, single strand oligonucleotides were annealed to create cohesive double stranded DNA fragments (as diagramed in Figure 10, Step 1). Specifically, oligonucleotides of about 80 bases in length corresponding to the sequences of interest with 20 base overlapping regions were synthesized using automated techniques of GenoSys Biotech Inc. The specific sequences of each of these oligonucleotides. The specific sequences of these oligonucleotides are presented in Figures 9 A and 9B. Figure 9A list the group often oligos used in engineering a heavy chain variable region gene called 2CAVHCOL1. 2CAVHCOL1 lacked 2 cysteine residues as compared to the consensus heavy chain variable gene. Figure 9B lists the group of 12 oligos used in the engineering of the light chain variable region gene called 2CAVLCOL1. 2CAVLCOL1 lacked two cysteine residues as compared to the consensus light chain variable region gene. In order to combine the oligos into the desired gene, groups of 10 or 12 oligos were combined as described below and as presented in Figure 10, where the identities of oligos 1 to 10 indicated in Figure 10 are provided in Table 5. Prior to combining, each oligonucleotide was 5' phosphorylated as follows: 25 μl of each oligo was incubated for 1 hour in the presence of T4 polynucleotide kinase and 50mM ATP at 37 °C. The reactions were stopped by heating for 5 minutes at
10 70 °C followed by ethanol precipitation. Once phosphorylated, complementary oligonucleotides (oligo 1 + oligo 10, oligo 2 + oligo 9, oligo 3 + oligo 8, oligo 4 + oligo 7, oligo 5 + oligo 6), as shown in Figure 10, were then mixed in sterile microcentrifuge tubes and annealed by heating the tube in a water bath at 65 °C for 5 minutes followed by cooling at room temperature for 30 minutes. Annealing resulted in short double strand DNA
15 fragments with cohesive ends.
Next, the cohesive double stand DNA fragments were ligated into longer strands (Figure 10, Steps 2-4), until the engineered variable region gene was assembled. Specifically, cohesive double strand DNA fragments were ligated in the presence of T4 DNA ligase and lOmM ATP for 2 hours in a water bath maintained at 16°C. Annealed
20 oligo 1/10 was mixed with annealed oligo 2/9, and annealed oligo 3/8 was mixed with annealed oligo 4/7. The resulting oligos were labeled oligo 1/10/2/9 and oligo 3/8/4/7. Next, oligo 3/8/4/7 was ligated to oligo 5/6. The resulting oligo 3/8/4/7/5/6 was then ligated to oligo 1/10/2/9 resulted in a full length variable region gene.
Alternatively, when groups of 12 oligos were used, the order of addition was: 1+12 =
25 1/12, 2+11=2/11, 3+10=3/10, 4+9=4/9, 5+8=5/8, 6+7=6/7, 1/12+2/11=1/12/2/11, 3/10+4/9=3/10/4/9, 5/8+6/7=5/8/6/7, 1/12/2/11+3/10/4/9 = 1/12/2/11/3/10/4/9, 1/12/2/11/3/10/4/9+5/8/6/7= full length variable region gene. The names of oligonucleotides used in construction of the engineered genes are listed in Table 5. The modified heavy chain variable region gene was denoted as 2CAVHCOL1. The modified
30 light chain variable region gene was denoted as 2CAVLCOL1.
The resulting modified variable region genes were then purified by gel electrophoresis. To remove unligated excess of oligos and other incomplete DNA fragments, ligated product was run on 1% low melting agarose gel at constant 110 V for 2 hours. The major band containing full length DNA product was cut out and placed in a
35 sterile 1.5 ml centrifuge tube. To release the DNA from the agarose, the gel slice was digested with f3-Agrase I at 40°C for 3 hours. The DNA was recovered by precipitation with 0.3 M NaOAc and isopropanol at — 20 °C for 1 hour followed by centrifugation at 12,000 rpm for 15 minutes. The purified DNA pellet was resuspended in 50 μl of TE buffer, pH 8.0. The engineered variable region gene was then amplified by PCR. Specifically, 100 ng of the engineered variable region gene was mixed with 25mM dNTPs, 200 ng of primers and 5 U of high fidelity thermostable Pfu DNA polymerase in buffer. Resulting PCR product was analyzed on 1% agarose gel.
Each purified DNA corresponding to the engineered variable region gene was subsequently inserted into the pUC19 bacterial vector. pUC19, is a 2686 base pair, a high copy number E. coli plasmid vector containing a 54 base pair polylinker cloning site in lacZ and an Amp selection marker. In order to prepare the vector for insertion of the engineered variable region gene, lOμg of pUC19 was linearized with Hinc II (50 U) for 3 hours at 37°C resulting in a vector with blunt end sequence 5' GTC. To prevent self re-ligation, linear vector DNA was dephosphorylated with 25 U of calf intestine alkaline phosphatase (CIP) for 1 hour at 37 °C. In order to insert the engineered variable region gene into the pUC19 vector, approximately 0.5 μg of dephosphorylated linear vector DNA was mixed with 3 μg of phosphorylated variable region gene in the presence of T4 DNA ligase (1000 U), and incubated at 16 °C for 12 hours.
The bacterial vector containing the engineered variable region gene was then used to transform bacterial cells. Specifically, freshly prepared competent DH5-α cells, 50 μl, were mixed with 1 μg of pUC19 containing the engineered variable region gene and transferred to an electroporation cuvette (0.2 cm gap; Bio-Rad). Each cuvette was pulsed at 2.5 kV/200 ohm/25 μF in an electroporator (Bio-Rad Gene Pulser). Immediately thereafter, 1 ml of SOC media was added to each cuvette and cells were allowed to recover for 1 hour at 37°C in centrifuge tubes. An aliquot of cells from each transformation was removed, diluted 1:100, then 100 μl plated onto LB plates containing ampicillin (Amp 40 μg/ml). The plates were incubated at 37 °C overnight due to the presence of the Amp marker. Only transformants containing pUC19 vector grew on LB/ Amp plates.
A single transformant colony was picked and grown overnight in a 3 ml LB/Amp sterile glass tube with constant shaking at 37°C. The plasmid DNA was isolated using Easy Prep columns (Pharmacia Biotech.) and suspended in 100 μl of TE buffer, pH 7.5. To confirm the presence of gene insert in pUC19, 25 μl of plasmid DNA from each colony was digested with a restriction endonuclease for 1 hour at 37 °C, and was analyzed on a 1% agarose gel. By this method plasmid DNA containing gene insert was resistant to enzyme cleavage due to loss of restriction site ( 5'..GTCGAC. 3') and migrated as closed circular (CC) DNA, while those plasmids without insert were cleaved and migrated as linear (L) double strand DNA fragment on gel.
In order to confirm correct gene sequences of the engineered variable region genes and to eliminate the possibility of unwanted mutations generated during the construction procedure, DNA sequencing was performed using M13/pUC reverse primer
(5'AACAGCTATGACCATG 3') for the clones as well as PCR gene products using 5' end 20 base primer ( 5' GAATT CATGGCTTG GGTGTG 3') on automated ABI 377 DNA Sequencer. All clones were confirmed to contain correct sequences.
Table 5. Construction of gene encoding modified antibodies containing CDRs from Mab 31.1
Oligo 1 Oligo 2 Oligo 3 Oligo 4 Oligo 5 Oligo Oligo 7 Oligo 8 Oligo 9 Oligo 10
2CAVHC VHC1 VHC2 VHC3 VHC4 VHC5 VHC VHC7 VHC8 VHC9 VHC10
OLI
2CAVLC VLC1 VLC2 VLC3 VLC4 VLCS VLC VLC7 VLC8 VLC9 VLC10 5 O I
6.3. INSERTION OF THE ENGINEERED VARIABLE REGION GENE INTO A MAMMALIAN EXPRESSION VECTOR
A complete antibody light chain has both a variable region and a constant region. A Q complete antibody heavy chain contains a variable region, a constant region, and a hinge region. A modified variable region genes 2CAVHCOL1 or 2CAVLCOL1 were inserted into vectors containing appropriate constant regions. Engineered variable region genes lacking cysteine residues in the light chain, were inserted into the pMRROlO.l vector Figure 6 A. The pMRROlO.l vector contained a human kappa light chain constant region. f Insertion of the engineered light chain variable region into this vector gave a complete light chain sequence. Alternatively, the engineered variable region gene lacking cysteine residues in the heavy chain, were inserted into the pGAMMAl vector Figure 6B. The pGAMMAl vector contained human and IgGl constant region and hinge region sequences. Insertion of the engineered heavy chain variable region gene into this vector gave a complete heavy Q chain sequence.
