AU1459899A - Modified antibodies with enhanced ability to elicit an anti-idiotype response - Google Patents

Description

WO 99/25379 PCT/US98/24303 MODIFIED ANTIBODIES WITH ENHANCED ABILITY TO ELICIT AN ANTI-IDIOTYPE RESPONSE 5 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. 10 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 15 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 20 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 25 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 30 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 (CH1, CH2, CH3...CHx), the number depending upon the particular antibody class. The CH1 region is separated from the CH2 region by a hinge region which allows flexibility in the antibody. 35 The variable region of each light chain aligns with the variable region of each heavy chain, -1- WO 99/25379 PCT/US98/24303 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, 5 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., Immunolovgy, 3rd Edition, London; Mosby, 1993, 10 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. 15 2.2. IMMUNOTHERAPY AND ANTI-IDIOTYPE ANTIBODIES In modem 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 20 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 25 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 30 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. 35 -2- WO 99/25379 PCT/US98/24303 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 5 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 10 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 15 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 20 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 25 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 30 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 Ab1 has been structurally damaged (Madiyalakan et al., 1995, Hybridoma 14:199-203), rendering the antibody more 35 foreign. There has been direct administration to the subject of exogenously produced anti -3- WO 99/25379 PCT/US98/24303 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 5 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 10 generate an anti-idiotypic response is that, Ab 1, 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. 15 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 20 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 25 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 30 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 35 corresponding to one or more cysteine residues that form a disulfide bond in said second -4- WO 99/25379 PCT/US98/24303 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. 5 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 10 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 15 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 20 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 25 amino terminal region (H 2 N-) 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 V L and Vs 30 respectively). The constant region domains are indicated as CL for the light chain constant domain and CH,, 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 CL and CH, domains. Fc indicates the constant region fragment containing the CH 2 and CH 3 domains. 35 -5- WO 99/25379 PCT/US98/24303 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 1 , 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 5 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 (Ab I) directed against a ligand in an anti-idiotypic cascade. Figure 5. Modification of the variable region of an immunoglobulin by replacing 10 cysteine residues in the variable regions with alanine residues to break a variable region intrachain disulfide bond. CH1, 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 pMRRO 10.1, which contains a human kappa light chain constant region sequence. (B). The structure of the 15 expression vector pGammal that contains a sequence encoding a human IgG1 constant region (CH1, 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. 20 Figures 7A and B. (A) The amino acid sequence and corresponding nucleotide sequence including the leader sequence for the consensus light chain variable region ConVL1. (B) The amino acid and corresponding nucleotide sequences for the consensus heavy chain variable region ConVH1 including the leader sequence. Figures 8A-B. (A) Amino acid and corresponding nucleotide sequence of 25 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 2CAVHCOL 1, which is the heavy chain variable region sequence of an antibody derived from mAb31.1, in which alanine residues have been substituted for 30 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. 35 (C) Oligonucleotide sequences for the oligonucleotides used to assemble the light chain -6- WO 99/25379 PCT/US98/24303 consensus region referred to as ConVL1. (D) Oligonucleotide sequences for the oligonucleotides used to assemble the heavy chain consensus region referred to as ConVL1. 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 5 colon cancer antigen. Figure 11. Dot blot showing the result of an assay for the competition of binding of the antibody derived from mnAB31.1, 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. 10 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, 15 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, 2CAVHCOL 1, and 2CAVLCOL 1. (C) 20 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 30 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 35 participates in forming an intrachain disulfide bond in the variable region of the -7- WO 99/25379 PCT/US98/24303 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 5 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. 