In order to engineer a mammalian vector comprising both heavy chain and light chain genes, the complete light chain sequence and complete heavy chain sequence were inserted into mammalian expression vector pNEPuDGV as shown in Figure 6C (Bebbington, C.R., 1991, In METHODS: A Companion to Methods in Enzymology, vol. 2, 5 pp. 136-145). The resulting vector encoding both light chain and the heavy chain of the modified antibody.
6.4. EXPRESSION OF SYNTHETIC MODIFIED ANTIBODIES IN MAMMALIAN CELLS
To examine the production of assembled antibodies the mammalian expression vector was transfected into COS cells. COS cells (an African green monkey kidney cell line, CV-1, transformed with an origin-defective SV40 virus) were used for short-term transient expression of the synthetic antibodies because of their capacity to replicate circular plasmids containing an SV40 origin of replication to very high copy number. The antibody expression vector was transferred to C0S7 cells (obtained from the American Type Culture Collection). The transfected cells were grown in Dulbecco's modified Eagle's Medium and transfected with the expression vectors using calcium precipitation (Sullivan et al., FEBS Lett. 285:120-123, 1991). The transfected cells were cultured for 72 hours after which supernatants were collected. Supernatants from transfected COS cells were assayed using ELISA method for assembled IgG. ELISA involves capture of the samples and standards onto a 96-well plate coated with an anti-human IgG Fc. Bound assembled IgG was detected with an anti-human Kappa chain linked to horse radish peroxidase (HRP) and the substrate tetramethylbenzidine (TMB). Color development was proportional to the amount of assembled antibody present in the sample.
6.5. MODIFIED ANTIBODY IMMUNOSPECIFICALLY BINDS TO HUMAN COLON CARCINOMA CELLS AND ANTIGENS PRODUCED BY THESE CELLS
The modified antibody was expressed and isolated as indicated in Section 6.4, supra. The binding capacity and specificity were then assayed using LS-174T cells WiDR cells (a human colon cancer cell line) and an antigen derived from these cells.
In order to examine the binding potency as well as specificity of MA31.1 binding, a dot blot analysis was performed (see Figure 11). Membrane preparations from LS-174T cells was applied to a nitrocellulose membrane using a Bio-Blot apparatus (Bio-Rad). The wells were blocked for non-specific binding using skim milk. Biotinylated antibody derived from Mab31.1 was incubated in all wells. Unlabelled antibody at concentrations of 0.003 to 20 nM was then applied to the nitrocellulose membrane and allowed to incubate. Unbound antibody was removed from the membrane by washing and a second antibody, alkaline phosphatase labeled antihuman IgG, was added. Finally, an alkaline phosphatase substrate was added which generated a dark purple precipitate, indicating the presence of bound labeled antibody. Figure 11 provides the results of the dot blot analysis. The figure demonstrated that the labeled antibody bound to the LS-174T cells. Additionally, the unlabeled antibody competed with biotinylated antibody binding, indicating specificity of binding of the antibody derived from Mab31.1 to tumor cell antigens.
6.6. ANTI-IDIOTYPE RESPONSE
The effect on binding of modified antibody to LS-174T cells was examined in a competition binding assay. LS-174T cells are human colon carcinoma cells which express antigen recognized by the Mab31.1 antibody. Peptides containing the sequence of one of the CDRs of the Mab31.1 antibody were generated. These peptides, the modified antibody and the control antibody derived from Mab31.1 were administered to mice in order to generate antisera against the CDR regions of Mab31.1 and the antibodies. Blood samples from mice were drawn two weeks and four weeks following injection. Antisera from the immuized mice were used in binding competition assays presented in Figures 12A and B. Antisera and biotinylated antibodies were assayed for their ability to bind LS- 174T cells. As demonstrated in Figure 12A and B, antisera raised to the CDR3 and CDR4 peptides dramatically competed for control antibody (antibody derived from Mab31.1) binding to LS-174T cells. Additionally, antisera raised against CDR1 and CDR2 also significantly competed for control antibody binding to LS-174T cells. Additionally, antisera from nice injected with the 2CAVHCOL1 and 2CAVLCOL1 antibodies (i.e., the modified antibodies having the cysteine to alanine change in the variable region) competed for binding with the biotinylated antibody derived from Mab31.1 better than antiserum from mice injected with the antibody derived from Mab31.1 (Figure 12B). This result indicates that administration of the antibodies having the cysteine to alanine change in the variable region elicit an anti-idiotype antibodies that recognize the colon carcinoma cell antigen better than antibodies with variable region intra-chain disulfide bonds.
Table 6. Biotin-Labeled Peptides Derived from CDR Sequences of Mab 31.1
Peptide ID Sequence
COL311 LI biotin-N-Thr-Ala-Lys-Ala-Ser-Gln-Ser-Val-Ser-Asn-Asp-Val-Ala
COL311 L2 biotin-N-Ile-Tyr-Tyr-Ala-Ser-Asn-Arg-Tyr-Thr
COL311 L3 biotin-N-Phe-Ala-Gln-Gln-Asp-Tyr-Ser-Ser-Pro-Leu-Thr
COL311 H 1 biotin-N-Phe-Thr- Asn-Tyr-Gly-Met- Asn
COL311 H2 biotin-N-Ala-Gly-T -Ile-Asn-Thr-Tyr-Thr-Gly-Glu-Pro-Thr-Tyr-Ala-Asp-
Asp-Phe-Lys-Gly COL311 H3 biotin-N-Ala-Arg-Ala-Tyr-Tyr-Gly-Lys-Tyr-Phe-Asp-Tyr
EXAMPLE 7: PRODUCTION OF A SYNTHETIC MODIFIED ANTIBODY CONTAINING HMFG-1 SEQUENCE
Antiidiotype antibodies were constructed which immunospecifically bound to the HMFG-1 antibody. HMFG-1 was an antibody to known to bind polymorphic epithelial mucin (PEM) (Stewart et al., 1990, J Clin Oncol 8:1941-50; Kosmas et al., 1994, Cancer 73:3000-3010). The antigenic determinant of PEM with the sequence ProAspThrArgPro was inserted into the variable chain region by methods of the invention. This short sequence is a highly immunogenic region of human polymorphic epithelial mucin (Gendler et al.,
1988, J. Biol. Chem. 263:12820-12823). Residues 27A-27E (SerLeuLeuTyrSer) of HMFG-
1 (Table 6) were replaced with ProAspThrArgPro using the oligonucleotide method described in section 6 supra, also in Figure 10. Antiidiotype synthetic HMFG-1 antibodies were produced which immunospecifically bound to HMFG-1, using the known sequences for the variable regions of the light and heavy chains of the HMFG-1. The oligos were added in the order 1 + 8 = 1/8, 2 + 7 = 2/7, 3 + 6 = 3/6, 4 + 5 = 4/5, 1/8 + 2/7 = 1/8/2/7, 3/6
+ 4/5 = 3/6/4/5, 1/8/2/7 + 3/6/4/5 = 1/8/2/7/3/6/4/5. Table 7 shows sequence comparison between HMFG-1 and various consensus CDR sequences. Information concerning HMFG-
1 and related monoclonal antibodies is set forth in WO 09/05142 (Imperial Cancer Research Technology, Ltd.) and humanized HMFG-1 is set forth in WO 92/04380 (Unilever).
Polymerase chain reaction (PCR) were used to amplify the assembled sequence as shown in Figure 13. The engineered variable regions gene constructed to contain nucleotide sequence encoding HMFG-1 is shown in Figure 13. The engineered variable region gene was inserted into appropriate vectors for antibody production, such as the pNEPuDGV vector, as described in Section 6, supra. Other methods for constructing engineered genes may be used, including but not limited to those methods described by Jayaraman et al., 1989, Nucleic Acids Res. 17:4403; Sandhu et al., 1992, BioTechniques 12:14; Barnett and Erfie, 1990, H. Nucleic Acids Res 18:3094; Ciccarelli et al., 1991, Nucleic Acids Res 19:6007; Michaels et al., 1992, BioTechniques 12:45, incorporated by reference herein.
Table 7. Sequence comparison between HMFG-1 antibody and various consensus CDR sequences
VHl CDRl Sequences
Residue 31 32 33 34 35 35A 35B
Human I Ser Tyr Ala He Ser
Human II Ser Tyr Ser/Tyr Trp Ser Trp Asn
Human HI Ser Tyr Ala Met Ser
Mouse IA Ser Gly Tyr Trp Asn Asn Ser
Mouse IB Ser Tyr Gly Val His Val Ser
Mouse IIA Asp/Ser Tyr Tyr Met Asn Asn
Mouse IIB Ser Tyr Trp Met His
I n Mouse IIC Asp/Ser Thr Tyr Met His
I Mouse IIIA Asp/Ser Phe/Tyr Tyr Met Glu
Mouse IIIB Arg Tyr Trp Met Ser
Mouse IIIC Arn Tyr Trp Met Asn
Mouse HID Ser Tyr Ala Met Ser
Mouse VA Ser Tyr Gly He Asn
Mouse VB Ser Tyr Gly Leu Tyr
HMFG-1 Ala Tyr Trp He Glu
VHl CDR2 Sequences
Residue 50 51 52 52A 52B 52C 53 54 55 56 57 58 59 60 61 62 63 64 65
Human I Trp He Asn Pro Tyr Gly Asn Gly Asp Thr Asn Tyr Ala Gin Lys Phe Gin Gly
Human II Arg He Tyr Tyr Arg Ala Tyr Ser Gly Ser Thr Asp/ Tyr Asn Pro Ser Leu Lys Ser
Asn
Human III Val He Ser Gly Lys Thr Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly
Mouse IA Tyr He Ser + Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser
Mouse IB Val He Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met Ser
Mouse IIA Asp He Asn Pro Gly Asn Gly Gly Thr Ser Tyr Asn Gin Lys Phe Lys Gly
Mouse IIB Arg He Asp Pro Asn Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys Ser
Mouse IIC Arg He Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gin Gly
Mouse IIIA Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu Tyr Ser Ala Ser Val Lys Gly
Mouse MB Glu He Asn Pro Lys Ala Asp Ser Ser Thr He Aan Tyr Thr Pro Ser Leu Lys Asp
Mouse IIIC Glu He Arg Leu Lys Ser Asp Asn Tyr Ala Thr His Tyr Ala Glu Ser Val Lys Gly
Mouse HID Thr He Ser Ser Lys Ser Gly Gly Gly Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly cn CO Mouse VA Tyr He Asn Pro Gly Asn Gly Tyr Thr Lys Tyr Asn Glu Lys Phe Lys Gly
Mouse VB Tyr He Ser Ser Ser Ser Ala Tyr Pro Asn Tyr Ala Gin Lys Phe Gin Gly
HMFG-1 Glu He Leu Pro Gly Ser Asn Asn Ser Arg Tyr Asn Glu Lys Phe Lys Gly
VHl CDR3 sequences
Residue 95 96 97 98 99 100 100A 100B 100C 100D 100E 100F 100G 100H 1001 100J 100K 101 102
Human I Ala Pro Gly Tyr Gly Ser Gly Gly Gly Cys Tyr Arg Gly Asp Tyr + Phe Asp Tyr
Human II Glu Leu Pro Gly Gly Tyr + Gly Asp Asp Tyr Tyr Tyr + + Gly Phe Asp Val
Human III + Arg + + + Ser Leu Ser Gly + Tyr Tyr Tyr Tyr His Tyr Phe Asp Tyr
Mouse IA Gly Gly Tyr Gly Tyr Gly Tyr Tyr Tyr Tyr Asp + Tyr Tyr Tyr Tyr Phe Asp Tyr
Mouse IB Asp Arg Gly Arg Tyr Tyr Tyr + Ser Gly + + + Tyr Tyr Ala Met Asp Tyr
Mouse IIA Gly + Tyr Tyr Ser Ser Ser Tyr Met + Ala + + Tyr Tyr Ala Phe Asp Tyr
Residue 95 96 97 98 99 100 100A 100B 100C 100D 100E 100F 100G 100H 1001 100J 100K 101 102
Mouse IIB Tyr Tyr Tyr Gly Gly Ser Ser + + Val Tyr + Tyr Tφ Tyr Phe Asp Tyr
Mouse IIC Gly Tyr Tyr Tyr Tyr Asp Ser + Val Gly Tyr Tyr Ala Met Asp Tyr
Mouse MA Asp Tyr Tyr Tyr Gly Ser Ser Tyr Tyr Glu Gly Pro Val Tyr Tφ Tyr Phe Asp Val
Mouse MB Leu Gly Gly Tyr Gly Tyr Phe Gly Ser Ser Tyr Tyr Ala Met Asp Tyr
Figure imgf000061_0001
Mouse IIIC Gly Gly Tyr Gly Gly + Arg Arg Ser + Tφ Phe Ala Tyr
Mouse HID Gly Gly Tyr Tyr Tyr Leu + Gly Ser Ala Pro Phe Asp Tyr Ala Met Asp Tyr
Mouse VA Ser Asn Tyr Tyr Gly Gly Ser Tyr Tyr Tyr + Phe Ala Tyr Tyr Tyr Phe Asp Tyr
Mouse VB Arg Val He Ser Arg Tyr Phe Asp Gly
HMFG-1 Ser Tyr Asp Phe Ala Tφ Phe Ala Tyr
VL CDRl Sequences residues 24 25 26 27 27A 27B 27C 27D 27E 27F 28 29 30 31 32 33 34
Human kappa I Arg Ala Ser Gin Ser Leu Val + + Ser He Ser Asn/Ser Tyr Leu Ala
Human kappa II Arg Ser Ser Gin Ser Leu Leu His Ser + Asp Gly Asn/Asp Asn/Thr Tyr Leu +
Human kappa III Arg Ala Ser Gin Ser Val Ser Ser Ser Tyr Leu Ala
Human kappa IV Lys Ser Ser Gin Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala
Mouse kappa I Lys Ser Ser Gin Ser Leu Leu Asn Ser Gly Asn Gin Lys Asn Tyr Leu
Mouse kappa II Arg Ser Ser Gin Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu Gin
Mouse kappa HI Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Met His
Mouse kappa IV Ser Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His
Mouse kappa V Arg Ala Ser Gin Asp Asp He Ser Asn Tyr Leu Asn
Mouse kappa VI Ser Ala Ser Ser Ser Val Ser Tyr Met His
Mouse kappa VII
HMFG-1 Lys Ser Ser Gin Ser Leu Leu Tyr Ser Ser Asn Gin Lys He Tyr Leu Ala
VL CDR2 Sequences residues 50 51 52 53 54 55 56
Human kappa I Ala Ala Ser Ser Leu Glu Ser
Human kappa II Leu Val Ser Asn Arg Ala Ser
Human kappa HI Gly Ala Ser Ser Arg Ala Thr
Human kappa IV TΦ Ala Ser Thr Arg Glu Ser
Mouse kappa I Tφ Ala Ser Thr Arg Glu Ser
Mouse kappa II Lys Val Ser Asn Arg Phe Ser
Mouse kappa III Ala Ala Ser Asn Leu Glu Ser
Mouse kappa IV Arg Thr Ser Asn Leu Ala Ser
Mouse kappa V Tyr Ala Ser Arg Leu His Ser
Mouse kappa VI Asp Tin- Ser Lys Leu Ala Ser
Mouse kappa VII
HMFG-1 Tφ Ala Ser Thr Arg Glu Ser
VL CDR3 Sequences residues 89 90 91 92 93 94 95 95A 95B 95C 95D 95E 95F 96 97
Human kappa I Gin Gin Tyr + Ser Leu Pro Glu Tφ Thr
Human kappa II Met Gin Ala Leu Gin + Pro Arg + Thr
Human kappa III Gin Gin Tyr Gly Ser Ser Pro Pro Leu Thr
Human kappa IV Gin Gin Tyr Tyr Ser Thr Pro + Thr
Mouse kappa I Gin Asn Asp Tyr Ser Tyr Pro Leu Thr
Mouse kappa II Phe Gin Gly Thr His Val Pro Pro Tyr Thr
Mouse kappa III Gin Gin Ser Asn Glu Asp Pro Pro T Thr
Mouse kappa IV Gin Gin Tφ Ser Ser Tyr Pro + Gly Leu Thr
Mouse kappa V Gin Gin Gly Asn Thr Leu Pro Pro Arg Thr
Mouse kappa VI Gin Gin TΦ Ser Ser Asn Pro Pro Met Pro Leu Thr
residues 89 90 91 92 93 94 95 95A 95B 95C 95D 95E 95F 96 97
Mouse kappa VII Leu Gin Tyr Asp Glu Phe Ala Tyr Thr
HMFG-1 Gin Gin Tyr Tyr Arg Tyr Pro Arg Thr
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
Various references are cited herein, the disclosures of which are incoφorated by reference in their entireties.