10 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 15 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, 20 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 30 antigen and have been modified to enhance their ability to elicit an anti-idiotype response. Such imnimunoglobulins 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 35 that is identical, except for one or more amino acid substitutions in the variable region, to a 35 -8- WO 99/25379 PCT/US98/24303 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 5 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 10 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 20 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 25 Human lambda light I 23,88 Human lambda light II 23,88 Human lambda light IIII 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 30Mouse 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 35 Mouse kappa light Miscellaneous 23,88 -9- WO 99/25379 PCT/US98/24303 Disulfide bond-forming Variable domain cysteines Species Subgroup (positions) Mouse lambda light 23,88 5 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) 10 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 15 Human heavy I 22,92 Human heavy II 22,92 Human heavy III 22,92 Mouse heavy I (A) 22,92 Mouse heavy I (B) 22,92 Mouse heavy II (A) 22,92 Mouse heavy II (B) 22,92 20 Mouse heavy II (C) 22,92 Mouse heavy III (A) 22,92 Mouse heavy III(B) 22,92 Mouse heavy III (C) 22,92 Mouse heavy III (D) 22,92 Mouse heavy V (A) 22,92 Mouse heavy V (B) 22,92 25 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) 30 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) -10- WO 99/25379 PCT/US98/24303 Disulfide bond-forming Variable domain cysteines Species Subgroup (positions) Caiman heavy 22, 92 5 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 0() indicate that the protein was not sequenced to that position, 10 10 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. 15 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 7A and B, respectively ("corresponding" means as determined by aligning the particular antibody 20 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 25 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 30 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 35 nonclassical amino acid or chemical amino acid analog that does not contain a sulfhydryl -11- WO 99/25379 PCT/US98/24303 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, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, y-Abu, E-Ahx, -amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, 5 ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, t-butylglycine, t butylalanine, phenylglycine, cyclohexylalanine, P-alanine, fluoro-amino acids, designer amino acids such as p-methyl amino acids, Ca-methyl amino acids, Na-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. 10 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). 15 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 20 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 25 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 30 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 35 Immunological Interest, 5th Ed., U.S. Department of Health and Human Services, Bethesda, -12- WO 99/25379 PCT/US98/24303 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 5 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 10 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 15 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 20 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, 25 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 30 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 35 -13- WO 99/25379 PCT/US98/24303 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 5 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. 10 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 15 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 20 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, 25 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 30 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 35 making mutations in the member and determining which mutations prevent binding. -14- WO 99/25379 PCT/US98/24303 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 5 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 15 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. 131: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 76:253-262; 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 35 immunospecifically bind a receptor and are antagonists the ligand that binds that receptor, -15- WO 99/25379 PCT/US98/24303 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 5 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 10 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 15 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, 20 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 25 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 30 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 7A 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 35 -16- WO 99/25379 PCT/US98/24303 of Proteins of Immunological Interest, 51 h 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 5 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. 10 (1991, Sequences of Proteins of Immunological Interest, 51 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. 15 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, 20 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 25 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 30 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 35 -17- WO 99/25379 PCT/US98/24303 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 5 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 10 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 15 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 20 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. 25 Table 2 CDR Number of residues L1 10-17 L2 7 L3 7-11 30 H1 5-7 H2 9-12 H3 2-25 (compiled from data in Kabat and Wu, 1971, Ann. NYAcad. Sci. 190:382-93) 35 -18- WO 99/25379 PCT/US98/24303 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 5 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, L 1, 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, H1, the CDR is 5 to 7 amino acid residues; if it is the second 10 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 15 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 20 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 25 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, 30 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, 35 in a preferred embodiment, the antigenicity of the idiotype of the immunoglobulin molecule -19- WO 99/25379 PCT/US98/24303 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, 5 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. 