Claims

WHAT IS CLAIMED IS:
1. A vaccine composition comprising an amount of a first immunoglobulin molecule sufficient to induce an anti-idiotype response, said first immunoglobulin molecule comprising a variable region and being identical, except for one or more amino acid substitutions in said variable region, to a second immunoglobulin molecule, said second immunoglobulin molecule being capable of immunospecifically binding an antigen, said one or more amino acid substitutions being the substitution of one or more amino acid residues that do not have a sulfhydryl group at one or more positions corresponding to one or more 0 cysteine residues that form a disulfide bond in said second immunoglobulin molecule; and a pharmaceutically acceptable carrier.
2. The vaccine composition according to claim 1, wherein said antigen is a tumor antigen. 5
3. The vaccine composition according to claim 2, wherein said antigen is a cancer antigen.
4. The vaccine composition according to claim 3, wherein said antigen is a 0 polymoφhic epithelial mucin antigen.
5. The vaccine composition according to claim 3, wherein said antigen is a human colon carcinoma-associated protein antigen.
5 6. The vaccine composition according to claim 3, wherein said antigen is a human colon carcinoma-associated carbohydrate antigen.
7. The vaccine composition according to claim 1, wherein said variable region is a light chain variable region and said amino acid residue that does not have sulfhydryl
_π group is at a position corresponding to position 23 or 88 of the light chain variable region of said second immunoglobulin molecule.
8. The vaccine composition according to claim 1, wherein said variable region is a heavy chain variable region and said amino acid residue that does not have a sulfhydryl
35 group is at a position corresponding to position 22 or 92 of the heavy chain variable region of said second immunoglobulin molecule.
9. The vaccine composition according to claim 1, 7 or 8, wherein said amino acid residue that does not have a sulfhydryl group is alanine.
10. The vaccine composition according to claim 1, wherein said second immunoglobulin molecule is Mab 31.1, Mab33.28. or Mab HMFG-1 and wherein said one or more amino acid substitutions include a substitution with alanine at position 23 and/or 88 of the light chain variable region.
11. The vaccine composition according to claim 1 , wherein said second immunoglobulin molecule is Mab 31.1, Mab 33.28, or Mab HMFG-1 and wherein said one or more amino acid substitutions include a substitution with alanine at position 22 and/or 92 of the heavy chain variable region.
12. The vaccine composition according to claim 3, wherein said antigen is a human milk fat globule antigen.
13. The vaccine composition according to claim 3, wherein said antigen is an antigen of a cancer of the breast, ovary, uterus, prostate, bladder, lung, skin, colon, pancreas, gastrointestinal tract, B cells or T cells.
14. The vaccine composition according to claim 3, wherein said antigen is said antigen is selected from the group consisting of KS 1/4 pan-carcinoma antigen, ovarian carcinoma antigen, prostatic acid phosphate, prostate specific antigen, melanoma-associated antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen, prostate specific membrane antigen, carcinoembryonic antigen, polymoφhic epithelial mucin antigen, human milk fat globule antigen, colorectal tumor-associated antigen TAG-72, CO17-1A, GICA 19-9, CTA-1, LEA, Burkitt's lymphoma antigen-38.13, CD19, human B- lymphoma antigen-CD20, CD33, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside GM3, tumor-specific transplantation type of cell-surface antigen, oncofetal antigen-alpha-fetoprotein L6, human lung carcinoma antigen L20, human leukemia T cell antigen-Gp37, neoglycoprotein, sphingolipids, EGFR, HER2 antigen, polymoφhic epithelial mucin, malignant human lymphocyte antigen-APO-1, 1 antigen Ml 8, M39, SSEA- 1, VEP8, VEP9, Myl, VIM-D5, D,56-22, TRA-1-85, C14, F3, AH6, Y hapten, Ley, TL5,
FC10.2, gastric adenocarcinoma antigen, CO-514, NS-10, CO-43, MH2, 19.9 found in colon cancer, gastric cancer mucins, T5A7, R24, 4.2, GD3, Dl.l, OFA-1, G^, OFA-2, GD2,
Ml:22:25:8, SSEA-3, SSEA-4 and T-cell receptor derived peptides.
15. The vaccine composition according to claim 1, wherein said antigen is an antigen of an infectious disease agent.
16. The vaccine composition according to claim 15, wherein said antigen is selected from the group consisting of influenza virus hemagglutinin, human respiratory syncytial virus G glycoprotein, core protein of Dengue virus, matrix protein of Dengue virus, measles virus hemagglutinin, heφes simplex virus type 2 glycoprotein gB, poliovirus I VP1, envelope glycoproteins of HIV I, hepatitis B surface antigen, diptheria toxin, streptococcus 24M epitope, gonococcal pilin, pseudorabies virus g50, pseudorabies virus glycoprotein H, pseudorabies virus glycoprotein E, transmissible gastroenteritis glycoprotein 195, transmissible gastroenteritis matrix protein, swine rotavirus glycoprotein 38, swine parvovirus capsid protein, Serpulina hydodysenteriae protective antigen, bovine viral diarrhea glycoprotein 55, Newcastle disease virus hemagglutinin-neuraminidase, swine flu hemagglutinin, swine flu neuraminidase, infectious bovine rhinotracheitis virus glycoprotein E, infectious laryngotracheitis virus glycoprotein G or glycoprotein I, a glycoprotein of La Crosse virus, neonatal calf diarrhea virus, hepatitis B virus core protein, hepatitis B virus surface antigen, equine influenza virus type A/ Alaska 91 neuraminidase, equine influenza virus type A/Miami 63 neuraminidase, equine influenza virus type A/Kentucky 81 neuraminidase, equine heφesvirus type 1 glycoprotein B, equine heφesvirus type 1 glycoprotein D, bovine respiratory syncytial virus attachment protein, bovine respiratory syncytial virus fusion protein, bovine respiratory syncytial virus nucleocapsid protein, bovine parainfluenza virus type 3 fusion protein, bovine parainfluenza virus type 3 hemagglutinin neuraminidase, bovine viral diarrhea virus glycoprotein 48, and bovine diarrhea virus glycoprotein 53.
17. The vaccine composition according to claim 1, wherein said antigen is a cellular receptor for an infectious disease agent.
18. The vaccine composition according to claim 17, wherein said cellular receptor is selected from the group consisting of LPV receptor, adenylate cyclase, BDV surface glycoproteins, N-acetyl-9-O-acetylneuraminic acid receptor, CD4+, highly sulphated type heparin sulphate, p65, Gal alpha 1-4-Gal-containing isoreceptors, CD 16b, integrin VLA-2 receptor, EV receptor, CD 14, glycoconjugate receptors, alpha/beta T-cell receptor, decay-accelerating factor receptor, extracellular envelope glycoprotein receptor, immunoglobulin Fc receptor poxvirus M-T7, GALV receptor, CD 14 receptor, Lewis(b) blood group antigen receptor, T-cell receptor, heparin sulphate glycoaminoglycans receptor, fibroblast growth factor receptor, CD1 la, CD2, G-protein coupled receptor, CD4, heparin sulphate proteoglycan, annexin II, CD 13 (aminopeptidase N), human aminopeptidase N receptor, hemagglutinin receptor, CR3 receptor, protein kinase receptor, galactose N- acetylgalactosamine-inhibitable lectin receptor, chemokine receptor, annexin I, actA protein, CD46 receptor, meningococcal virulence associated opa receptors, CD46 receptor, carcinoembryonic antigen family receptors, carcinoembryonic antigen family Bgla receptor, gamma interferon receptor, glycoprotein gp70, rmc-1 receptor, human integrin receptor alpha v beta 3, heparin sulphate proteoglycan receptor, CD66 receptor, integrin receptor, membrane cofactor protein, CD46, GMl, GM2, GM3, CD3, ceramide, hemagglutinin- neuraminidase protein, erythrocyte P antigen receptor, CD36 receptor, glycophorin A receptor, interferon gamma receptor, KDEL receptor, mucosal homing alpha4beta7 receptor, epidermal growth factor receptor, alpha5betal integrin protein, non-glycosylated J774 receptor, CXCR1-4 receptor, CCRl-5 receptor, CXCR3 receptor, CCR5 receptor, gp46 surface glycoprotein, TNFR p55 receptor, TNFp75 receptor, soluble interleukin-1 beta receptor.
19. The vaccine composition according to claim 15 or 17, wherein said infectious disease agent is a bacterium.
20. The vaccine composition according to claim 19, wherein said bacterium is selected from the group consisting of mycobacteria rickettsia, mycoplasma, Neisseria spp., legionella, Shigella spp., Vibrio cholerae, Streptococci, corynebacteria diphtheriae, clostridium tetani, bordetella pertussis, Haemophilus spp., Chlamydia spp., and Escherichia coli or causes Syphilis or Lyme's disease.
21. The vaccine composition according to claim 15 or 17 , wherein said infectious disease agent is a virus.
22. The vaccine composition according to claim 21 , wherein said virus is selected from the group consisting of hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, heφes simplex type I, heφes simplex type II, rindeφest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus,
-* cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsachie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I, human immunodeficiency virus type II, picornaviruses, enteroviruses, caliciviridae, Norwalk group of viruses, togaviruses, alphaviruses, flaviviruses, coronaviruses, rabies virus, Marburg viruses, ebola viruses, parainfluenza virus, orthomyxoviruses, bunyaviruses, arenaviruses,
10 reoviruses, rotaviruses, orbiviruses, human T cell leukemia virus type I, human T cell leukemia virus type II, simian immunodeficiency virus, lentiviruses, polyomaviruses, parvoviruses, Epstein-Barr virus, human heφesvirus-6, cercopithecine heφes virus 1, and poxviruses.
15 23. The vaccine composition according to claim 15 or 17 , wherein said infectious disease agent is a parasite.
24. The vaccine composition according to claim 23, wherein said parasite is selected from a group consisting of plasmodia, eimeria, leishmania, kokzidioa, and trypanosoma and fungi.
25. The vaccine composition according to claim 1 , in which said first immunoglobulin molecule is of a type selected from the group consisting of IgG, IgE, IgM,
IgD and IgA.
25
26. A vaccine composition comprising an amount of a fragment of a first immunoglobulin molecule sufficient to induce an anti-idiotype response, said fragment comprising a variable region and being identical, except for one or more amino acid substitutions in said variable region, to the corresponding fragment of a second
30 immunoglobulin molecule, said fragment of said second immunoglobulin molecule being capable of immunospecifically binding an antigen, said one or more amino acid substitutions being the substitution of an amino acid residue that does not have a sulfhydryl group at one or more positions corresponding to one or more cysteine residues that form a disulfide bond in said second immunoglobulin molecule; and a pharmaceutically acceptable carrier.
35
27. The vaccine composition according to claim 26, in which said fragment is a single chain immunoglobulin.
28. The vaccine composition according to claim 26, in which said fragment is a Fab fragment, (Fab')2 fragment, a heavy chain dimer, a light chain dimer, or a Fv fragment.
29. The vaccine composition according to claim 26, in which said fragment further comprises a constant region.
0 30. The vaccine composition according to claim 26, in which the variable region is from a mouse immunoglobulin, and the constant region is from a human immunoglobulin.
31. The vaccine composition according to claim 1, in which the variable region has framework regions from a human antibody and complementarily determining regions 5 (CDRs) from a mouse immunoglobulin.
32. The vaccine composition according to claim 1 in which the first immunoglobulin molecule is linked via a covalent bond to an amino acid sequence of a protein selected from the group consisting of 11-2, 11-4, 11-5, 11-, 11-7, 11-10, ╬│-interferon or 0 MHC-derived peptide, G-CSF, TNF, porin, NK cell antigen or cellular endocytosis receptor.
33. The vaccine composition according to claim 26 in which the fragment of the first immunoglobulin molecule is linked via a covalent bond to an amino acid sequence of a protein selected from the group consisting of 11-2, 11-4, 11-5, 11-, 11-7, 11-10, ╬│-interferon or 5 MHC-derived peptide, G-CSF, TNF, porin, NK cell antigen or cellular endocytosis receptor.
34. A method of generating an anti-idiotype response in a subject comprising administering to said subject an amount of a first immunoglobulin molecule sufficient to induce an anti-idiotype response, said first immunoglobulin molecule comprising a variable
-n region and being identical, except for one or more amino acid substitutions in said variable region, to a second immunoglobulin molecule, said second immunoglobulin molecule being capable of immunospecifically binding an antigen, said one or more amino acid substitutions being the substitution of an amino acid residue that does not have a sulfhydryl group at one or more positions corresponding to one or more cysteine residues that form a disulfide bond in said second immunoglobulin molecule.
35. The method according to claim 34 which further comprises isolating an antibody from said subject, said antibody recognizing the idiotype of said second immunoglobulin molecule and administering said antibody to a second subject.
36. The method according to claim 34, wherein said antigen is a tumor antigen.
37. The method according to claim 34, wherein said antigen is a cancer antigen.
38. The method according to claim 36, wherein said antigen is a polymoφhic epithelial mucin antigen.
39. The method according to claim 36, wherein said antigen is a human colon carcinoma-associated protein antigen.
40. The method according to claim 36, wherein said antigen is a human colon carcinoma-associated carbohydrate antigen.
41. The method according to claim 35, wherein said variable region is a light chain variable region and said amino acid residue that does not have sulfhydryl group is at a position corresponding to position 23 or 88 of the light chain variable region of said second immunoglobulin molecule.
42. The method according to claim 35, wherein said variable domain is a heavy chain variable region and said amino acid residue that does not have a sulfhydryl group is at position corresponding to position 22 or 92 of the heavy chain variable region of said second immunoglobulin molecule.
43. The method according to claim 34, wherein said amino acid residue that does not have a sulfhydryl group is alanine.
44. The method according to claim 34, wherein said second immunoglobulin molecule is Mab 31.1, Mab 33.28, and HMFG-1 and wherein said amino acid substitutions include a substitution with alanine at position 23 and/or 88 of the light chain variable region.
45. The method according to claim 34, wherein said second immunoglobulin molecule is Mab 31.1, Mab 33.28, and HMFG-1 and wherein said amino acid substitutions include a substitution with alanine at position 22 and/or 92 of the heavy chain variable region.