10 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 CH1 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 15 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. 20 Techniques have been 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 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 25 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 30 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 35 framework (Winter et al. U.S. Patent No. 5,225,539). CDR-grafted antibodies have been -2035 -20- WO 99/25379 PCT/US98/24303 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. 5 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 10 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 15 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 20 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, y-interferon, MHC derived peptide, G-CSF, TNF, porins, NK cell antigens, or cellular endocytosis receptor. 25 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 30 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 35 -21- WO 99/25379 PCT/US98/24303 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 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 10 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 15 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) 20 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 30 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 35 identical, except for one or more amino acid substitutions in said variable region, to a second -22- WO 99/25379 PCT/US98/24303 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 5 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 10 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 15 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 20 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 25 (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. 30 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 35 analog thereof) of the invention, or a nucleic acid encoding the modified immunoglobulin, or -23- WO 99/25379 PCT/US98/24303 functionally active fragment, derivative or analog thereof), which modified immunoglobulin is derived from an immunoglobulin 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 5 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 10 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 p 9 7 (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), CO17 lA (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 30 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, 35 human leukemia T cell antigen-Gp37 (Bhattacharya-Chatterjee et al., 1988, J.1 ofl Immun. -2435 -24- WO 99/25379 PCT/US98/24303 141:1398-1403), neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR (Epidermal growth factor receptor), HER2 antigen (p 185"), 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 5 antigen (Feizi, 1985, Nature 314:53-57) such as I antigen found in fetal erthrocytes and primary endoderm, I(Ma) found in gastric adencarcinomas, M18 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' found in 10 embryonal carcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells, El 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 15 mucins, TA 7 found in myeloid cells, R 2 4 found in melanoma, 4.2, GD 3 , D1.1, OFA-1, GM, OFA-2, GD 2 , M1:22:25:8 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 20 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, 25 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 30 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 -25- WO 99/25379 PCT/US98/24303 TABLE 3 MALIGNANCIES AND RELATED DISORDERS Leukemia 5 acute leukemia acute lymphocytic leukemia acute myelocytic leukemia myeloblastic promyelocytic myelomonocytic monocytic 10 erythroleukemia chronic leukemia chronic myelocytic (granulocytic) leukemia chronic lymphocytic leukemia Polycythemia vera Lymphoma Hodgkin's disease non-Hodgkin's disease 15 Multiple myeloma Waldenstr6m'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 35 papillary carcinoma 35 papillary adenocarcinomas -26- WO 99/25379 PCT/US98/24303 cystadenocarcinoma medullary carcinoma bronchogenic carcinoma renal cell carcinoma hepatoma bile duct carcinoma 5 choriocarcinoma seminoma embryonal carcinoma Wilms' tumor cervical cancer uterine cancer testicular tumor 10 lung carcinoma small cell lung carcinoma bladder carcinoma epithelial carcinoma glioma astrocytoma medulloblastoma craniopharyngioma 15 ependymoma pinealoma hemangioblastoma acoustic neuroma oligodendroglioma meningioma melanoma neuroblastoma 20 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. -27- WO 99/25379 PCT/US98/24303 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 5 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 10 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 15 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, 20 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 25 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 30 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 chronic 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 35 myeloma), and a first degree kinship with persons having a cancer or precancerous disease -28- WO 99/25379 PCT/US98/24303 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 5 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 10 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 15 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 20 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 30 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 35 as gpl20(Putney et al., 198, Science 234:1392-1395), hepatitis B surface antigen (Itoh et al., -2935 -29- WO 99/25379 PCT/US98/24303 198, Nature 308:19; Neurath et al., 198, Vaccine 4:34), diptheria toxin (Audibert et al., 1981, Nature 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 gIII (gpC), 5 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 10 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 15 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 ofNegative Strand Viruses, Bishop and Compans (eds.), Elsevier, NY, p. 17), murine leukemia virus (Steeves et al., 1974, J. Virol. 14:187), mouse mammary tumor virus (Massey and Schochetman, 1981, Virology 115:20), 20 hepatitis B virus core protein and/or hepatitis B virus surface antigen (see, e.g., U.K. Patent Publication No. GB 2034323A published June 4, 1980; Ganem and Varmus, 1987, Ann. Rev. Biochem. 5:51-93; Tiollais et al., 1985, Nature 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 25 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 N), bovine parainfluenza virus type 3 fusion protein, and 30 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 35 -30- WO 99/25379 PCT/US98/24303 limitation, such cellular receptors, along with their corresponding pathogens are listed in Table 4. Table 4 5 Pathogen Cellular Receptor B-lymphotropic papovavirus LPV receptor on B-cells (LPV) Bordatella pertussis Adenylate cyclase 10 Borna Disease virus (BDV) BDV surface glycoproteins Bovine coronavirus N-acetyl-9-O-acetylneuraminic acid receptor Choriomeningitis virus CD4 + Dengue virus Highly sulphated type heparin sulphate 15 p 6 5 E. coli Gal-alpha-1-4Gal-containing isoreceptors Ebola CD16b Echovirus 1 Integrin VLA-2 receptor Echovirus-11 (EV) EV receptor 20 Endotoxin (LPS) CD14 Enteric bacteria Glycoconjugate receptors Enteric Orphan virus alpha/beta T-cell receptor Enteroviruses Decay-accelerating factor receptor 25 Feline leukemia virus Extracellular envelope glycoprotein (Env-SU) receptor Foot and mouth disease virus Immunoglobulin Fc receptor Gibbon ape leukemia virus GALV receptor 30 (GALV) Gram-negative bacteria CD 14 receptor Heliobacter pylori Lewis(b) blood group antigen receptor Hepatitis B virus (HBV) T-cell receptor 35 -31- WO 99/25379 PCT/US98/24303 Pathogen Cellular Receptor Herpes Simplex Virus Heparin sulphate glycosaminoglycan receptor Fibroblast growth factor receptor 5 HIV-1 CC-Chemokine receptor CCR5 CD11a CD2 G-protein coupled receptor CD4 10 Human cytomegalovirus Heparin sulphate proteoglycan Annexin II CD13 (aminopeptidase N) Human coronovirus Human aminopeptidase N receptor Influenza A, B & C Hemagglutinin receptor 15 Legionella CR3 receptor Protein kinase receptor Galactose N-acetylgalactosamine (Gal/GalNAc) inhibitable lectin receptor Chemokine receptor 20 Leishmania mexicana Annexin I Listeria monocytogenes ActA protein Measles virus CD46 receptor Meningococcus Meningococcal virulence associated Opa receptors 25 25 Morbilliviruses CD46 receptor Mouse hepatitis virus Carcinoembryonic antigen family receptors Carcinoembryonic antigen family Bgla receptor Murine leukemia virus Envelope glycoproteins 30 Murine gamma herpes virus gamma interferon receptor Murine retrovirus Glycoprotein gp70 Rmc-1 receptor Murine coronavirus mouse Carcinoembryonic antigen family receptors hepatitis virus 35 -32- WO 99/25379 PCT/US98/24303 Pathogen Cellular Receptor Mycobacterium avium-M Human Integrin receptor alpha v beta 3 Neisseria gonorrhoeae Heparin sulphate proteoglycan receptor 5 CD66 receptor Integrin receptor Membrane cofactor protein CD46 'GM1 10 GM2 GM3 CD3 Ceramide Newcastle disease virus Hemagglutinin-neuraminidase protein 15 Fusion protein Parvovirus B 19 Erythrocyte P antigen receptor Plasmodium falciparum CD36 receptor Glycophorin A receptor Pox Virus Interferon gamma receptor 20 Pseudomonas KDEL receptor Rotavirus Mucosal homing alpha4beta7 receptor Samonella typhiurium Epidermal growth factor receptor Shigella alpha5betal integrin protein 25 Streptococci Nonglycosylated J774 receptor T-helper cells type 1 Chemokine receptors including: 6. CXCR1-4 7. CCR1-5 30 8. CXCR3 9. CCR5 T-cell lymphotropic virus 1 gp46 surface glycoprotein 35 -33- WO 99/25379 PCT/US98/24303 Pathogen Cellular Receptor Vaccinia virus TNFRp55 receptor TNFRp75 receptor 5 Soluble Interleukin-1 beta receptor 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 10 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 15 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. 20 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 25 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. 30 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. 35 -34- WO 99/25379 PCT/US98/24303 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 5 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 10 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 15 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, 20 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 30 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, 35 encapsulation in biopolymers (e.g., poly-B- -1->4-N-acetylglucosamine polysaccharide; see -35- WO 99/25379 PCT/US98/24303 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, 5 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 30 234. 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 -36- WO 99/25379 PCT/US98/24303 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 5 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 10 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 15 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-(l1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine. 20 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 25 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 30 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). -37- WO 99/25379 PCT/US98/24303 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 5 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 10 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 15 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 20 (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 25 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. 30 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 35 techniques. -38- WO 99/25379 PCT/US98/24303 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 5 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. 