46. The method according to claim 34, wherein said antigen is a human milk fat globule antigen.
47. The method according to claim 34, wherein said antigen is an antigen for a 0 cancer of the breast, ovary, uterus, prostate, bladder, lung, skin, pancreas, colon, gastrointestinal tract, B cell or T cell.
48. The method according to claim 36, wherein said antigen is selected from the group consisting of KS 1/4 pan-carcinoma antigen, ovarian carcinoma antigen, prostatic 5 acid phosphate, prostate specific antigen, melanoma-associated antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen, prostate specific membrane antigen, carcinoembryonic antigen, polymoφhic epithelial mucin antigen, human milk fat globule antigen, colorectal tumor-associated antigen TAG-72, CO 17-1 A, GICA 19-9, CTA- 1, LEA, Burkitt's lymphoma antigen-38.13, CD 19, human B-lymphoma antigen-CD20, 0 CD33, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside GM3, tumor- specific transplantation type of cell-surface antigen, oncofetal antigen-alpha-fetoprotein L6, human lung carcinoma antigen L20, human leukemia T cell antigen-Gp37, neoglycoprotein, sphingolipids, EGFR, HER2 antigen, polymoφhic epithelial mucin, malignant human lymphocyte antigen-APO-1, 1 antigen M18, M39, SSEA-1, VEP8, VEP9, Myl, VIM-D5,
25 D,56-22, TRA-1-85, C14, F3, AH6, Y hapten, Ley, TL5, FC10.2, gastric adenocarcinoma antigen, CO-514, NS-10, CO-43, MH2, 19.9 found in colon cancer, gastric cancer mucins, T5A7, R24, 4.2, GD3, Dl.l, OFA-1, G^, OFA-2, GD2, Ml:22:25:8, SSEA-3, SSEA-4, and T- cell receptor derived peptides.
30 49. The method according to claim 34, wherein said antigen is an antigen of an infectious disease agent.
50. The method according to claim 49, wherein said antigen is selected from the group consisting of influenza virus hemagglutinin, human respiratory syncytial virus G 35 glycoprotein, core protein of Dengue virus, matrix protein of Dengue virus, measles virus hemagglutinin, heφes simplex virus type 2 glycoprotein gB, poliovirus I VP1, envelope glycoproteins of HIV I, hepatitis B surface antigen, diptheria toxin, streptococcus 24M epitope, gonococcal pilin, pseudorabies virus g50, pseudorabies virus glycoprotein H, pseudorabies virus glycoprotein E, transmissible gastroenteritis glycoprotein 195, transmissible gastroenteritis matrix protein, swine rotavirus glycoprotein 38, swine parvovirus capsid protein, Serpulina hydodysenteriae protective antigen, bovine viral diarrhea glycoprotein 55, Newcastle disease virus hemagglutinin-neuraminidase, swine flu hemagglutinin, swine flu neuraminidase, infectious bovine rhinotracheitis virus glycoprotein E, infectious laryngotracheitis virus glycoprotein G or glycoprotein I, a glycoprotein of La Crosse virus, neonatal calf diarrhea virus, hepatitis B virus core protein, hepatitis B virus surface antigen, equine influenza virus type A/ Alaska 91 neuraminidase, equine influenza virus type A/Miami 63 neuraminidase, equine influenza virus type A/Kentucky 81 neuraminidase, equine heφesvirus type 1 glycoprotein B, equine heφesvirus type 1 glycoprotein D, bovine respiratory syncytial virus attachment protein, bovine respiratory syncytial virus fusion protein, bovine respiratory syncytial virus nucleocapsid protein, bovine parainfluenza virus type 3 fusion protein, bovine parainfluenza virus type 3 hemagglutinin neuraminidase, bovine viral diarrhea virus glycoprotein 48, and bovine diarrhea virus glycoprotein 53.
51. The method according to claim 34, wherein said antigen is a cellular receptor for an infectious disease agent.
52. The method according to claim 51 , wherein said cellular receptor is selected from the group consisting of LPV receptor, adenylate cyclase, BDV surface glycoproteins, N-acetyl-9-O-acetylneuraminic acid receptor, CD4+, highly sulphated type heparin sulphate, p65, Gal alpha 1-4-Gal-containing isoreceptors, CD 16b, integrin VLA-2 receptor, EV receptor, CD 14, glycoconjugate receptors, alpha/beta T-cell receptor, decay-accelerating factor receptor, extracellular envelope glycoprotein receptor, immunoglobulin Fc receptor poxvirus M-T7, GALV receptor, CD 14 receptor, Lewis(b) blood group antigen receptor, T- cell receptor, heparin sulphate gly coaminogly cans receptor, fϊbroblast growth factor receptor, CD1 la, CD2, G-protein coupled receptor, CD4, heparin sulphate proteoglycan, annexin II, CD 13 (aminopeptidase N), human aminopeptidase N receptor, hemagglutinin receptor, CR3 receptor, protein kinase receptor, galactose N-acetylgalactosamine-inhibitable lectin receptor, chemokine receptor, annexin I, actA protein, CD46 receptor, meningococcal virulence associated opa receptors, CD46 receptor, carcinoembryonic antigen family receptors, carcinoembryonic antigen family Bgla receptor, gamma interferon receptor, glycoprotein gp70, rmc-1 receptor, human integrin receptor alpha v beta 3, heparin sulphate proteoglycan receptor, CD66 receptor, integrin receptor, membrane cofactor protein, CD46, GMl, GM2, GM3, CD3, ceramide, hemagglutinin-neuraminidase protein, erythrocyte P antigen receptor, CD36 receptor, glycophorin A receptor, interferon gamma receptor, KDEL receptor, mucosal homing alpha4beta7 receptor, epidermal growth factor receptor, alpha5betal integrin protein, non-glycosylated J774 receptor, CXCR1-4 receptor, CCRl-5 receptor, CXCR3 receptor, CCR5 receptor, gp46 surface glycoprotein, TNFR p55 receptor, TNFp75 receptor, soluble interleukin-1 beta receptor.
53. The method according to claim 49 or 51 , wherein said infectious disease agent is a bacterium.
54. The method according to claim 53, wherein said bacterium is selected from the group consisting of mycobacteria rickettsia, mycoplasma, Neisseria spp., legionella,
Vibrio cholerae, Shigella spp., Streptococci, corynebacteria diphtheriae, clostridium tetani, bordetella pertussis, Haemophilus spp., Chlamydia spp., and Escherichia coli, or causes Syphilis or Lyme's disease.
55. The method according to claim 49 or 51 , wherein said infectious disease agent is a virus.
56. The method according to claim 53, wherein said virus is selected from the group consisting of hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, heφes simplex type I, heφes simplex type II, rindeφest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsachie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I, human immunodeficiency virus type II, picornaviruses, enteroviruses, caliciviridae, Norwalk group of viruses, togaviruses, alphaviruses, flaviviruses, coronaviruses, rabies virus, Marburg viruses, ebola viruses, parainfluenza virus, orthomyxoviruses, bunyaviruses, arenaviruses, reoviruses, rotaviruses, orbiviruses, human T cell leukemia virus type I, human T cell leukemia virus type II, simian immunodeficiency virus, lentiviruses, polyomaviruses, parvoviruses, Epstein-Barr virus, human heφesvirus-6, cercopithecine heφes virus 1 , and poxviruses.
57. The method according to claim 49 or 51 , wherein said infectious disease agent is a parasite.
58. The method according to claim 57, wherein said infectious paraste is selected from a group consisting of plasmodia, eimeria, leishmania, kokzidioa, and trypanosoma and fungi.