10 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 15 (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 20 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 25 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, 30 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 35 the immunoglobulin molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication -3935 -39- WO 99/25379 PCT/US98/24303 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 immunoglobulin can be modified to introduce the 5 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 10 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 15 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 20 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 25 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 30 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 DNA technology using techniques well known in the art. 35 The modified immunoglobulin molecule can then be recombinantly expressed and isolated -40- WO 99/25379 PCT/US98/24303 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 5 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 immunoglobulin molecule coding sequences and 10 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 cytomegalovirus 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 -41- WO 99/25379 PCT/US98/24303 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 5 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 adenovirus late promoter; the vaccinia virus 7.5K promoter). In bacterial systems, a number of expression vectors may be advantageously selected 10 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 15 (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. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S 20 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 pGEX 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. 25 In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodopterafrugiperda 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). 30 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 35 non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant -4235 -42- WO 99/25379 PCT/US98/24303 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. 5 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, 10 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 15 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. 20 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 25 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 30 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 35 immunoglobulin molecule. -43- WO 99/25379 PCT/US98/24303 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 5 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 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 30 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 immunoglobulin 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 -44- WO 99/25379 PCT/US98/24303 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 5 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, Nature 322:52; Koher, 1980, Proc. Natl. Acad. Sci. USA 10 77:2197). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA. 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 30 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 35 techniques, e.g., enzyme linked immunosorbent assay (ELISA), immunoblots, -45- WO 99/25379 PCT/US98/24303 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 5 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 10 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. 15 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 20 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. 25 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 30 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 3 H-thymidine incorporation, by direct cell count, by detecting changes 35 in transcriptional activity of known genes such as proto-oncogenes (e.g.,fos, myc) or cell -4635 -46- WO 99/25379 PCT/US98/24303 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. 5 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. 10 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 15 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 20 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 25 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. 30 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 35 antigen, the largest dimensions of both erythema and induration are measured with a ruler. -47- WO 99/25379 PCT/US98/24303 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 5 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 10 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 5 "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). In other aspects, the modified immunoglobulins may be tested for efficacy by 15 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 20 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. 25 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 30 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 35 infectious disease agent may also be determined by measuring the levels of the antigen -4835 -48- WO 99/25379 PCT/US98/24303 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. 5 6. EXAMPLE: ANTI-IDIOTYPIC VACCINE INDUCER FOR COLON CANCER This example describes the construction of a modified antibody derived from the 10 monoclonal antibody MAb31.1 (hybridoma secreting Mab31.1 is available from the American Type Tissue Collection as accession No. HB 12314). 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 15 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 20 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 25 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 30 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 9A and 9B. Figure 9A list the group of ten oligos 35 used in engineering a heavy chain variable region gene called 2CAVHCOL 1. -49- WO 99/25379 PCT/US98/24303 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 5 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: 25gl of each oligo was incubated for 1 hour in the presence of T4 polynucleotide kinase and 50mM ATP at 37oC. The reactions were stopped by heating for 5 minutes at 10 70oC 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 0 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 10mM ATP for 2 hours in a water bath maintained at 16 0 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 -50- WO 99/25379 PCTIUS98/24303 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 -20oC for 1 hour followed by centrifugation at 12,000 rpm for 15 minutes. The purified DNA pellet was resuspended in 50 il of TE buffer, pH 8.0. The engineered variable region gene was then amplified by PCR. Specifically, 100 5 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 10 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, 10g of pUC19 was linearized with Hinc H (50 U) for 3 hours at 37 0 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) 15 for 1 hour at 370 C. In order to insert the engineered variable region gene into the pUC19 vector, approximately 0.5 pg of dephosphorylated linear vector DNA was mixed with 3 ptg of phosphorylated variable region gene in the presence of T4 DNA ligase (1000 U), and incubated at 16 0 C for 12 hours. The bacterial vector containing the engineered variable region gene was then used to 20 transform bacterial cells. Specifically, freshly prepared competent DH5-a cells, 50 jil, were mixed with 1 pg of pUC 19 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 pF 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 37oC 25 in centrifuge tubes. An aliquot of cells from each transformation was removed, diluted 1:100, then 100 pl plated onto LB plates containing ampicillin (Amp 40 pg/ml). The plates were incubated at 37oC 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 30 sterile glass tube with constant shaking at 37 0 C. The plasmid DNA was isolated using Easy Prep columns (Pharmacia Biotech.) and suspended in 100 pl of TE buffer, pH 7.5. To confirm the presence of gene insert in pUC19, 25 gl ofplasmid DNA from each colony was digested with a restriction endonuclease for 1 hour at 37oC, and was analyzed on a 1% agarose gel. By this method plasmid DNA containing gene insert was resistant to enzyme 35 cleavage due to loss of restriction site ( 5'..GTCGAC.. 3') and migrated as closed circular -51- WO 99/25379 PCT/US98/24303 (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 5 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. 10 Table 5. Construction of gene encoding modified antibodies containing CDRs from Mab 31.1 Oligol Oligo 2 Oligo 3 Oligo 4 Oligo 5 Oligo Oligo 7 Oligo 8 Oligo 9 Oligol0 2CAVHC VHC1 VHC2 VHC3 VHC4 VHCS VHC VHC7 VHC8 VHC9 VHC10 OLI 2CAVLC VLC1 VLC2 VLC3 VLC4 VLCS VLC VLC7 VLC8 VLC9 VLC10 15 OLI 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 20 complete antibody heavy chain contains a variable region, a constant region, and a hinge region. A modified variable region genes 2CAVHCOL 1 or 2CAVLCOL 1 were inserted into vectors containing appropriate constant regions. Engineered variable region genes lacking cysteine residues in the light chain, were inserted into the pMRRO 10.1 vector Figure 6A. The pMRRO 10.1 vector contained a human kappa light chain constant region. 25 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 pGAMMA1 vector Figure 6B. The pGAMMA1 vector contained human and IgG1 constant region and hinge region sequences. Insertion of the engineered heavy chain variable region gene into this vector gave a complete heavy 30 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, 35 -52- WO 99/25379 PCT/US98/24303 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 5 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 10 plasmids containing an SV40 origin of replication to very high copy number. The antibody expression vector was transferred to COS7 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 15 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 20 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, 25 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 30 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 35 was added which generated a dark purple precipitate, indicating the presence of bound -53- WO 99/25379 PCT/US98/24303 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. 5 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 10 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. 15 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 20 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 25 region elicit an anti-idiotype antibodies that recognize the colon carcinoma cell antigen better than antibodies with variable region intra-chain disulfide bonds. 30 35 -54- WO 99/25379 PCT/US98/24303 Table 6. Biotin-Labeled Peptides Derived from CDR Sequences of Mab 31.1 Peptide ID Sequence 5 COL311 L1 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 H1 biotin-N-Phe-Thr-Asn-Tyr-Gly-Met-Asn 10 COL311 H2 biotin-N-Ala-Gly-Trp-Ile-Asn-Thr-Tyr-Thr-Gly-Glu-Pro-Thr-Tyr-Ala-Asp 10 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 15 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 20 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 25 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 30 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 35 vector, as described in Section 6, supra. Other methods for constructing engineered genes -55- WO 99/25379 PCT/US98/24303 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. 5 10 15 20 25 30 35 -56- WO 99/25379 PCT/US98/24303 0CA t) Q> c2 tn d) En a-F 4 "-4 u 0 -4 P ~~<~<E 0-4 P-10 rJ> 00000000000~ -57- 4 WO 99/25379 -PCTIUS98/24303 - 4 4 - )------------------- ~~"-4 7-04 0 0 : 1=4:Z r.0* HW " ~. - 4 =- - - - - -. a. a. - - > + - w HH H HHtCn~H H w-- tn V) C >N >, EnH >N H ch Q 03 in ~ c-- Q).- Cn C)l 4) - cn en 2'4 H/ En cn0HEn oo 2o r-, o 0 0 00 t q &- "0 0 " 0 . tn 04 00O -4 < 914 C 94U) 4) - =- Q4 P4 0 -q A HOH" cn cn 0n 9L toH to<~< 0 H 0d on. 0< GM >C" :C ~q *u -58- WO 99/25379 PCTIUS98/24303 o ~ c ccl en(~ # cn cn 3 5=4 3-3-3 0c 0 ri C3 ct ~ - j - ~i2 3- ~ Cd a. e o2 3+ " 3- 3 5 3co o OD to to OD-t ~~~~~~ >~- - 3- OHOOOHO<~~ > 000~~ CC~~~~c m m- m- >03 m- ) ~ . - - ------ 3.3 cn 0- " " 43Q.. 0 0 0 q- WO 99/25379 -PCT/US98/24303 ~+ ~ M0 C.' 0 0 0 0 0 \,z tn + $.. I. 1.. 1-4 U-4 -4 cn C/ Cn c cn c w C/ t00 0 00 0 0 0 0 3-~ u to~ W~ =1 = t -c ~3-4 .4 -rA c.' +n c. n c~U i (A &n rA 3-c-n - ~ n elH<H0~0 C) CJ <~ (, (D 0 c 0-60 WO 99/25379 PCT[US98/24303 tn ON w*~C 'tn ) ; tnF tn in~ < F c7NF tn* en bb 00 WO 99/25379 PCT/US98/24303 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 5 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 incorporated by reference in their entireties. 10 15 20 25 30 35 -62-