59. The method according to claim 34, in which said first immunoglobulin is of a type selected from the group consisting of IgG, IgE, IgM, IgD and IgA. 0
60. A method of generating an anti-idiotype response in a subject comprising administering an amount of a fragment of a first immunoglobulin molecule sufficient to induce an anti-idiotype response, said fragment comprising a variable region and being identical, except for one or more amino acid substitutions in said variable region, to a 5 corresponding fragment of a second immunoglobulin molecule, said fragment of said second immunoglobulin molecule being capable of immunospecifically binding an antigen, said one or more amino acid substitutions being the substitution of an amino acid residue that does not have a sulfhydryl group at one or more positions corresponding to one or more cysteine residues that form a disulfide bond in said second immunoglobulin molecule. 0
61. The method according to claim 60 which further comprises isolating an antibody from said subject, said antibody recognizing the idiotype of said second immunoglobulin molecule and administering said antibody to a second subject.
25 62. The method according to claim 60, wherein said antigen is a tumor antigen.
63. The method according to claim 60, wherein said antigen is a cancer antigen.
64. The method according to claim 60, wherein said antigen is a polymoφhic -0 epithelial mucin antigen.
65. The method according to claim 62, wherein said antigen is a human colon carcinoma-associated protein antigen.
35
66. The method according to claim 62, wherein said antigen is a human colon carcinoma-associated carbohydrate antigen.
67. The method according to claim 60, wherein said variable region is a light chain variable region and said amino acid residue that does not have a sulfhydryl group at position corresponding to position 23 or 88 of the heavy chain variable region of said second immunoglobulin molecule.
68. The method according to claim 60, wherein said variable domain is a heavy chain variable region and said amino acid residue that does not have a sulfhydryl group is at position corresponding to position 22 or 92 of the heavy chain variable region of said second immunoglobulin molecule.
69. The method according to claim 60, 67, or 68, wherein said amino acid residue that does not have a sulfhydryl group is alanine.
70. The method according to claim 63, wherein said antigen is a human milk fat globule.
71. The method according to claim 63, wherein said antigen is an antigen for a cancer of the breast, ovary, uterus, prostate, bladder, lung, skin, pancreas, colon, gastrointestinal tract, B cell or T cell.
72. The method according to claim 60, wherein said antigen is an antigen of an 5 infectious disease agent.
73. The method of claim 72, wherein said antigen is selected from the group consisting of influenza virus hemagglutinin, human respiratory syncytial virus G glycoprotein, core protein of Dengue virus, matrix protein of Dengue virus, measles virus Q hemagglutinin, heφes simplex virus type 2 glycoprotein gB, poliovirus I VP1, envelope glycoproteins of HIV I, hepatitis B surface antigen, diptheria toxin, streptococcus 24M epitope, gonococcal pilin, pseudorabies virus g50, pseudorabies virus glycoprotein H, pseudorabies virus glycoprotein E, transmissible gastroenteritis glycoprotein 195, transmissible gastroenteritis matrix protein, swine rotavirus glycoprotein 38, swine <- parvovirus capsid protein, Serpulina hydodysenteriae protective antigen, bovine viral diarrhea glycoprotein 55, Newcastle disease virus hemagglutinin-neuraminidase, swine flu hemagglutinin, swine flu neuraminidase, infectious bovine rhinotracheitis virus glycoprotein E, infectious laryngotracheitis virus glycoprotein G or glycoprotein I, a glycoprotein of La Crosse virus, neonatal calf diarrhea virus, hepatitis B virus core protein, hepatitis B virus surface antigen, equine influenza virus type A Alaska 91 neuraminidase, equine influenza virus type A/Miami 63 neuraminidase, equine influenza virus type A/Kentucky 81 neuraminidase, equine heφesvirus type 1 glycoprotein B, equine heφesvirus type 1 glycoprotein D, bovine respiratory syncytial virus attachment protein, bovine respiratory syncytial virus fusion protein, bovine respiratory syncytial virus nucleocapsid protein, bovine parainfluenza virus type 3 fusion protein, bovine parainfluenza virus type 3 hemagglutinin neuraminidase, bovine viral diarrhea virus glycoprotein 48, and bovine diarrhea virus glycoprotein 53.
74. The method according to claim 60, wherein said antigen is a cellular receptor for an infectious disease agent.
75. The method according to claim 74, wherein said cellular receptor is selected from the group consisting of LPV receptor, adenylate cyclase, BDV surface glycoproteins, N-acetyl-9-O-acetylneuraminic acid receptor, CD4+, highly sulphated type heparin sulphate, p65, Gal alpha 1-4-Gal-containing isoreceptors, CD 16b, integrin VLA-2 receptor, EV receptor, CD 14, glycoconjugate receptors, alpha/beta T-cell receptor, decay-accelerating factor receptor, extracellular envelope glycoprotein receptor, immunoglobulin Fc receptor poxvirus M-T7, GALV receptor, CD 14 receptor, Lewis(b) blood group antigen receptor, T- cell receptor, heparin sulphate glycoaminoglycans receptor, fibroblast growth factor receptor, CD1 la, CD2, G-protein coupled receptor, CD4, heparin sulphate proteoglycan, annexin II, CD 13 (aminopeptidase N), human aminopeptidase N receptor, hemagglutinin receptor, CR3 receptor, protein kinase receptor, galactose N-acetylgalactosamine-inhibitable lectin receptor, chemokine receptor, annexin I, actA protein, CD46 receptor, meningococcal virulence associated opa receptors, CD46 receptor, carcinoembryonic antigen family receptors, carcinoembryonic antigen family Bgla receptor, gamma interferon receptor, glycoprotein gp70, rmc-1 receptor, human integrin receptor alpha v beta 3, heparin sulphate proteoglycan receptor, CD66 receptor, integrin receptor, membrane cofactor protein, CD46, GMl, GM2, GM3, CD3, ceramide, hemagglutinin-neuraminidase protein, erythrocyte P antigen receptor, CD36 receptor, glycophorin A receptor, interferon gamma receptor, KDEL receptor, mucosal homing alpha4beta7 receptor, epidermal growth factor receptor, alpha5betal integrin protein, non-glycosylated J774 receptor, CXCR1-4 receptor, CCRl-5 receptor, CXCR3 receptor, CCR5 receptor, gp46 surface glycoprotein, TNFR p55 receptor, TNFp75 receptor, soluble interleukin-1 beta receptor.
-> 76. The method according to claim 72 or 74, wherein said infectious disease agent is a bacterium.
77. The method according to claim 76, wherein said bacterium is selected from the group consisting of mycobacteria rickettsia, mycoplasma, Neisseria spp., legionella,
10 Vibrio cholerae, Shigella spp., Streptococci, corynebacteria diphtheriae, clostridium tetani, bordetella pertussis, Haemophilus spp., Chlamydia spp., and Escherichia coli, or causes Syphilis or Lyme's disease.
78. The method according to claim 72 or 74, wherein said infectious disease 15 agent is a virus.
79. The method according to claim 78, wherein said virus is selected from the group consisting of hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, heφes simplex type I, heφes simplex type II, rindeφest, rhinovirus, echovirus,
20 rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsachie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I, human immunodeficiency virus type II, picornaviruses, enteroviruses, caliciviridae, Norwalk group of viruses, togaviruses, alphaviruses, flaviviruses, coronaviruses, rabies virus, Marburg viruses, ebola viruses, 5 parainfluenza virus, orthomyxoviruses, bunyaviruses, arenaviruses, reoviruses, rotaviruses, orbiviruses, human T cell leukemia virus type I, human T cell leukemia virus type II, simian immunodeficiency virus, lentiviruses, polyomaviruses, parvoviruses, Epstein-Barr virus, human heφesvirus-6, cercopithecine heφes virus 1, and poxviruses.
30 80. The method according to claim 72 or 74, wherein said infectious disease agent is a parasite.
81. The method according to claim 80, wherein said parasite is selected from a group consisting of plasmodia, eimeria, leishmania, kokzidioa, trypanosoma, and fungi.
35
82. The method according to claim 60, in which said fragment is a single chain immunoglobulin.
83. The method according to claim 82, in which the fragment is a Fab fragment, (Fab')2 fragment, a heavy chain dimer, a light chain dimer, or an Fv fragment.