Claims (47)

1. A vaccine composition comprising an amount of a first immunoglobulin molecule sufficient to induce an anti-idiotype response, said first immunoglobulin molecule 5 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 10 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. 15

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 20 polymorphic epithelial mucin antigen.

5. The vaccine composition according to claim 3, wherein said antigen is a human colon carcinoma-associated protein antigen. 25 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 30 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 -63- WO 99/25379 PCT/US98/24303 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 5 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 10 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 15 of the heavy chain variable region.

12. The vaccine composition according to claim 3, wherein said antigen is a human milk fat globule antigen. 20 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 25 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, polymorphic epithelial mucin antigen, human milk fat globule antigen, colorectal tumor-associated antigen TAG-72, 30 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, polymorphic 35 epithelial mucin, malignant human lymphocyte antigen-APO-1, I antigen M18, M39, SSEA -64- WO 99/25379 PCT/US98/24303 1, VEP8, VEP9, Myl, VIM-D5, D 1

56-22, TRA-1-85, C14, F3, AH6, Y hapten, Le y , TL5, FC10.2, gastric adenocarcinoma antigen, CO-514, NS-10, CO-43, MH2, 19.9 found in colon cancer, gastric cancer mucins, TsA 7 , R 24 , 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD 2 , M1:22:25:8, SSEA-3, SSEA-4 and T-cell receptor derived peptides. 5 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 10 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, herpes simplex virus type 2 glycoprotein gB, poliovirus I VP 1, envelope glycoproteins of HIV I, hepatitis B surface antigen, diptheria toxin, streptococcus 24M epitope, gonococcal pilin, pseudorabies virus g50, pseudorabies virus 15 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 20 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 herpesvirus type 1 glycoprotein B, equine herpesvirus type 1 25 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. 30 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 35 receptor is selected from the group consisting of LPV receptor, adenylate cyclase, BDV -65- WO 99/25379 PCT/US98/24303 surface glycoproteins, N-acetyl-9-O-acetylneuraminic acid receptor, CD4', highly sulphated type heparin sulphate, p65, Gal alpha 1-4-Gal-containing isoreceptors, CD16b, integrin VLA-2 receptor, EV receptor, CD14, glycoconjugate receptors, alpha/beta T-cell receptor, decay-accelerating factor receptor, extracellular envelope glycoprotein receptor, 5 immunoglobulin Fc receptor poxvirus M-T7, GALV receptor, CD14 receptor, Lewis(b) blood group antigen receptor, T-cell receptor, heparin sulphate glycoaminoglycans receptor, fibroblast growth factor receptor, CD11 a, CD2, G-protein coupled receptor, CD4, heparin sulphate proteoglycan, annexin II, CD13 (aminopeptidase N), human aminopeptidase N receptor, hemagglutinin receptor, CR3 receptor, protein kinase receptor, galactose N 10 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 Bg 1 a receptor, gamma interferon receptor, glycoprotein gp70, rmc-1 receptor, human integrin receptor alpha v beta 3, heparin sulphate proteoglycan receptor, CD66 receptor, integrin receptor, 15 membrane cofactor protein, CD46, GM1, 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, CCR1-5 receptor, CXCR3 receptor, CCR5 receptor, gp46 20 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. 25 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 30 coli or causes Syphilis or Lyme's disease. 21. The vaccine composition according to claim 15 or17 , wherein said infectious disease agent is a virus. 35 -66- WO 99/25379 PCT/US98/24303 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, herpes simplex type I, herpes simplex type II, rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, 5 cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsachie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I, human immunodeficiency virus type II, picomaviruses, 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 herpesvirus-6, cercopithecine herpes 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 20 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 -67- WO 99/25379 PCT/US98/24303 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 5 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. 10 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 15 (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, II-, 11-7, 11-10, y-interferon or 20 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, Il-, 11-7, 11-10, y-interferon or 25 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 30 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 35 in said second immunoglobulin molecule. -6835 -68- WO 99/25379 PCT/US98/24303 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. 5 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 polymorphic 10 epithelial mucin antigen. 39. The method according to claim 36, wherein said antigen is a human colon carcinoma-associated protein antigen. 15 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 20 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 25 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. 30 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. 35 -69- WO 99/25379 PCT/US98/24303 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. 5 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 10 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 15 acid phosphate, prostate specific antigen, melanoma-associated antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen, prostate specific membrane antigen, carcinoembryonic antigen, polymorphic epithelial mucin antigen, human milk fat globule antigen, colorectal tumor-associated antigen TAG-72, CO 17-1A, GICA 19-9, CTA 1, LEA, Burkitt's lymphoma antigen-38.13, CD19, human B-lymphoma antigen-CD20, 20 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, polymorphic epithelial mucin, malignant human lymphocyte antigen-APO-1, I antigen M18, M39, SSEA-1, VEP8, VEP9, Myl, VIM-D5, 25 D 1 56-22, TRA-1-85, C14, F3, AH6, Y hapten, Le y , TL5, FC10.2, gastric adenocarcinoma antigen, CO-514, NS-10, CO-43, MH2, 19.9 found in colon cancer, gastric cancer mucins, T 5 A 7 , R 24 , 4.2, GD 3 , D1.1, OFA-1, GM2, OFA-2, GD 2 , M1:22:25:8, 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 -70- WO 99/25379 PCT/US98/24303 hemagglutinin, herpes simplex virus type 2 glycoprotein gB, poliovirus I VP 1, 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, 5 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 10 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 herpesvirus type 1 glycoprotein B, equine herpesvirus type 1 glycoprotein D, bovine respiratory syncytial virus attachment protein, bovine respiratory 15 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. 20 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, 25 N-acetyl-9-O-acetylneuraminic acid receptor, CD4', highly sulphated type heparin sulphate, p65, Gal alpha 1-4-Gal-containing isoreceptors, CD16b, integrin VLA-2 receptor, EV receptor, CD14, glycoconjugate receptors, alpha/beta T-cell receptor, decay-accelerating factor receptor, extracellular envelope glycoprotein receptor, immunoglobulin Fc receptor poxvirus M-T7, GALV receptor, CD14 receptor, Lewis(b) blood group antigen receptor, T 30 cell receptor, heparin sulphate glycoaminoglycans receptor, fibroblast growth factor receptor, CD11 a, CD2, G-protein coupled receptor, CD4, heparin sulphate proteoglycan, annexin II, CD13 (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 35 virulence associated opa receptors, CD46 receptor, carcinoembryonic antigen family -71- WO 99/25379 PCT/US98/24303 receptors, carcinoembryonic antigen family Bg 1 a 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, GM1, GM2, GM3, CD3, ceramide, hemagglutinin-neuraminidase protein, erythrocyte P 5 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, CCR1-5 receptor, CXCR3 receptor, CCR5 receptor, gp46 surface glycoprotein, TNFR p55 receptor, TNFp75 receptor, soluble interleukin-1 beta receptor. 10 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 15 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. 20 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, 25 adenovirus, herpes simplex type I, herpes simplex type 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, human immunodeficiency virus type II, picornaviruses, enteroviruses, caliciviridae, Norwalk group of viruses, togaviruses, 30 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-6, cercopithecine herpes virus 1, and poxviruses. 35 -72- WO 99/25379 PCT/US98/24303

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 5 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. 10

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 15 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. 20

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 polymorphic 30 epithelial mucin antigen.

65. The method according to claim 62, wherein said antigen is a human colon carcinoma-associated protein antigen. 35 -73- WO 99/25379 PCT/US98/24303

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 5 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 10 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 15 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. 20 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 25 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 30 hemagglutinin, herpes 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 35 parvovirus capsid protein, Serpulina hydodysenteriae protective antigen, bovine viral -74- WO 99/25379 PCT/US98/24303 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 5 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 herpesvirus type 1 glycoprotein B, equine herpesvirus type 1 glycoprotein D, bovine respiratory syncytial virus attachment protein, bovine respiratory syncytial virus fusion protein, bovine respiratory syncytial virus nucleocapsid protein, 10 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 15 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, 20 p65, Gal alpha 1-4-Gal-containing isoreceptors, CD16b, integrin VLA-2 receptor, EV receptor, CD14, glycoconjugate receptors, alpha/beta T-cell receptor, decay-accelerating factor receptor, extracellular envelope glycoprotein receptor, immunoglobulin Fc receptor poxvirus M-T7, GALV receptor, CD14 receptor, Lewis(b) blood group antigen receptor, T cell receptor, heparin sulphate glycoaminoglycans receptor, fibroblast growth factor 25 receptor, CD1 la, CD2, G-protein coupled receptor, CD4, heparin sulphate proteoglycan, annexin II, CD13 (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 30 receptors, carcinoembryonic antigen family Bg 1 a 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, GM1, GM2, GM3, CD3, ceramide, hemagglutinin-neuraminidase protein, erythrocyte P antigen receptor, CD36 receptor, glycophorin A receptor, interferon gamma receptor, KDEL 35 receptor, mucosal homing alpha4beta7 receptor, epidermal growth factor receptor, -75- WO 99/25379 PCT/US98/24303 alpha5betal integrin protein, non-glycosylated J774 receptor, CXCR1-4 receptor, CCR1-5 receptor, CXCR3 receptor, CCR5 receptor, gp46 surface glycoprotein, TNFR p55 receptor, TNFp75 receptor, soluble interleukin-1 beta receptor. 5 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, herpes simplex type I, herpes simplex type II, rinderpest, 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, 25 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-6, cercopithecine herpes virus 1, and poxviruses. 30 80. The method according to claim 72 or74, 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 -76- WO 99/25379 PCT/US98/24303

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 5 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. 10 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 15 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 20 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 25 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 30 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 35 amino acid substitutions being the substitution of an amino acid residue that does not have a -7735 -77- WO 99/25379 PCT/US98/24303 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. 5 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, 10 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 15 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, gonorrhea, AIDS, malaria, shigella, salmonella, hepatitis A, hepatitis C, lyme disease, encephalitis, herpes, gram negative 20 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: 30 (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 -78- WO 99/25379 PCT/US98/24303 (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. 5 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 10 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 15 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 20 (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. 25 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 30 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 35 corresponding to one or more cysteine residues that form a disulfide bond in said second -79- WO 99/25379 PCT/US98/24303 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 5 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 10 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 corresponding to one or more cysteine 15 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. 20

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 25 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, 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 30 cysteine residues that form said disulfide bond in said second immunoglobulin molecule are not stabilizing changes. 35 -80-