84. The method according to claim 60, in which said fragment further comprises a constant region.
0 85. A method according to claim 84, in which the variable region is from a mouse immunoglobulin, and the constant region is from a human immunoglobulin.
86. A method according to claim 60, in which the variable region has framework regions from a human antibody and complementarily determining regions (CDRs) from a 5 mouse immunoglobulin.
87. A method of treating or preventing cancer, in a subject in need of such treatment or prevention, said method comprising administering to a subject a vaccine composition comprising an amount of a first immunoglobulin molecule sufficient to induce 0 an anti-idiotype response, said first immunoglobulin molecule comprising a variable region and being identical, except for one or more amino acid substitutions in said variable region, to a second immunoglobulin molecule, said second immunoglobulin molecule being capable of immunospecifically binding a cancer antigen, said one or more amino acid substitutions being the substitution of an amino acid residue that does not have a sulfhydryl group at one 5 or more positions corresponding to one or more cysteine residues that form a disulfide bond in said second immunoglobulin molecule; and a pharmaceutically acceptable carrier.
88. A method of treating or preventing an infectious disease, in a subject in need of such treatment or prevention, said method comprising administering to a subject a vaccine
,ΓÇ₧ composition comprising an amount of a first immunoglobulin molecule sufficient to induce an anti-idiotype response, said first immunoglobulin molecule comprising a variable region and being identical, except for one or more amino acid substitutions in said variable region, to a second immunoglobulin molecule, said second immunoglobulin molecule being capable of immunospecifically binding an antigen of an infectious disease agent, said one or more amino acid substitutions being the substitution of an amino acid residue that does not have a sulfhydryl group at one or more positions corresponding to one or more cysteine residues that forms a disulfide bond in said second immunoglobulin molecule; and a pharmaceutically acceptable carrier.
89. A method of treating or preventing an infectious disease, in a subject in need of such treatment or prevention, said method comprising administering to a subject a vaccine composition comprising an amount of a first immunoglobulin molecule sufficient to induce an anti-idiotype response, said first immunoglobulin molecule comprising a variable region and being identical, except for one or more amino acid substitutions in said variable region, 0 to a second immunoglobulin molecule, said second immunoglobulin molecule being capable of immunospecifically binding a cellular receptor for an infectious disease agent, said one or more amino acid substitutions being the substitution of an amino acid residue that does not have a sulfhydryl group at one or more positions corresponding to one or more cysteine residues that forms a disulfide bond in said second immunoglobulin molecule; and a 5 pharmaceutically acceptable carrier.
90. The method according to claim 88 or 89 , in which said infectious disease is selected from the group consisting of Syphilis, gonoπhea, AIDS, malaria, shigella, salmonella, hepatitis A, hepatitis C, lyme disease, encephalitis, heφes, gram negative 0 bacterial infection, gram positive bacterial infection, and pneumococcus infection.
91. A method of preparing a first immunoglobulin molecule sufficient to induce an anti-idiotype response, said first immunoglobulin molecule comprising a variable region and being identical, except for one or more amino acid substitutions in said variable region, to a
25 second immunoglobulin molecule, said second immunoglobulin molecule being capable of immunospecifically binds an antigen, said one or more amino acid substitutions being the substitution of an amino acid residue that does not have a sulfhydryl group at one or more positions corresponding to one or more cysteine residues that form a disulfide bond in said second immunoglobulin molecule, said method comprising the steps of: -ΓÇ₧ (a) constructing a nucleic acid that encodes said first immunoglobulin molecule by replacing in a nucleic acid that encodes said second immunoglobulin the nucleotides encoding said one or more cysteine residues that form said disulfide bond with the nucleotides that encode said one or more amino acid residues that do not have a sulhydyl group;
35 (b) introducing the nucleic acid constructed in step (a) into a cell such that said first immunoglobulin molecule is expressed by the cell; and
(c) recovering the expressed first immunoglobulin molecule.
92. The method of claim 91 wherein said nucleotides are replaced by site- directed mutagenesis.
93. A method of preparing a first immunoglobulin molecule sufficient to induce an anti-idiotype response, said first immunoglobulin molecule comprising a variable domain 0 and being identical, except for one or more amino acid substitutions in said variable domain, to a second immunoglobulin molecule, said second immunoglobulin molecule being capable of immunospecifically binds an antigen, said one or more amino acid substitutions being the substitution of an amino acid residue that does not have a sulfhydryl group at one or more positions corresponding to one or more cysteine residues that form a disulfide bond in said 5 second immunoglobulin molecule, comprising the steps of:
(a) synthesizing a nucleic acid containing an artificial gene that encodes said first immunoglobulin molecule;
(b) introducing the nucleic acid synthesized in step (a) into a cell such that the encoded first immunoglobulin molecule is expressed by the cell; and 0 (c) recovering the expressed first immunoglobulin molecule.
94. The method of claim 91 or 93, in which said second immunoglobulin molecule is Mab 31.1 or Mab 33.28.
5 95. The method of claim 91 or 93, in which said second immunoglobulin molecule is HMFG-1.
96. A vaccine composition comprising an amount of a first immunoglobulin molecule sufficient to induce an anti-idiotype response, said first immunoglobulin molecule -π comprising a variable region and being identical, except for one or more amino acid substitutions in said variable region, to a second immunoglobulin molecule, said second immunoglobulin molecule being capable of immunospecifically binding an antigen, in which at least some of said one or more amino acid substitutions are the substitution of one or more amino acid residues that do not have a sulfhydryl group at one or more positions coπesponding to one or more cysteine residues that form a disulfide bond in said second immunoglobulin molecule, wherein the amino acid substitutions that are not the substitution of one or more amino acid residues that do not have a sulfhydryl group at said one or more positions corresponding to said one or more cysteine residues that form said disulfide bond in said second immunoglobulin molecule are not stabilizing changes; and a pharmaceutically acceptable carrier.
97. A method of generating an anti-idiotype response in a subject comprising administering to said subject an amount of a first immunoglobulin molecule sufficient to induce an anti-idiotype response, said first immunoglobulin molecule comprising a variable 0 region and being identical, except for one or more amino acid substitutions in said variable region, to a second immunoglobulin molecule, said second immunoglobulin molecule being capable of immunospecifically binding an antigen, in which at least some of said one or more amino acid substitutions are the substitution of an amino acid residue that does not have a sulfhydryl group at one or more positions coπesponding to one or more cysteine 5 residues that form a disulfide bond in said second immunoglobulin molecule, wherein the amino acid substitutions that are not the substitution of one or more amino acid residues that do not have a sulfhydryl group at said one or more positions coπesponding to said one or more cysteine residues that form said disulfide bond in said second immunoglobulin molecule are not stabilizing changes. 0
98. A first immunoglobulin molecule comprising a variable region and being identical, except for one or more amino acid substitutions in said variable region, to a second immunoglobulin molecule, said second immunoglobulin molecule being capable of immunospecifically binding an antigen, in which at least some of said one or more amino 5 acid substitutions are the substitution of an amino acid residue that does not have a sulfhydryl group at one or more positions coπesponding to one or more cysteine residues that form a disulfide bond in said second immunoglobulin molecule, wherein the amino acid substitutions that are not the substitution of one or more amino acid residues that do not have a sulfhydryl group at said one or more positions coπesponding to said one or more
_0 cysteine residues that form said disulfide bond in said second immunoglobulin molecule are not stabilizing changes.
35
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AU1459799A (en) 1999-06-07
CN1327388A (en) 2001-12-19
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AU2003252902A1 (en) 2003-11-06
AU737457B2 (en) 2001-08-23
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CA2310269A1 (en) 1999-05-27
EP1032420A1 (en) 2000-09-06
IL136114A0 (en) 2001-05-20
EP1030684A4 (en) 2004-09-15
WO1999025378A9 (en) 1999-08-12
CN1294517A (en) 2001-05-09
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AU1459899A (en) 1999-06-07
BR9815580A (en) 2002-01-29
WO1999025378A1 (en) 1999-05-27
BR9815289A (en) 2001-12-26
EP1032420A4 (en) 2004-09-15

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